Fig 2: CLCC1 and ORF3A physically interact.293T cells were transfected with the indicated plasmids (top). Forty-eight hours after transfection, the cells were lysed and immunoprecipiataion was performed with the indicated antibody. (A) Western blots for CLCC1 and HA are shown for the anti-HA immunoprecipitation. (B) Western blots for HA and FLAG are shown for the anti-FLAG precipitation. Both panels representative of three independent experiments. (C) 293T cells transfected with the indicated plasmids were stained for endogenous CLCC1, ORF3A (anti-HA) and the endoplasmic reticulum (anti-PDI) n = 2. (D) 293T cells were transfected with wild type CLCC1 of D25E CLCC1 and ORF3A-HA and stained for FLAG, HA, and PDI n = 3. (E) 293T cells were transfected with ORF3A-HA and stained for PDI and RCAS1 (golgi) n = 3. (F) 293T cells were transfected with ORF3A-HA and stained for HA and WGA (plasma membrane) n = 4. (G) 293T cells were transfected with ORF3A-HA and stained for HA, EEA1, and LAMP1 n = 4. (H) For each marker, the median ORF3A intensity in the listed organelle is plotted. Standard deviation is shown. The blue dashed line indicates the median intensity of anti-HA-ORF3A for all ORF3A positive masks, while the red dashed line represents the intensity outside the ORF3A mask within cells. n = 4 lysosome, golgi, ER, endo-some, n = 8 whole cell, nucleus, membrane, cytoplasm.

Fig 3: PDIA1 depletion inhibits NLRP3 inflammasome activation.A. Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocytes stably expressing shRNAs of the respective target PDI. were stimulated for 3 h with LPS (1 μg/mL) and 24 h with ATP (5 mM) or nigericin (1 μM). *p<0.05, ***p<0.001, ****p<0.0001 two-way ANOVA test with Dunnett correction for multiple comparisons. Error bars show SD. B. Relative number of ASC-GFP specks per cell detected using fluorescent microscopy in undifferentiated THP1 ASC-GFP monocyte cells stably expressing either a scramble shRNA or a PDIA1 shRNA (PDIA1KD). Cells were stimulated with LPS (1 μg/mL) for 16 h and nigericin (5 μM) for 3 h. Quantification is relative to the overall average number of specks / total cells across all wells in LPS (1 μg/mL) and nigericin (5 μM) stimulated wells. ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. C. Caspase-1 activity assessed using Caspase-1 Glo assay from PDIA1KD or scramble THP1 monocytes stimulated for 16 h with LPS (1 μg/mL) and 45 min nigericin (1 μM). Error bars show SD; each dot represents an individual replicate relative to LPS. ****p<0.0001 using a two-way ANOVA. D. Immunoblot of cell lysates from PDIA1KD or scramble THP1 cells stimulated with LPS (1 μg/mL) for 3 h and nigericin (5 μM) for 1 h. ZVAD-VAD-FMK (50 μM) was used as a positive control. E. Quantification of protein levels measured using immunoblots from cell lysates from PDIA1KD or scramble THP1 cells stimulated with LPS (1 μg/mL) for 3 h. *p<0.05, ***p<0.001. ****p<0.0001 using multiple unpaired two-tailed t-tests. Error bars show SEM for n=3.

Fig 4: Pharmacologic PDI inhibitors block NLRP3 inflammasome activation.A. Chemical structures of the PDI inhibitors KSC-34 and RB-11-CA.[41] B. Relative number of ASC-GFP specks per cell detected using fluorescent microscopy in undifferentiated THP1 ASC-GFP expressing monocyte cells pre-treated with KSC-34 (20 μM) and stimulated with LPS (1 μg/mL) for 16 h followed by a treatment of nigericin (5 μM) for 3 h. Quantification is relative to the overall average number of specks / total cells across all wells in LPS (1 μg/mL) and nigericin (5 μM) stimulated wells. ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. B. Rhodamine fluorescence and Coomassie staining of an SDS-PAGE gel of lysates from THP1 cells treated for 16 h with AA147alk (10 μM), KSC-34 (20 μM), or RB-11-CA (20 μM). C. Relative number of ASC-GFP specks per cell detected using fluorescent microscopy in undifferentiated THP1 ASC-GFP expressing monocyte cells pre-treated with KSC-34 (20 μM) for 16 h then stimulated with LPS (1 μg/mL) for 3 h and nigericin for 3 h (5 μM). ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. D. Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocytes pre-treated with KSC-34 (20 μM) or RB-11-CA (20 μM) and subsequently stimulated for 3 h with LPS (1 μg/mL) and 24 h with nigericin (1 μM) relative to an unstimulated, vehicle treated control. *p< 0.05, ***p<0.001, ****p<0.0001 with one-way ANOVA test with Dunnett correction for multiple comparisons. Error bars show SEM for n=3.