Figure 5 from SREBP-Dependent Regulation of Lipid Homeostasis Is Required for Progression and Growth of Pancreatic Ductal Adenocarcinoma

Site-1 protease activation of the SREBP pathway is required for PDAC cell growth in low-serum conditions. A, PDAC cells (Pa03c and Pa16c) were cultured in either 10% or 1% FBS with indicated concentrations of the Site-1 protease inhibitor PF-429242 for 72 hours, and cell growth was determined using an MTS assay with data normalized to 10% FBS untreated cells. Data are representative of two biological replicates with three technical replicates for each biological replicate. Error bar denotes SD. B, Immunoblot analysis of nuclear extracts from human Pa03c and Pa16c cells cultured for 16 hours in either 10% FBS, 1% FBS, or 1% FBS-containing PF-429242 (10 μmol/L). Blots were probed for either SREBP1-N or SREBP2-N (arrow), and lysine-specific histone demethylase 1A (KDM1A) served as a loading control. The result is representative of two biological replicates. C, Pa03c and Pa16c cells were cultured under the same conditions as in B, and quantitative real-time PCR was performed for target genes of SREBP1 (SCD, FASN, and INSIG1) and SREBP2 (HMGCR, HMGCS1, and LDLR). GAPDH served as a control. Data are representative of two biological replicates with three technical replicates for each biological replicate. Error bar denotes standard deviation. Statistical significance was determined using one-way ANOVA and Tukey honestly significant difference (HSD) test. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. D, Immunoblot analysis of whole cell lysates from Pa03c and Pa16c cells cultured as in B for indicated SREBP target protein expression. The asterisk indicates a nonspecific band present in Pa03c and Pa16c cell lines. Actin served as a loading control. The result is representative of two biological replicates.