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. Author manuscript; available in PMC: 2011 May 3.
Published in final edited form as: Mol Cancer Res. 2010 Jan 12;8(1):93–106. doi: 10.1158/1541-7786.MCR-08-0491

Figure 5. TRC8 knockdown upregulates SREBP-2.

Figure 5

(A) HEK293 cells cultured in DMEM (10% FCS) were transfected with the indicated siRNAs (mock, scrambled or TRC8-specific [Ambion #-136327]). Detergent lysates were analyzed 72h post-transfection for pre-SREBP-2 and actin. Densitometry was performed on serial dilutions of replicate (n = 6) transfected lysates, relative expression values are means, +/− s.d. Representative blots are shown. (B) Duplicate cultures of HEK293 cells stably transfected with the inducible shRNA-TRC8 construct were treated for 4 days with vehicle (−) or dox (+), as indicated, to knockdown endogenous TRC8 (Fig. S2C). Cells were then incubated with fresh 5% FCS (+ sterols) or sterol-depletion media (sterols) for 20 h, harvested and processed into membrane and nuclear fractions. Western blots were analyzed for precursor and nucSREBP-2 (1D2); calnexin and Coomassie stain provided loading controls; knockdown of TRC8 was verified using anti-RNF139 antibody. (C) Cell cultures identical to (B) were treated with excess sterols (12.5 μg/mL cholesterol/10 μg/mL 25-HC) for the final 6h prior to harvest to arrest processing and accumulate SREBP precursors. Membrane and nuclear fractions were analyzed for SREBP-2 (1D2); knockdown of TRC8 was verified using anti-RNF139 antibody. Calnexin and a Coomassie stained gel provided loading controls. (D) Cultures of TRC8-HA or control (vec) HEK293 cells in DMEM (10% FCS) were treated with vehicle or dox (100 ng/mL) to induce TRC8-HA and harvested for RNA 24 h later. qRT-PCR was conducted for the indicated genes. Ct values were normalized geometric means of 4 controls; HMBS is graphed as a negative control. Bars represent means +/− SEM; *p < 0.05; **p < 0.01 by Student’s t-test (n = 6). Vector – dox (open bars); vector + dox (grey bars); TRC8-HA – dox (striped bars); TRC8-HA + dox (black bars). (E). TRC8 knockdown cells, treated with dox as in (B, C) to reduce TRC8, were sterol-deprived for the indicated times. Quadruplicate samples were analyzed by qRT-PCR for SREBP target genes. Bars represent means +/− SEM; *p < 0.05; **p < 0.01 by Student’s t-test (n = 4). TRC8 k/d cells – dox (open bars); + dox (black bars).