The binding and effect of single- and double-stranded RNA on PSMα3.

(A) EMSA assay illustrating the interaction between PSMα3 and RNA. The assay compares the effects of increasing concentrations of PSMα3 (0 µM, 40 µM, 80 µM, 160 µM, and 320 µM), shown in a gradient from left to right, on the mobility shift of single-stranded Poly(A) RNA (left panel) and double-stranded Poly (AU) RNA (right panel) at ∼400ng/µL. (B) Transmission electron micrographs of 100 µM PSMα3 incubated for 2 hours in the presence of 400 ng/µL of either Poly(A) RNA (left) or Poly (AU) RNA (right). Scale bars represent 500 nm.

Colocalization, droplet formation and texture of PSMα3 mixed with varying Poly (AU) RNA concentrations.

(A-B) Widefield fluorescence microscopy images of 100 µM PSMα3-FITC (green) in the presence of 50 ng/µL (A) or 400 ng/µL (B) Poly (AU) RNA (red), showing their individual and composite channel images. (C-E) FRAP analysis of PSMα3-FITC with 50 ng/µL Poly (AU) RNA taken after 10 min (C) or 2-hours co-incubation (D), or with 400 ng/µL Poly (AU) RNA taken after 10 min co-incubation (E). The images in panels C-D were captured before bleaching, immediately after bleaching, and 40 seconds post-bleaching. Scale bars in all images represent 20 µm.

TEM and TIRF visualization of PSMα3 aggregation and morphology with different Poly (AU) RNA concentrations and incubation times.

(A) TEM micrographs of 100 µM PSMα3 incubated with or without Poly (AU) RNA at varying concentrations of 10 ng/µL, 50 ng/µL, and 400 ng/µL for 2 hours (top row) and 24 hours (bottom row). Scale bars represent 500 nm. (B) TIRF microscopy images showing 100 µM FITC-PSMα3 (green) co-incubated with 50 ng/µL Poly (AU) RNA and the amyloid indicator AT630 (magenta) for 30 minutes and 2 hours. Scale bars represent 20 μm. (C) TIRF microscopy images of 100 µM FITC-PSMα3 (green) co-incubated with 400 ng/µL Poly (AU) RNA and AT630 (magenta) for 30 min. Scale bars represent 20 µm.

Impact of Poly (AU) RNA on PSMα3 cytotoxicity and antibacterial activity.

Antimicrobial activity of PSMα3 against E. Coli using the PrestoBlue cell viability assay (A) and its cytotoxicity against HeLa cells using the LDH colorimetric assay (B) were assessed with and without Poly (AU) RNA at varying concentrations. The experiments were performed in at least three replicates and repeated across three independent days to ensure result reliability. Cytotoxicity and bacterial cell viability percentages were calculated as the mean of all replicates, with error bars representing the standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA for normally distributed data in GraphPad Prism (version 11). Significance levels are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Colocalization of PSMα3 with nucleic acids in HeLa cells.

(A) Confocal microscopy images showing the localization and colocalization of 20 µM PSMα3-FITC (green) and nucleic acids stained with PI (red) inside the nucleolus of HeLa cells (indicated in arrows). The left panel illustrates the distribution of PSMα3 within the cell. The middle panel shows the nucleic acids stained with PI. The right panel is a composite image that demonstrates the colocalization of PSMα3 with nucleic acids. Scale bars represent 15 µm. (B) FRAP analysis of 20 µM FITC-labeled PSMα3 inside the nucleolus (indicated by the arrow) of HeLa cells, showing fluorescence recovery after 60 seconds. Scale bars represent 10 µm.

EGCG modulates PSMα3 aggregation and reduces toxicity against HeLa cells.

(A) TEM micrographs of 100 μM PSMα3 incubated for 24 hours, without (left) and with (right) a fivefold molar excess of EGCG. Scale bars: 500 nm. (B) Live-cell confocal microscopy of HeLa cells treated with 20 μM PSMα3-FITC (green) without (top) and with EGCG (bottom), imaged immediately after preparation (t = 0) and after 10 minutes. Hoechst 33342 (blue) marks the nuclei, while PI staining (red) indicates membrane disruption. Scale bars: 10 μm. (C) Cytotoxicity of PSMα3 against HeLa cells in the presence and absence of EGCG and Poly (AU) RNA at two different concentrations, assessed via LDH assay. The experiment was performed in triplicate and repeated on three separate days for consistency. Cytotoxicity percentages were averaged across all replicates, with error bars representing the standard error of the mean. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA, GraphPad Prism v11).

Residue-specific interactions between PSMα3 and EGCG.

(A) One-dimensional (1D) 1H NMR spectra of 1.0 mM PSMα3 alone (red) and in complex with 0.5 mM EGCG (blue), recorded at 35 °C. Specific residues, including Glu8/Met1 and Glu2, show chemical shift changes suggestive of direct interaction with EGCG (highlighted in the upper left). Dashed boxes mark proton signals corresponding to EGCG. Peak broadening of H1/H2 protons, compared to the EGCG-only reference sample (light gray), indicates interaction from the EGCG side. Slight opalescence observed in the sample suggests potential aggregate formation. (B) Two-dimensional (2D) 1H–1H TOCSY and NOESY spectra of the PSMα3:EGCG complex at a 2:1 ratio, recorded at 35 °C. The cross-peaks in the TOCSY spectrum allow the identification of the spin system and direct connections through the scalar coupling between the proton amide (HN) and the alpha protons (Hα) of the same residue. The cross-peaks in the NOESY spectrum establish a sequential connection between neighboring residues (i+1), allowing spectral assignment. The remaining cross-peaks (i+3 or i+4) in the spectrum support PSMα3 secondary structure in the experimental conditions. (C) Temporal stability of the PSMα3:EGCG sample over 3 days.

Effect of RNA concentration on LL37 phase separation, aggregation, and activity at pH 7 after heat shock.

Fluorescence microscopy images showing LL37 (FITC-labeled, green) in the presence of increasing concentrations: 100 ng/μL (A), 200 ng/μL (B) and 400 ng/μL (C) of Poly (AU) RNA (PI-labeled, red) after heat shock at 65°C for 15 minutes at pH 7. Scale bars represent 20 µm. (D) LL37 cytotoxicity, with and without Poly (AU) RNA and EGCG at varying concentrations, was assessed in HeLa cells using the LDH colorimetric assay. The experiment was performed in triplicate and repeated on three separate days for reproducibility. Cytotoxicity percentages represent the average of all replicates, with error bars indicating the standard error of the mean. (E) The antimicrobial activity of LL37, with and without Poly (AU) RNA and EGCG at varying concentrations, was evaluated against E. coli using the PrestoBlue Cell Viability assay. The experiment was performed in triplicate and repeated on three separate days for reproducibility. Bacterial viability percentages represent the average of all replicates, with error bars indicating the standard error of the mean. Statistical significance (D-E): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA, GraphPad Prism v11).

Encapsulation of Poly (AU) RNA within PSMα3-FITC droplets.

FITC-PSMα3 (green) was mixed with 50 ng/µL Poly (AU) RNA labeled with PI. The left panel shows the FITC green channel, the middle panel displays the PI red channel, and the right panel is a composite image showing the overlap of PSMα3-FITC and Poly (AU) RNA-PI signals. Scale bars represent 20 µm.

Turbidity measurements of PSMα3 with increasing Poly (AU) RNA concentrations.

PSMα3 concentration was maintained at 100 µM, and Poly (AU) RNA concentrations varied across 0, 10, 20, 50, 100, 200, and 400 ng/µL. Data points represent the average optical density at 400 nm (OD400) recorded within the first 30 minutes post-resuspension, with each condition tested in triplicate. Error bars indicate the standard error of the mean.

RNA stabilizes an α-helical conformation of PSMα3 over time.

Solid-state circular dichroism (ssCD) spectra of PSMα3 incubated alone or with polyAU RNA at concentrations of 50 ng/µL and 400 ng/µL. Spectra were collected immediately after preparation (A), and after 2 hours of incubation (B). Measurements were recorded in the far-UV range (180–250 nm) to assess changes in secondary structure under the indicated conditions.

EGCG inhibits PSMα3 Fibrillation.

Fibrillation kinetics of 100 µM freshly dissolved PSMα3, monitored by Thioflavin-T (ThT) fluorescence. The data compares PSMα3 fibrillation in the absence (blue curve) and presence (red curve) of EGCG. The graph displays the mean fluorescence intensities from triplicate ThT measurements, with error bars representing the standard error of the mean.

EGCG reduces the antimicrobial activity of PSMα3 against E. coli.

(A) Super-resolution light microscopy images of E. coli treated with 20 μM PSMα3-FITC (green) in the absence (left) and presence (right) of EGCG at a 1:5 molar ratio. Scale bars: 5 μm. (B) Antimicrobial activity of PSMα3 against E. coli, evaluated using the PrestoBlue cell viability assay, with and without EGCG (1:5 molar ratio). The experiments were performed in at least three replicates and repeated across three independent days to ensure result reliability. Cytotoxicity and bacterial cell viability percentages were calculated as the mean of all replicates, with error bars representing the standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA for normally distributed data in GraphPad Prism (version 11). Significance levels are indicated as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

LL37 undergoes aggregation with Poly (AU) RNA before heat shock.

Light microscopy images showing LL37-FITC (green) forming aggregates in the presence of 100 ng/μL Poly (AU) RNA, stained with propidium iodide (red). The composite image (right) highlights the colocalization of LL37 and RNA (yellow), indicating RNA-induced aggregation. Scale bars: 20 μm.

PSMα3 aggregates with Poly (AU) RNA after heat shock at physiological pH.

Light microscopy images showing PSMα3-FITC (green) forming aggregates with 50 ng/μL Poly (AU) RNA, stained with propidium iodide (red), after heat shock at 60°C for 15 minutes in 50 mM HEPES, 150 mM NaCl, at physiological pH (7.4). The composite image (right) highlights the colocalization of PSMα3 and RNA (yellow), indicating RNA-induced aggregation under heat stress. Scale bars: 20 μm.

Effect of RNA concentration on PSMα3 phase separation and aggregation at pH 4 after heat shock.

Fluorescence microscopy images showing the effects on PSMα3-FITC (green) in the presence of increasing concentrations of Poly (AU) RNA (labeled with Propidium Iodide, red) following heat shock at 65°C for 15 minutes at pH 4. at 50 ng/μL (A), 100 ng/μL (B), 200 ng/μL (C) and 400 ng/μL (D). At a low RNA concentration of 50 ng/μL (A), clear phase separation of PSMα3 was observed, as evidenced by the presence of well-defined, spherical droplets. As the RNA concentration increased to 100 ng/μL, the droplets began to lose their spherical structure and showed more morphological irregularities (B). At RNA concentrations of 200 ng/μL and 400 ng/μL, a distinct transition to aggregation with irregular, amorphous clusters were observed (C and D, respectively). The composite images revealed an overlap between the PSMα3 and RNA, indicating co-localization during both phase separation and amorphous cluster formation. Scale bars in all images represent 20 µm.

Poly (AU) RNA Modulates LL-37 Aggregation Dynamics Before and After Heat Shock.

TEM micrographs of 100 µM LL-37 incubated alone or with 100 ng/µL or 400 ng/µL Poly (AU) RNA for 2 hours or 24 hours. (A) Samples imaged before heat shock at the indicated point. (B) Samples subjected to a 65°C heat shock for 15 minutes, followed by further incubation for 2 or 24 hours before imaging.