Differential effects of moderate and strong dsRNA-sensing pathway inhibition on saRNA transgene expression and cell loss.

a, Schematic of the native saRNA, E3, and E3-NSs-L* constructs designed to inhibit dsRNA-sensing pathways and report saRNA transgene expression. The native saRNA construct lacks dsRNA-sensing inhibitors. The E3 construct expresses vaccinia virus E3, a pleiotropic inhibitor of dsRNA sensing, expected to provide moderate inhibition. The E3-NSs-L* construct expresses vaccinia virus E3, and additionally includes Toscana virus NSs and Theiler’s virus L*, which target the PKR and OAS/RNase L pathways, expected to provide strong inhibition. EGFP is expressed via an IRES to report cap-independent translation, while a subgenomic promoter (depicted with an angled arrow) enables transcription of an RNA transcript that expresses mScarlet3 via cap-dependent translation. saRNA constructs were transfected into primary mouse FLS, which were labeled with BioTracker to monitor cell number. b, Representative images of EGFP (green) and mScarlet3 (red) expression in FLS transfected with native saRNA, E3, or E3-NSs-L* over 3 weeks. Scale bar = 5 mm. c, Representative images of FLS transfected with the same constructs, showing BioTracker intensity over time. Scale bar = 5 mm. d, Quantification of EGFP fluorescence over time (n = 11 biological replicates). The E3 construct provided the greatest EGFP expression, while the E3-NSs-L* construct showed intermediate levels. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. e, Quantification of mScarlet3 fluorescence over time (n = 11 biological replicates). The E3 construct provided the greatest mScarlet3 expression, while the E3-NSs-L* showed intermediate levels. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. f, Quantification of BioTracker fluorescence over time (n = 11 biological replicates). The native saRNA and E3 constructs reduced BioTracker fluorescence, indicating cell loss. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data are presented as mean ± SEM. For panels (d-f): Data were normalized to starting cell number, indicated by BioTracker intensity on day 0, prior to transfection. The mock transfection control data is also presented in Figure 5c-e. Acronyms: saRNA, self-amplifying RNA; dsRNA, double-stranded RNA; nsP, Venezuelan equine encephalitis virus non-structural protein; IRES, encephalomyocarditis virus internal ribosome entry site; moxBFP, monomeric oxidizing environment-optimized blue fluorescent protein; E3, Vaccinia virus E3 protein; NSs, Toscana virus non-structural NSs protein; L*, Theiler’s murine encephalomyelitis virus L* protein; T2A, Thosea asigna virus 2A peptide; P2A, porcine teschovirus-1 2A peptide; PKR, protein kinase R; OAS, oligoadenylate synthase; AUC, area under the curve.

saRNA induces increased phosphatidylserine staining and reduced viability, which is prevented by E3-NSs-L*.

a, Representative cropped images of Annexin V-CF800 staining, indicating phosphatidylserine exposure or loss of membrane integrity, performed daily over 6 days using a microplate imager. Scale bar = 1.5 mm. b, Representative cropped images of calcein AM staining, indicating viability, on day 7 post-treatment. Scale bar = 1.5 mm. c, Annexin V staining, quantified as the area of positive pixels determined using Li thresholding (n = 6 biological replicates). Staurosporine, native saRNA, and the E3 construct significantly increased annexin V staining, while E3-NSs-L* did not. Data are normalized to the average of the mock transfection group. Statistical significance of treatment effects at each time point compared to mock transfection was determined using two-way RM ANOVA with Bonferroni’s multiple comparisons test. Data are presented as mean ± SEM. d, Calcein AM intensity measured on day 7 post-treatment (n = 6 biological replicates). Native saRNA, and E3 significantly reduced cell viability compared to mock transfection, while E3-NSs-L* did not. Cell viability in the E3-NSs-L* group was significantly higher than the E3 group. Connecting lines indicate responses from the same biological replicate. All groups differed significantly from staurosporine; these comparisons are omitted from the figure for clarity due to the large number of statistical comparisons. Data are normalized to cell number on day 0 as determined by BioTracker staining before transfection. Statistical significance was determined by one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test comparing all groups. saRNA constructs used are shown in Fig. 1a. For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

dsRNA-sensing pathway inhibitors suppress saRNA-induced eIF2α phosphorylation but do not affect saRNA-induced reductions in eIF4E phosphorylation.

a, Phosphorylated eIF2α levels examined day 2 post-transfection by in-cell western assay (n = 6 biological replicates). Both E3 and E3-NSs-L* constructs significantly reduced eIF2α phosphorylation. Data are presented as fold-change relative to mock-transfected cells. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test. b, eIF2α levels examined day 2 post-transfection by in-cell western assay (n = 5 biological replicates). E3-NSs-L* significantly increased total eIF2α levels compared to native saRNA transfection. Data are presented as fold-change relative to mock-transfected cells. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test. c, Phosphorylated eIF4E levels examined day 2 post-transfection by in-cell western assay (n = 6 biological replicates). All saRNA constructs tested significantly reduced eIF4E phosphorylation levels compared to mock transfection. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test. d, eIF4E levels examined day 2 post-transfection by in-cell western assay (n = 6 biological replicates). One-way RM ANOVA revealed no significant differences between groups (F(3,15) = 1.207, P = 0.3410). saRNA constructs used are shown in Figure 1a. Connecting lines indicate responses from the same biological replicate. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001. The mock transfection control data are also used in Figure 6a–d.

Inhibition of NF-κB signalling attenuates saRNA-induced secretion of antiviral cytokine production.

a, FLS were transfected with the saRNA constructs depicted in Fig. 1a, and antiviral cytokines in cell culture supernatant were quantified two days later using a bead-based immunoassay (n = 6 biological replicates). Inhibition of dsRNA-sensing pathways did not broadly suppress cytokine secretion, but significantly reduced saRNA-induced production of TNF, IFN-α and IFN-β. Cytokine levels were normalized to pre-transfection cell number (measured using BioTracker), scaled within each biological replicate (highest value set to 100%), and displayed as a heatmap of group means. Detailed plots for individual cytokines are shown in Supplemental Figure S4. Statistical significance was assessed using one-way repeated-measures ANOVA on unscaled data, with multiple comparisons controlled using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (FDR = 5%). All cytokines passed significance thresholds for discovery. Treatment effects were analyzed using Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. mock transfection; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. native saRNA. The mock transfection control is shared with panel (c). b, Schematic of the moxBFP, srIκBα, and srIκBα-Smad7-SOCS1 saRNA constructs designed to inhibit inflammatory signalling. All constructs include dsRNA-sensing pathway inhibitors (vaccinia virus E3, Toscana virus NSs, and Theiler’s virus L*). The moxBFP construct, used as a control, lacks inflammatory signalling inhibitors. The srIκBα construct co-expresses srIκBα to block NF-κB signalling, representing moderate inflammatory signalling inhibition. The srIκBα-Smad7-SOCS1 construct co-expresses srIκBα, Smad7 and SOCS1 to additionally suppress TGF-β and IFN pathways, representing strong inhibition. The angled arrow denotes the subgenomic promotor. c, FLS were transfected with the saRNA constructs shown in (b), and antiviral cytokines were measured two days post-transfection (n = 6 biological replicates). Inhibition of NF-κB signalling with srIкBα broadly suppressed cytokine secretion. Data normalization and visualization were performed as described in (a), but scaling was applied independently to account for the different constructs tested. Detailed cytokine plots are shown in Supplemental Figure S5. Statistical significance was assessed using one-way repeated-measures ANOVA on unscaled data, with multiple comparisons controlled using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (FDR = 5%). All cytokines met discovery thresholds. Treatment effects were assessed using Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. mock transfection; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. moxBFP. The mock transfection control is shared panel (a). Acronyms: nsP, Venezuelan equine encephalitis virus non-structural protein; IRES, encephalomyocarditis virus internal ribosome entry site; moxBFP, monomeric oxidizing environment-optimized blue fluorescent protein; E3, Vaccinia virus E3 protein; NSs, Toscana virus non-structural NSs protein; L*, Theiler’s murine encephalomyelitis virus L* protein; T2A, Thosea asigna virus 2A peptide; P2A, porcine teschovirus-1 2A peptide, E2A, equine rhinitis A virus 2A peptide, srIкBα, super-repressor inhibitor of κBα; smad7, mothers against decapentaplegic homolog 7; SOCS1, suppressor of cytokine signalling 1; IFN-γ, interferon-γ; CXCL1, C-X-C motif chemokine ligand 1; TNF, tumor necrosis factor; MCP-1, monocyte chemoattractant protein-1; IL-12p70, interleukin-12; CCL5, chemokine ligand 5; IL-1β, interleukin-1β; CXCL10, C-X-C motif chemokine ligand 10; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-10, interleukin-10; IFN-β, interferon-β; IFN-α, interferon-α; IL-6, interleukin-6.

Differential effects of srIκBα and srIκBα-Smad7-SOCS1 on cell number and transgene expression.

a, Representative images of BioTracker staining over 3 weeks in FLS transfected with different saRNA constructs. Scale bar = 5 mm. b, Representative images of EGFP (green) and mScarlet3 (red) expression over 3 weeks in FLS transfected with different saRNA constructs. Scale bar = 5 mm. c, Quantification of BioTracker fluorescence intensity over time (n = 11 biological replicates). srIκBα induces reduction in cell number, which is prevented by srIκBα-Smad7-SOCS1. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. d, Quantification of EGFP fluorescence intensity over time (n = 11 biological replicates). All constructs showed low levels of EGFP expression. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. e, Quantification of mScarlet3 fluorescence intensity over time (n = 11 biological replicates). The srIκBα-Smad7-SOCS1 produced 2-5 times more mScarlet3 fluorescence than either moxBFP or srIκBα constructs. Statistical significance of treatment effects at each time point compared to mock transfection was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. saRNA constructs used are shown in Figure 4b. For reference, a dotted line shows the responses to native saRNA in panels c–f. For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data were normalized to cell number (BioTracker intensity) on day 0, prior to transfection. The mock transfection control data used in this figure is also presented in Figure 1d–f. Data are presented as mean ± SEM.

srIκBα reduces eIF2α phosphorylation and total eIF4E levels, effects reversed by co-expression of Smad7 and SOCS1

In-cell western assays were performed 2 days post-transfection. Data are presented as fold change relative to mock-transfected cells. a, Phosphorylation of eIF2α is significantly reduced by srIκBα, and this reduction is reversed by co-expression of Smad7 and SOCS1 (n = 6 biological replicates). Statistical significance was determined by one-way RM ANOVA and Holm-Šídák’s multiple comparisons test to compare all groups. b, Total eIF2α levels are not significantly affected by srIκBα or srIκBα-Smad7-SOCS1 (n = 6 biological replicates). One-way RM ANOVA revealed no significant differences among groups. F(2,8)=3.683, P=0.0735. c, Phosphorylation of eIF4E is not significantly affected by srIκBα or srIκBα-Smad7-SOCS1 (n = 6 biological replicates). One way RM ANOVA revealed no significant difference among groups. F(2,10)=1.336, P=0.3059. d, Total eIF4E levels are significantly reduced by srIκBα, an effect reversed by co-expression of Smad7 and SOCS1 (n = 6 biological replicates). Statistical significance was determined by one-way RM ANOVA and Holm-Šídák’s multiple comparisons test to compare all groups. saRNA constructs are described in Figure 4b. Connecting lines indicate responses from the same biological replicate. For all statistical reporting, *P < 0.05, **P < 0.01 and ***P < 0.001. Mock transfection data used for normalization are the same as in Fig. 3.

Prolonged transfection with srIκBα or srIκBα-Smad7-SOCS1 significantly reduces basal fibroblast activation factor-α (FAP-α) levels.

a, Representative in-cell western images showing FAP-α expression. Columns show different biological replicates, and rows show different treatments. The montage on the right shows FAP-α signal normalized to CellTag signal (FAP-α/CellTag). b, In-cell western assay of FAP-α expression (n = 8 biological replicates). Both srIκBα and srIκBα-Smad7-SOCS1 significantly reduce FAP-α levels compared to mock transfection, while moxBFP does not differ significantly from mock. Statistical significance was determined by one-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test to compare groups to mock transfection. Connecting lines indicate responses from the same biological replicate. **P<0.01.

ML336 enables reversible external control of transgene expression from the srIκBα-Smad7-SOCS1 construct.

a, FLS were transfected with the srIκBα-Smad7-SOCS1 construct and treated with increasing concentrations of ML336. mScarlet3 fluorescence was measured two days post-transfection and normalized to vehicle-treated controls (n = 5 biological replicates). The concentration-response curve was fit using a variable-slope sigmoidal model, yielding an IC50 of 8.5 nM (95% CI: 6.1–11.9 nM). b, ML336 was removed from the media on day 2 post-transfection, and mScarlet3 fluorescence was measured on day 3 (n = 5 biological replicates). Fluorescence changes were normalized to the maximum response within each biological replicate and fit to a bell-shaped concentration-response curve. ML336 removal at concentrations between 10 nM and 1 μM led to disinhibition of the RdRp and increased mScarlet3 expression. c, Representative images of EGFP (green) and mScarlet3 (red) expression over 13 days in FLS transfected with srIкBα-smad7-SOCS1. Cultures were treated with vehicle or 1 μM ML336, starting 1 day post-transfection. Scale bar = 5 mm. d, Representative images of calcein AM staining on day 13 post-transfection. Scale bar = 5 mm. e, Quantification of EGFP fluorescence intensity in srIκBα-Smad7-SOCS1-transfected FLS (n = 6 biological replicates). Cultures were treated with vehicle or 1 μM ML336 (treatment period indicated by shading). EGFP fluorescence was significantly lower in ML336-treated cultures compared to vehicle-treated cultures on day 7. Statistical significance relative to vehicle-treated cells was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. #P < 0.05, ###P < 0.01. f, Quantification of mScarlet3 fluorescence intensity in srIκBα-Smad7-SOCS1-transfected FLS (n = 6 biological replicates). Cultures were treated with vehicle or 1 μM ML336 (treatment period indicated by shading). mScarlet3 fluorescence was significantly lower in ML336-treated cultures compared to vehicle-treated cultures beginning on day 3. Statistical significance relative to vehicle-treated cells was determined by two-way RM ANOVA with Greenhouse–Geisser correction and Dunnett’s multiple comparisons test. #P < 0.05, ##P < 0.01, ###P < 0.001. g, Quantification of calcein AM staining on day 13 post-transfection, following 12 days of vehicle or ML336 treatment (n = 6 biological replicates). ML336-treated cultures showed no significant difference in calcein AM signal compared to mock transfection, while vehicle-treated cultures exhibited significantly lower calcein AM signals compared to both mock-transfected and ML336-treated cultures. Connecting lines indicate responses from the same biological replicate. Statistical significance was assessed by one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test. **P<0.01, ***P<0.001. All fluorescence data were normalized to BioTracker intensity on day 0 and are presented as mean ± SEM.

Blocking dsRNA-sensing pathways or saRNA replication inhibits saRNA-induced cell loss.

a, AUC analysis of BioTracker fluorescence data shown in Figure 1f, summarizing cumulative effects over the time course (n = 11 biological replicates). Increasing dsRNA-sensing pathway inhibition prevents saRNA-induced reductions in integrated BioTracker signal. Statistical analysis was performed using one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test to compare all groups. Mock transfection data is also presented in Supplemental Figure S5a. b, Quantification of mock transfection normalized CellTag signal (n = 29 biological replicates). Increasing dsRNA-sensing pathway inhibition mitigates saRNA-induced reductions in CellTag signal. Statistical analysis was performed using one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test to compare all groups. Assays were performed 2 days post-transfection. Data in this panel were pooled from in-cell western assays, incorporating data presented in Figure 3, Figure 3—figure supplement 1c, Figure 6, and additional data not shown elsewhere. c, Quantification of calcein AM viability dye signal before and one day after transfection with native saRNA or mock transfection, with or without the RdRp inhibitor ML336 (n = 4 biological replicates). Transfection with native saRNA reduced cell viability, an effect that was prevented by co-treatment with ML336. Statistical analysis was performed using two-way repeated-measures ANOVA with Greenhouse– Geisser correction, followed by Dunnett’s multiple comparisons test relative to the mock-transfected, vehicle-treated group. For all statistical reporting, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data are presented as mean ± SEM.

The E3-NSs-L* construct protects against saRNA-induced PKR upregulation, RNA degradation, and reduced global protein synthesis.

a, PKR levels examined day 2 post-transfection by in-cell western assay (n = 4 biological replicates). E3-NSs-L* significantly reduced PKR levels compared to both native saRNA and E3 transfection. Data are presented as fold-change relative to mock-transfected cells. Statistical significance was determined by one-way RM ANOVA with Tukey’s multiple comparisons test. b, rRNA integrity of FLS transfected with E3-NSs-L* is significantly higher than that of E3-transfected FLS (n = 5 biological replicates). rRNA integrity was assessed using the RNA Integrity Number (RIN) algorithm, which ranges from 1 to 10, with 10 indicating fully intact rRNA. Total RNA was extracted from FLS 1 day post-transfection. Data are shown as a Gardner-Altman comparison plot, with the right panel illustrating the mean effect size ± 95% CI. Statistical significance was assessed using a paired t-test (P = 0.0054). Dotted lines indicate group means. c, Translation rates were assessed two days post-transfection by measuring puromycin incorporation by in-cell western assay (n = 5 biological replicates). Transfection with native saRNA or the E3 construct significantly reduced the rate of protein synthesis, whereas the E3-NSs-L* construct had no effect. Statistical significance was determined by one-way repeated-measures ANOVA with Tukey’s multiple comparisons test. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Cadherin-11, a marker of fibroblast-like synoviocytes (FLS), stains cells isolated from mouse knee explants.

Representative microscopy images of FLS isolated from mouse knee explants, stained with cadherin-11 (CDH11), an FLS marker. From left to right, columns show nuclear staining (Hoechst 33342, cyan), lipophilic membrane dye labelling (Biotracker NIR680, magenta), and Alexa 488 anti-rabbit secondary immunostaining (yellow), followed by a merged image. The top row shows cells treated with rabbit anti-CDH11 primary antibody, confirming CDH11 expression in isolated cells. The bottom row presents a no primary antibody control, showing no signal in the 488 channel. Scale bar = 200 μm.

Linear unmixing corrects spectral overlap of EGFP and mScarlet3 fluorescent signals imaged using the Odyssey M laser scanner.

Schematic of the saRNA constructs used for linear unmixing, each expressing a different fluorescent protein: moxBFP, EGFP, and mScarlet3. These constructs were transfected into tSA201 cells. b, Representative imaging results showing fluorescence in the 488 and 520 channels, 1 day after transfection. moxBFP-transfected cells were not detected in either channel. EGFP was primarily detected in the 488 channel, with 11.32% bleedthrough into the 520 channel. mScarlet3 was primary detected in the 520 channel, with 0.94% bleedthrough into the 488 channel. Background signal was determined from mock transfected cells. c, Corrected images using linear unmixing to negate spectral bleedthrough from mScarlet3 and EGFP signals. Scale bars = 5 mm.

BioTracker fluorescence indicates cell number following staurosporine-induced apoptosis, despite an increase after mock transfection.

Validation of BioTracker fluorescence as a marker for cell number in the presence of apoptotic stimuli. a, Representative images of FLS stained with BioTracker under three conditions: no transfection control, mock transfection, and mock transfection with 1 μM staurosporine. BioTracker fluorescence intensity increases after mock transfection but decreases following staurosporine treatment, indicating cell loss. Scale bar = 5 mm. b, Quantification of BioTracker fluorescence over time for the three conditions (n = 3). Statistical significance was determined by two-way RM ANOVA with Dunnett’s multiple comparisons test comparing groups to the no treatment control. c, Representative images of the same FLS stained with Calcein AM. Unlike BioTracker, Calcein AM fluorescence does not increase after mock transfection but decreases after staurosporine treatment, reflecting apoptotic cell death. Scale bar = 5 mm. d, Quantification of BioTracker fluorescence over time for the three conditions (n = 3). Fluorescence decreases following staurosporine treatment, consistent with findings in (a, b). Statistical significance was determined by two-way RM ANOVA with Dunnett’s multiple comparisons test comparing groups to the no treatment control. For all statistical reporting, **P < 0.01 and ***P < 0.001. Data are presented as mean ± SEM.

Select cytokines show variable responses to dsRNA-sensing pathway inhibition following saRNA transfection.

a-m, FLS were transfected with the saRNA constructs described in Figure 1a or mock transfected. Cell culture supernatants were collected two days post-transfection, and cytokine levels were quantified by bead-based immunoassay and normalized to cell number using the BioTracker signal measured on day 0 (n = 6 biological replicates). Native saRNA triggered robust secretion of multiple antiviral cytokines. Co-expression of viral innate immune inhibitors reduced some cytokine responses and enhanced others. Both E3 and E3-NSs-L* significantly reduced secretion of IFN-α and IFN-β, and E3-NSs-L* additionally suppressed TNF. In contrast, E3 increased MCP-1, and E3-NSs-L* increased GM-CSF levels relative to native saRNA. Statistical significance was assessed using one-way repeated-measures ANOVA for each cytokine. Multiple comparisons were controlled using the Benjamini–Krieger–Yekutieli false discovery rate (FDR) procedure (Q = 5%). All cytokines passed the discovery threshold. Treatment effects were analyzed using Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± SEM. Mock transfection controls are shared with Supplemental Figure S5. Acronyms: IFN-γ, interferon-γ; CXCL1, C-X-C motif chemokine ligand 1; TNF, tumor necrosis factor; MCP-1, monocyte chemoattractant protein-1; IL-12p70, interleukin-12; CCL5, chemokine ligand 5; IL-1β, interleukin-1β; CXCL10, C-X-C motif chemokine ligand 10; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-10, interleukin-10; IFN-β, interferon-β; IFN-α, interferon-α; IL-6, interleukin-6.

Inhibition of inflammatory signalling pathways broadly reduces saRNA-induced secretion of anti-viral cytokines.

a-m, FLS were transfected with the saRNA constructs described in Figure 4b or mock transfected. Cell culture supernatants were collected two days post-transfection, and cytokine levels were quantified by bead-based immunoassay and normalized to cell number using the BioTracker signal measured on day 0 (n = 6 biological replicates). The moxBFP construct triggered secretion of antiviral cytokines, which were broadly reduced by expression of srIκBα or srIκBα-Smad7-SOCS1. Statistical significance was assessed using one-way repeated-measures ANOVA for each cytokine. Multiple comparisons were controlled using the Benjamini–Krieger–Yekutieli false discovery rate (FDR) procedure (Q = 5%). All cytokines passed the discovery threshold. Treatment effects were analyzed using Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as mean ± SEM. Mock transfection controls are shared with Supplemental Figure S4. Acronyms: IFN-γ, interferon-γ; CXCL1, C-X-C motif chemokine ligand 1; TNF, tumor necrosis factor; MCP-1, monocyte chemoattractant protein-1; IL-12p70, interleukin-12; CCL5, chemokine ligand 5; IL-1β, interleukin-1β; CXCL10, C-X-C motif chemokine ligand 10; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-10, interleukin-10; IFN-β, interferon-β; IFN-α, interferon-α; IL-6, interleukin-6.

Reduced antiviral gene expression and replicon activity observed with co-expression of inflammatory signalling inhibitors.

a, Antiviral and proinflammatory transcripts were quantified by qPCR 2 days post-transfection (n = 3 biological replicates). Statistical comparisons were performed on ΔCT values normalized to 18S rRNA using one-way ANOVA with multiple comparisons controlled using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (FDR = 5%). All investigated transcripts passed significance thresholds for discovery. Treatment effects were analyzed using Holm-Šídák’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. mock transfection; #P < 0.05 vs. native saRNA. Mean-ΔΔCT values normalized to mock transfection are shown on the heatmap (larger values indicate higher expression). b, EGFP transcript levels were quantified by qPCR at 2 days post-transfection (n = 3 biological replicates). Expression was normalized to 18S rRNA, and data are shown as ΔCT values. FLS transfected with the srIκBα-Smad7-SOCS1 construct had higher ΔCT values (indicating lower EGFP transcript levels) compared to those transfected with native saRNA or the E3 construct. Statistical comparisons were performed on ΔCT values using one-way ANOVA with Tukey’s multiple comparisons test. c, In vitro transcribed saRNA constructs were resolved by denaturing gel electrophoresis. Native saRNA (11,181 nt), E3 (11,030 nt), and E3-NSs-L* (12,562 nt) migrated as single bands corresponding to their expected sizes. In contrast, the moxBFP (13,282 nt), srIκBα (13,614 nt), and srIκBα-Smad7-SOCS1 (15,664 nt) constructs each exhibited two bands: one at the expected size and a smaller lower intensity band of consistent size across all three constructs, suggestive of a common truncated transcript. d, Band intensity plots of moxBFP, srIκBα and srIκBα-Smad7-SOCS1 derived from the gel shown in panel (c).

srIκBα reduces cell number and viability, while srIκBα-Smad7-SOCS1 preserves both.

a, AUC analysis of BioTracker fluorescence data shown in Figure 5c, summarizing cumulative effects over the time course (n = 11). srIκBα induces reductions in integrated BioTracker signal, which is prevented by srIκBα-Smad7-SOCS1. Statistical analysis was performed using one-way RM ANOVA with Greenhouse–Geisser correction and Tukey’s multiple comparisons test to compare all groups. Mock transfection data is also presented in Figure 1—figure supplement 1a. b, Quantification of mock transfection normalized CellTag signal (n = 20). The srIκBα construct reduces cell number, whereas this reduction is prevented by the srIκBα-Smad7-SOCS1 construct. Data were normalized to mock transfection. Statistical significance was determined by one-way RM ANOVA with Greenhouse–Geisser correction, and Tukey’s multiple comparisons test. Data in this panel were pooled from in-cell western assays performed on day 2 post-transfection, incorporating data presented in Figure 3 and Figure 6 as well as additional data not shown elsewhere. ***P < 0.001. c, Calcein AM staining performed 3 days post-transfection (n = 4). The srIκBα-Smad7-SOCS1 construct protects against reductions in cell viability induced by srIκBα. Data were normalized to the moxBFP construct. Statistical significance was determined by a paired t test. *P<0.05. Data are presented as mean ± SEM. saRNA constructs used are shown in Figure 4b.