Figures and data
ORMDL3 negatively regulates RLR induced type I IFN signaling pathway.
(A) HEK293T cells were transfected with an empty vector (EV) or a ORMDL3 plasmid for 12 h and were then infected with VSV (MOI = 0.01) or transfected with polyI:C. The expression of the mRNAs encoded by the indicated genes was detected using qRT-PCR. (B) HEK293T-EV and HEK293T–Flag-ORMDL3 cells were transfected with or without polyI:C, and immunoblot analyses of phosphorylated IRF3 (p-IRF3), total IRF3, GAPDH and ORMDL3 levels were performed. (C) Results of the qRT-PCR assays showing mRNA levels of IFNB1 in A549 cells transfected with control or ORMDL3 followed by transfected with polyI:C or polyG:C. (D) Results of the qRT-PCR assays showing mRNA levels of Ifnb1 in mice primary BMDM cells infected with control or psc-AAV-ORMDL3 virus followed by transfected with polyI:C or polyG:C. (E) Results of the luciferase assay showing IFN-β-Luc activity in HEK293T cells transfected with EV or ORMDL3 plasmids together with individual EV, RIG-I, cGAS plus STING, and TRIF plasmids for 24 h. (F) Results of the luciferase assay showing ISRE-Luc activity in HEK293T cells transfected with EV or ORMDL3 plasmids together with individual EV, RIG-I, cGAS plus STING, and TRIF plasmids for 24 h. (G) Wild-type and ORMDL3 stable knockdown and overexpression A549 cells were infected with VSV-GFP (MOI = 0.01) for 12 hours, and viral infectivity was detected using fluorescence microscopy. (H) Wild-type and ORMDL3 stable knockdown A549 cells were infected with HSV-1 (MOI = 0.1) for 24 h, and viral infectivity was detected using fluorescence microscopy. *p < 0.05, **p < 0.01, ****p < 0.0001. Data are representative of at least two independent experiments.
ORMDL3 regulates the protein abundance of RIG-I.
(A,B) RT-PCR analyses of the expression of the indicated mRNAs in HEK293T cells transfected with EV or ORMDL3 plasmids combined with co-transfection of individual plasmids encoding EV, RIG-I, MAVS, TBK1,IRF3-5D. (C) Results of the qRT-PCR assays showing mRNA levels of IFNB1 CCL5 and ISG54 in HEK293T cells transfected with control or ORMDL3-specific siRNAs followed by secondary transfection with EV or RIG-I-N plasmids. (D) Results of the WB assays show protein levels in HEK293T cells transfected with control or ORMDL3-specific siRNAs followed by secondary transfection with EV or RIG-I-N plasmids. (E) Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag-RIG-I,RIG-I-N-Myc and Flag-MDA5 IRF3,HA-TBK1, and increasing doses of expression vector for Flag-ORMDL3. (F) Immunoblot analysis of extracts of 293T cells transfected with expression vector for RIG-I-N-Myc and RIG-I-C-Myc and increasing doses of expression vector for Flag-ORMDL3. (G) HEK293T cells were transfected with Flag-RIG-I and ORMDL3-Myc plasmids, as indicated, with or without polyI:C co-transfection. Cell lysates were immunoblotted with a-Flag and a-Myc antibodies. (H) Results of the qRT-PCR.assays showing IFNB1,CCL5,ISG54,IL-6 and TNF mRNA in HEK293T cells transfected with EV or ORMDL3 plasmids together with individual RIG-I-N plasmids for 24 h. *p < 0.05, **p < 0.01, ****p < 0.0001. Data are representative of at least two independent experiments.
ORMDL3 promotes the proteasome degradation of RIG-I.
(A) HEK293T cells were transfected with plasmids encoding RIG-I-N-Myc together with increasing amounts of Flag-ORMDL3 plasmid treated with MG132 (10 μM), chloroquine (CQ) (50 μM) for 6h and the cell lysates were analyzed by immunoblot. (B) HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with an GFP antibody (a-GFP) followed by immunoblots using GFP (a-GFP) and a-HA antibodies. (C) 293T cells were transfected with RIG-I-N-Myc (WT or KR) and increasing doses of expression vector for Flag-ORMDL3. The expression levels of RIG-I-N-Myc were analyzed by immunoblot. (D) 293T cells were transfected with Rig—N,RIG-I-N-Myc (WT, KR,mutant1,mutant2), and increasing doses of expression vector for Flag-ORMDL3. The expression levels of RIG-I-N-Myc were analyzed by immunoblot. (E) 293T cells were transfected with RIG-I-N-Myc (WT, K146R, K154R, K164R, K172R) with or without Flag-ORMDL3. The expression levels of RIG-I-N-Myc and its mutant forms were analyzed by immunoblot. (F) HEK293T cells were transfected with RIG-I-N-Myc (WT or 4KR) and increasing doses of expression vector for Flag-ORMDL3. The expression levels of RIG-I-N-Myc (WT or 4KR) were analyzed by immunoblot. (G) HEK293T cells were transfected with RIG-I-N-GFP and HA-KUb,HA-K48Ub and HA-K63Ub with EV or Flag-ORMDL3, and cell lysates were immunoprecipitated with GFP antibody (a-GFP) followed by immunoblots using GFP (a-GFP) and a-HA antibodies. (H) BMDM cells were infected with psc-AAV-GFP and psc-AAV-ORMDL3-Flag virus,followed by immunoblot analysis of RIG-I, Flag and Tubulin.
ORMDL3 interacts with signaling adaptor MAVS.
(A) HEK293T cells were transfected with EV or Flag-RIG-I/MAVS/MDA5/TBK1/IRF3/IKKE with ORMDL3-GFP, and cell lysates were immunoprecipitated with Flag antibody (a-Flag) followed by immunoblots using GFP (a-GFP) and a-Flag antibodies. (B) HEK293T cells were transfected with different MAVS truncations with EV or Flag-ORMDL3, and cell lysates were immunoprecipitated with Flag antibody (a-Flag) followed by immunoblots using HA (a-HA) and a-Flag antibodies. (C) HEK293T cells were transfected with EV or Flag-MAVS with different ORMDL3 truncations, and cell lysates were immunoprecipitated with Flag antibody (a-Flag) followed by immunoblots using GFP (a-GFP) and a-Flag antibodies. (D) HEK293T cells were transfected with EV or Flag-MAVS with different ORMDL3 truncations, followed by RT-PCR analysis of IFNB1,CCL5,ISG54,ISG56. (E) HEK293T cells were transfected with RIG-I-N-Myc with EV or different ORMDL3 truncations, followed by immunoblots using GFP (a-GFP) and a-Myc antibodies. (F) FRET experiment of YFP-MAVS and CFP-ORMDL3 in Hela cells,YFP-MAVS is the donor and CFP-ORMDL3 is the acceptor,FRET efficiency is 24.4019%. *p < 0.05, **p < 0.01, ****p < 0.0001. Data are representative of at least two independent experiments.
USP10 induces RIG-I stabilization.
(A) Protein interacted with ORMDL3 screened from mass spectrometry results. (B) HEK293T cells were transfected with ORMDL3-GFP and Flag-USP10 plasmids, as indicated, with or without polyI:C cotransfection. Cell lysates were immunoprecipitated with the a-Flag antibody, and immunoblotted with a-Flag and a-GFP antibodies. (C) HEK293T cells were transfected with RIG-I-MYC and EV or Flag-USP10 plasmids, Cell lysates were immunoprecipitated with the a-Flag antibody, and immunoblotted with a-Flag and a-Myc antibodies. (D) Immunoblot the protein level of RIG-I in USP10 stable overexpression HEK293T cell line. (E) Immunoblot the protein level of RIG-I in USP10 stable knockdown HEK293T cell line. (F) IP and immunoblot analysis of 293T cells transfected with vectors expressing Flag-RIG-I and HA-K48 linked ubiquitin with or without USP10-Myc. (G) HEK293T cells were transfected with RIG-I-N-Myc (WT or 4KR) and increasing doses of expression vector for Flag-USP10. The expression levels of RIG-I-N-Myc were analyzed by immunoblot. (H) USP10 stable knockdown HEK293T cell line was transfected with RIG-I-N-Myc and Flag-ORMDL3. The expression levels of RIG-I-N-Myc were analyzed by immunoblot.
ORMDL3 disturbs USP10 induced RIG-I stabilization.
(A) HEK293T cells were transfected with Flag-USP10 and RIG-I-Myc plasmids, as indicated, with or without ORMDL3 co-transfection. Cell lysates were immunoprecipitated with the a-Flag antibody, and immunoblotted with a-Flag and a-Myc antibodies. (B) Immunoblot analysis of extracts of HEK293T cells transfected with Flag-USP10 and Flag-MAVS and Flag-IRF3 and RIG-I-N-Myc with or without ORMDL3 co-transfection, the cell lysates were analyzed by immunoblot. (C) 293T cells were transfected with plasmids encoding RIG-I-N-Myc together with Flag-USP10 with or without Flag-ORMDL3 plasmid treated with MG132 (10 μM), chloroquine (CQ) (50 μM) for 6 h The cell lysates were analyzed by immunoblot. (D) HEK293T cells were transfected with Flag-USP10 and RIG-I-N-Myc(WT,KR,mutant1,mutant2) plasmids, as indicated, with or without Flag-ORMDL3 co-transfection. Cell lysates were immunoblotted with a-Flag and a-Myc antibodies. (E) 293T cell were transfected with RIG-I-N-Myc (WT or K146R K154R K164R K172R) and Flag-USP10 with or without Flag-ORMDL3. The expression levels of RIG-I-N-Myc(WT or K146R K154R K164R K172R) were analyzed by immunoblot. (F) HEK293T cells were transfected with Flag-USP10 and RIG-I-N-Myc(WT and 4KR) plasmids, as indicated, with or without Flag-ORMDL3 co-transfection. Cell lysates were immunoblotted with a-Flag and a-Myc antibodies. (G) IP and immunoblot analysis of 293T cells transfected with vectors expressing RIG-I-N-GFP/RIG-I-N-4KR-GFP and HA-K48 Linked ubiquitin with or without USP10 transfection. (H) HEK293T cells were transfected with Flag-USP10 and RIG-I-N-Myc and with or without ORMDL3 co-transfection followed by RT-PCR analysis of IFNB1,CCL5.ISG54. *p < 0.05, **p < 0.01, ****p < 0.0001. Data are representative of at least two independent experiments.
Knockdown of LLC/MC38 ORMDL3 enhances anti-tumor immunity.
(A to C) LLC tumor representative images on day 21 after tumor inoculation with shNC, shORMDL3-1, shORMDL3-2 LLC stable knockdown cell line (A) tumor weight (B) tumor growth (C) on day 21 after subcutaneous injection of 1.5×10^6LLC in C57BL/6. (D) Results of the qRT-PCR assays showing mRNA levels of Ccl5, Cxcl10, Tnf, Il-6 of LLC tumor. (E,F) Flow cytometry assay of CD3+ T cells in CD45+, CD107a+ CD8+ T cells percentages. (G to I) MC38 tumor representative images on day 27 after tumor inoculation with shNC, shORMDL3-1, shORMDL3-2 MC38 stable knockdown cell line (G) tumor weight (H) tumor growth (I) on day 27 after subcutaneous injection of 5×10^5 MC38 in C57BL/6. (J) Results of the qRT-PCR assays showing mRNA levels of Ifnb1,Ccl5.Cxcl10 of MC38 tumor. (K) Results of the IHC assay showing expression levels of ORMDL3,RIG-I and CD8 in MC38 tumor.*p < 0.05, **p < 0.01, ****p < 0.0001. Data are representative of at least two independent experiments.
ORMDL3 expression under different treatments.
(A) Results of the WB and qRT-PCR assays showing ORMDL3 protein and RNA levels HCT15 DLD1 SW480 SW620 CRC cell line and A549 293T THP1 infected with HSV (MOI = 0.1) for indicated times. (B) Results of the WB and qRT-PCR assays showing ORMDL3 protein and RNA levels HCT15 DLD1 SW480 SW620 CRC cell line and A549 293T THP1 infected with VSV-1 (MOI = 0.01) for indicated times. (C) Results of the WB and qRT-PCR assays showing ORMDL3 protein and RNA levels HCT15 DLD1 SW480 SW620 CRC cell line and A549 293T THP1 treated with RIG-I agonist SB9200 for indicated times.
ORMDL3 downregulation is conservative in both human and murine cells.
(A) RT-PCR analysis of CXCL10 when ORMDL3 was co-expressed with RIG-I MAVS TBK1 or IRF3-5D. (B) Results of the qRT-PCR.assays showing IFNB1, CCL5, ISG54 mRNA in HEK293T cells transfected with EV or ORMDL3 plasmids together with individual RIG-I-N or MDA5-N plasmids for 24 h. (C) Immunoblot assay of exogenous RIG-I expression in HEK293T transfected with ORMDL3. (D) Immunoblot assay of exogenous Rig-I expression in HEK293T transfected with Ormdl3. (E) Immunoblot assay of endogenous RIG-I expression in HEK293T transfected with ORMDL3. (F) The diagram of RIG-I truncations.
ORMDL3 promotes the degradation of RIG-I.
(A) RT-PCR assay of RIG-I-N mRNA levels in HEK293T cells transfected with RIG-I-N and increasing amounts of ORMDL3. (B) Different annotations of KR mutation of RIG-I-N.
ORMDL3 constrains IFN pathway by its domains.
(A) HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with an HA antibody (a-HA) followed by immunoblots using GFP (a-GFP) and a-HA antibodies. (B) Scheme of the truncations of ORMDL3. (C) HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with a Flag antibody (a-Flag) followed by immunoblots using GFP (a-GFP) and a-Flag antibodies. (D) Results of the luciferase assays showing IFNβ activity in HEK293T cells transfected with ORMDL3 truncations with RIG-I-N. (E) Results of the qRT-PCR assays showing mRNA levels of IFNB1, CCL5 in HEK293T cells transfected with ORMDL3 truncations with MAVS. (F) HEK293T cells were transfected with RIG-I-N-Myc and different truncation of ORMDL3 and followed by immunoblot assay.
Inhibiting ORMDL3 enhances the abundance of RIG-I.
(A,B) Flow cytometry assay of CD8+T cells in CD3+ and CD44+CD8+ T cells percentages. (C) Immunoblot assay of LLC tumor in group shNC, shOrmdl3-1,shOrmdl3-2, followed by analysis of protein level of ORMDL3 and RIG-I. (D) RT-PCR showing the shRNA knockdown of ORMDL3 or shRNA control in LLC lung cancer cells. (E) Immunoblot showing the shRNA knockdown of ORMDL3 or shRNA control in LLC lung cancer cells. (F) RT-PCR showing the shRNA knockdown of ORMDL3 or shRNA control in MC38 colon cancer cells. (G) Immunoblot showing the shRNA knockdown of ORMDL3 or shRNA control in MC38 colon cancer cancer cells.
ORMDL3 expression is associated with poor survival and reduced immune cell infiltration and ISGs.
(A) ORMDL3 expression between tumor and adjacent normal tissues in The Cancer Genome Atlas(TCGA) pan-cancer cohorts. (B to D) Association of ORMDL3 level with overall survival(OS),progression free survival(PFS) and disease-specific survival(DSS) in TCGA-LUAD. (E) Correlation among ORMDL3 expression with stromalscore and CD8T cell infiltration in LUAD cohort from TCGA datasets. (F) Correlation among ORMDL3 expression with CD8 activation markers: CD8A, PRF1, GZMA, GZMB and ISG: CCL5, CXCL10, CD274 and PDCD1LG2 in The Cancer Genome Atlas (TCGA) pan-cancer cohorts form the TIMER2.0 database.
Schematic show that ORMDL3 promotes the degradation of RIG-I and attenuates IFN in cancer.
