Fig. 1: HSP27 downregulation alone or even combined with HSP27/TGF-β1 downregulation is not sufficient as a targetable molecule in sorafenib-resistant HCC.

A Validation of TGF-β1 and HSP27 mRNA downregulation in SNU449 and sorafenib-resistant SNU449 (SNU449-SR)cells was examined by quantitative real-time PCR after 36 h of infection with adenovirus expressing shRNA targeting TGF-β1, shRNA targeting HSP27 or shRNAs targeting HSP27/TGF-β1 at an MOI of 100. B (top) Cell viability of SNU449 and sorafenib-resistant SNU449 (SNU449-SR) cells was examined after 24 h of infection with adenovirus expressing shRNA targeting TGF-β1, shRNA targeting HSP27 or shRNAs targeting HSP27/TGF-β1 at an MOI of 100, followed by sorafenib (15 μM) treatment for 24 h in HCC and sorafenib-resistant HCC. The cells were infected with adenovirus expressing nonsense shRNA as a negative control at an MOI of 100. Cell viability was tested by Trypan blue. The error bars represent the standard errors from three independent experiments. (bottom) Clonogenic assays of SNU449 and SNU449-SR cells were performed after infection with adenovirus expressing shRNA of TGF-β1 or shRNA of HSP27 or shRNAs of HSP27/TGF-β1 followed by sorafenib (15 μM) treatment in HCC and sorafenib-resistant HCC and incubation for an additional 14 days after appropriate dilution of treated samples. MOI multiplicity of infection, NC negative control. C Transcriptome profiling analysis of SNU449 and SNU449-SR through shRNA of HSP27 or shRNA of TGF-β1 or shRNAs of HSP27/TGF-β1. The expression values of the DEGs are shown in a heatmap. Gene expression levels are visualized as row-standardized z-scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of SNU449 and SNU449-SR (shRNA of HSP27 or shRNA of TGF-β1 or shRNAs of HSP27/TGF-β1). D SNU449 or SNU449-SR cells were infected with adenovirus expressing 100 MOI of shRNA of HSP27 or shRNA of TGF-β1 or shRNAs of HSP27/TGF-β1. Scatter plots of the relative probe set intensities after infection were subsequently generated. Normalized probe set intensities of various infections are plotted. The numbers indicate over 2-fold variation in intensities outside of two diagonal lines. E Cellular levels of GRP78 or vimentin mRNA (left) or protein (right) before and after acquired sorafenib resistance in SNU449 cells were examined by real-time PCR or western blotting, respectively. F SNU449 or SNU449-SR cells were infected with adenovirus expressing shRNAs of HSP27/TGF-β1 at 100 MOI, and the endogenous cellular level of GRP78 or vimentin mRNA was subsequently examined via real-time PCR after RNA extraction. The cells were infected with adenovirus expressing nonsense shRNA as a negative control (NC) at an MOI of 100. G SNU449 or SNU449-SR cells were infected with adenovirus expressing shRNA targeting HSP27, shRNA targeting TGF-β1 or shRNAs targeting HSP27/TGF-β1 at an MOI of 100. The cells were infected with adenovirus expressing nonsense shRNA as a negative control at an MOI of 100. After 48 h, the expression levels of vimentin, GRP78 and β-actin were detected via western blot analysis. The statistical significance was determined by Student’s t tests (A, E, F) or two-way analysis of variance (ANOVA)(B). Data are shown as mean values ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant.