Fig. 2.

Genetic or chemical inhibition of HSP72 decreases collagen expression in vitro. (a) Western blot analysis of Hs578T cell lysates stably expressing HSPA1A-targeting shRNAs or a non-silencing control (shCtrl) showing the relative levels of the indicated proteins. Densitometry analysis represents fold-change in COL1A1 and HSP72 levels normalized to β-ACTIN relative to shRNA ctrl, n = 3. (b) RT-qPCR analysis of Hs578T cell lysates stably expressing HSPA1A-targeting shRNAs or a shCtrl showing the relative levels of the indicated mRNAs normalized to TMEM11, n = 1 biological replicate, 3 technical replicates shown. (c) Western blot analysis of cultured WT and Hsp72^−/− immortalized MEF cell lysates. Densitometry analysis shows fold change in COL1A1 levels normalized to β-ACTIN relative to respective WT MEF samples, n = 4. (d) RT-qPCR analysis of cultured WT and Hsp72⁻ immortalized MEFs showing the relative levels of the indicated mRNAs normalized to Tmem11 relative to WT samples, n = 3. (e) Western blot analysis of cell lysates of Hs578T cells treated with a titration of JG98 for 72 h, the relative expression represents values normalized to the DMSO control sample. The number of replicates represented in the densitometry analysis is n = 3 (DMSO), 1 (0.01 µM), 1 (0.1 µM), 1 (0.25 µM), 2 (0.5 µM), 3 (1.0 µM). (f) RT-qPCR analysis of Hs578T cells treated with 1 µM JG98 for 72 h, the relative expression represents values normalized to TMEM11 relative to respective DMSO control samples, n = 3. A one-sample t-test was performed for all data normalized to control samples, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent ± SD. Abbreviations used: COL1A1, collagen, type I, alpha 1; HSP72, heat shock protein 72; MEF, mouse embryonic fibroblasts.