Fig. 4. CASPASE-3-induced SREBP1 activation but not cleavage is reduced in HCC cells with SPTBN1 knockdown.

(A) Western blot of the indicated proteins or protein cleavage products in THLE2 cells exposed to the indicated concentrations of TNFα or cycloheximide for 3 hrs.

(B) Abundance of V5-tagged N-SPTBN1 or C-SPTBN1 expressed in THLE2 cells in the cytoplasmic or nuclear fractions.

(C) Western blot of the indicated proteins or protein cleavage products in Huh7 cells transfected with the indicated siRNAs and exposed to vehicle or TNFα (20 ng/ml) + cycloheximide (10 μg/ml) for 3 hrs. Black vertical line indicates samples were analyzed on a same blot separated by other samples.

(D) Relative mRNA abundance for the SREBP1 target gene (SCD1) and the SREBP2 target gene (LDLR) in Huh7 cells as described in C. Data are normalized to 18S and shown as mean ± SEM (n = 3). Significance was determined by 2-sided t-test (**, p < 0.005).

(E) Western blot of the indicated proteins or protein cleavage products in Huh7 cells exposed to the indicated concentrations of PA or the caspase inhibitor Z-DEVD-FMK.

(F) Coimmunoprecipitation of SPTBN1 and detection of pre-SREBP1, N-SPTBN1, and SPTBN1 in Huh7 cells exposed to PA (0.33 mM) for the indicated times.

(G) Western blot of the indicated proteins or protein cleavage products in Huh7 cells treated with indicated siRNA and exposed to the indicated concentrations of PA or the caspase inhibitor Z-DEVD-FMK.

Western blot data are representative of 1 of 2–3 experiments. CHX, cycloheximide; IP, immunoprecipitate; siCtrl, control siRNA; siSPTBN1; siRNA against SPTBN1. For B, C, E, G, quantification of 2 independent experiments were shown on the right. Each band was quantified by Image J and normalization to loading control. Treatment group was set as 1 in panel C, E, G; cytoplasm group was set as 1 in panel B.