Fig 2: Caspase 1 expression is reduced in response to LXR activation.BMDM were treated with vehicle (DMSO), GW3965, LXR antagonist (LXR ant) or LPS as indicated in Fig. 3A. (a) Caspase 1 mRNA expression was analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in LPS-treated cells. Values represent the mean ± SEM (n = 3–4). t-test: *p > 0.05, ns p > 0.05. (b) Caspase 1 mRNA levels in BMDM from WT and LXRα β −/− mice were analyzed by RT-qPCR. Shown is a representative experiment performed in triplicate (mean ± SD) t-test: *p ≤ 0.05, ns p > 0.05. (c) BMDM were treated with or without GW3965 and LPS for the final 6 hours with addition of ATP for the last 2 hours. Pro-caspase 1, cleaved caspase 1 and NLRP3 protein content were analyzed by immunoblotting and Hsp90 levels were used as loading control. (d) NLRP3 mRNA expression in LXR-ligand treated BMDM was analysed as in B. Shown is a representative experiment of 3 (mean ± SD). (e) NLRP3 protein levels were analyzed by immunoblotting. For (c,e), representative experiments of 2–3 independent assays are shown.

Fig 3: hHO-1 eRNAs are enriched in the cell nucleus. (A) Fractionation of the cytoplasmic and nuclear portions of HeLa cells. HeLa cells were either untreated (lanes 1 and 3) or treated with 100 µM DEM for 6 h (lanes 2 and 4) or not, and cytoplasmic (lanes 1 and 2) and nuclear portions (lanes 3 and 4) were prepared. Fractionation efficiency was validated by immunoblotting using HSP90- (Santa Cruz, sc-59577) and Pol II- (Santa Cruz, sc-899) specific antibodies as a cytoplasmic and nuclear marker, respectively. The RNA content of the cyclophilin A gene (CycA) (B), HO-1 (C), eRNA E2-3 (D) and eRNA E1-4 (E) in the cytoplasm or the nucleus was measured by real-time PCR using either TaqMan® Gene Expression Assay system or specific primer sets with Universal Probe Library Probes. The values of HO-1(C), eRNA E2-3 (D) and eRNA E1-4 (E) were normalized to CycA expression. The value of lane 3 was arbitrarily set as 1 and the relative RNA level was expressed as the mean ± SEM of three independent assays. **P < 0.01 (two-tailed, unpaired Student's t-test), ##P < 0.01 compared to the value of lane 2 (one-way ANOVA followed by a Dunnett's post hoc test for multiple parameter comparisons).

Fig 4: Immunofluorescence for acetylate-lisine, Hsp60, and Hsp90. (a–c,g–i) Representative images of COVID-19 lung parenchyma. (a,b) Representative immunofluorescence for acetylate-lisine ((a), red) and Hsp60 ((b), green); nuclei stained with DAPI are shown in blue. (c) The panel shows the merged confocal laser scanning image of acetylate-lisine, Hsp60, and nuclei. (g,h) Representative immunofluorescence for acetylate-lisine ((g), red) and Hsp90 ((h), green); nuclei stained with DAPI are shown in blue. (i) The panel shows the merged confocal laser scanning image of acetylate-lisine, Hsp90, and nuclei. (d–f,j–l) Representative images of control lung parenchyma. (d,e) Representative immunofluorescence for acetylate-lisine ((d), red) and Hsp60 ((e), green); nuclei stained with DAPI are shown in blue. (f) The panel shows the merged confocal laser scanning image of acetylate-lisine, Hsp60, and nuclei. (j,k) Representative immunofluorescence for acetylate-lisine ((j), red) and Hsp90 ((k), green); nuclei stained with DAPI are shown in blue. (l) The panel shows the merged confocal laser scanning image of acetylate-lisine, Hsp90, and nuclei. (m,n) The bar graph shows the colocalization ratio (%) for acetylate-lisine /Hsp60 (m) and acetylate-lisine /Hsp90 (n). Open bar, control; shaded bar, COVID-19. Data are presented as the means ± SD. * significantly different from COVID-19 group (p = 0.0185). # significantly different from COVID-19 group (p = 0.0282). Scale bar 25 μm.

Fig 5: Immunohistochemistry for Hsp60 and Hsp90. (a,c,e) Representative images of immunohistochemical detection of Hsp60 in lung parenchyma of the COVID-19 and control groups. (g) Histogram showing the percentage of Hsp60-positive epithelial cells in the control and COVID-19 groups. (b,d,f) Representative images of immunohistochemical detection of Hsp90 in lung parenchyma of the COVID-19 and control groups. (h) Histogram showing the percentage of Hsp90-positive epithelial cells in the COVID-19 and control groups. Magnification 400×, scale bar 20 µm. Data are presented as the means ± SD. *: p < 0.0001.