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. 2022 Mar 28;30(7):2554–2567. doi: 10.1016/j.ymthe.2022.03.015

Figure 5.

Figure 5

Matrix stiffness modulates SCD1 expression

HCC cells expressing control (shControl) or SCD1 shRNA (shSCD1) were grown on polyacrylamide hydrogels with the indicated rigidities (1.6 and 25.6 kPa) and then switched from 1.6 to 25.6 kPa (1.6–25.6 kPa) or from 25.6 to 1.6 kPa (25.6–1.6 kPa) in 3D Matrigel overlay culture (A) Representative bright-field images of HCC cells. The in vitro invasion of HCC cells is quantified by the degree of cell scattering using ImageJ software. (B) SCD1 expression was analyzed by western blot. (C) qRT-PCR analysis of SCD1 mRNA expression in HCC cells on polyacrylamide hydrogels with the indicated rigidities (1.6, 3.2, 6.4, and 25.6 kPa) in 3D Matrigel overlay culture. (D) Effect of matrix stiffness on SCD1 protein stability was analyzed by western blot in the presence of cycloheximide (CHX). The half-life (T1/2) of SCD1 was calculated. HCC cells were collected at the indicated time points after exposure to CHX, and cell lysates were subjected to immunoblotting. (E) SCD1 protein expression was increased in HCC cells treated with the proteasome inhibitor MG-132. (F) The level of ubiquitinated SCD1 was reduced in HCC cells cultured on stiff matrix. (G and H) HCC cells were treated with the anti-β1-integrin antibody or p-FAK inhibitor PF562271 (PF). SCD1 expression was analyzed by western blot. ∗∗∗p < 0.001.