Fig 2: Hsp90 C-terminal inhibitor Novobiocin promotes VDAC1 oligomerization and apoptosis in tumor xenograft. HepG2-Luc Cells (1×107) were injected subcutaneously into nude mice to form xenograft tumors. (A) Tumor luminescence foci were visualized using IVIS Lumina XRMS living imaging system 14 days after injection. (B) The excised xenograft tumors from control, STA9090-treated and NB-treated mice 14 days after injection of HepG2-Luc cells (left panel) and the tumors’ weight (right panel). (C) Growth of the xenograft tumor volume every two days was recorded and calculated as V=0.5 × L × W2 (V: volume, L: length, W: width). (D) VDAC1 oligomerization of xenograft tumors were visualized by Western blot analysis after treatment with cross-linking reagent EGS (500 μM). Each lane represents different mouse. (E) The level of cleavage-PARP from xenograft tumors was determined by Western blot analysis. Each lane represents different mouse. (F) Immunohistochemistry staining with cleaved-PARP and cleaved-caspase 3 antibodies on dissection samples from xenografted tumors. Scale bar is 100 μm. Quantification of the immunohistochemistry images were analyzed by ImageJ software v1.8.0 (NIH, MD, USA) as average optical density (AOD) [AOD = Integrated Optical Density (IOD) SUM/Area SUM]. Quantitative data are mean ± SD of n = 6 mice per group. *p < 0.05, **p < 0.01.

Fig 3: Inhibition of Hsp90 activity posttranslationally decreases the amount of SARS-CoV2 proteins. Effect of overexpression (A) or knockout (B) of two Hsp90 isoforms on SARS-CoV-2 Orf7a, Orf7b, Orf3, M, and N protein expression. C and D, effect of overexpression of Hsp90β (C) or Hsp90α (D) on SARS-CoV-2 Orf7a, Orf7b, Orf3, M, and N protein expression in 90β-KO HEK293T cells. E, WT, 90α-KO, or 90β-KO HEK293T cells were transfected with M-mCherry or N-GFP vectors for 24 h and observed by fluorescence microscopy. F, cytotoxicity was measured in HEK293T cells after 12 h treatment with 17-DMAG. Cell viability was determined and calculated as a percentage of the viable cells after treatment with DMSO. G, HEK293T cells were transfected with pRK5-N or pRK5-M, and at 12 h post-transfection (hpt), fresh medium was added containing the indicated concentrations of 17-DMAG. At 24 hpt, cell lysates were subjected to immunoblot with the indicated antibodies. β-actin was used to normalize band intensity and the relative expression level of each viral protein was based on the levels of vehicle (0 μM 17-DMAG)-treated cells. H, HEK293T cells were transfected with pRK5-N or pRK5-M and fresh medium containing 10 μM 17-DMAG was added at the indicated times. At 24 hpt, cell lysates were subjected to immunoblot with the indicated antibodies. β-actin was used to normalize band intensity and the relative expression level of each viral protein was based on the levels of DMSO-treated cells; ∗p ≤ 0.05; ∗∗p ≤ 0.01; n.s.: Not significant. Hsp90, heat shock protein 90; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Fig 4: Hsp90 inhibition alleviates COVID-19 lung injury in a Syrian hamster model.A, Ad5-N/GFP-transduced hamsters were intranasally infected with 106.5 pfu/mL of SARS-CoV-2 GFP/ΔN virus. Animals were euthanized at 5 days postinfection (dpi) and lungs were harvested. B, photographs of gross pathology in lung specimens isolated from infected hamsters at 5 dpi. C, representative H&E staining of lungs from hamsters. D, histology scores determined from (C). E, qRT–PCR was used to quantify transcripts of key inflammatory factors in cytokine storms: TNF-α, IL-6, and IL-1β. Relative expression of target genes was normalized to GAPDH (n = 4–5 hamsters per group); ∗p ≤ 0.05; ∗∗p ≤ 0.01; n.s.: Not significant. COVID-19, coronavirus disease 2019; Hsp90, heat shock protein 90; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Fig 5: Effect of pregnancy and/or thermal treatment on endometrial transcript abundance of heat shock proteins. Endometrium from cyclic and pregnant gilts maintained under thermal neutral or heat stress conditions were assessed for changes in specific HSP transcript abundance using qPCR. No effect of pregnancy status or thermal treatment on (A) HSP90AA1 and (B) HSP90AB1 was observed. (C) HSPA1A was elevated in the pregnant endometrium irrespective of thermal treatment. (D) HSPA6 was increased in the pregnant endometrium under HS when compared with the other treatment groups. (E) Pregnancy status reduced HSPD1 transcript abundance whereas HS had no effect. Neither pregnancy status nor thermal treatment influenced (F) HSPB1 transcript abundance, but (G) HSPB8 was elevated in the pregnant endometrium under HS conditions. Normalized transcript abundances are reported as fold change. Different lowercase letters indicate statistical significance between treatment groups; P ≤ 0.05.