a. A Western blot probing for phosphorylated MAPK/p38 reveals that SEB-1 sebocytes do not phosphorylate p38 in response to IGF-1. C-6 glioma cells treated with anisomycin serve as a positive control for phosphorylated p38, while unstimulated C-6 glioma cells represent the negative control. The phospho p38 blot was stripped and re-probed with an antibody that recognizes total p38 as a loading control.
b. A Western blot probing for phosphorylated SAPK/JNK reveals that SEB-1 sebocytes do not phosphorylate SAPK/JNK in response to IGF-1. Total cell extracts from 293 cells treated with UV light (Cell Signaling Technology) serve as a positive control for phosphorylated JNK, while untreated 293 extracts serve as the negative control. The phospho SAPK/JNK was stripped and re-probed with an antibody that recognizes total SAPK/JNK as a loading control.

The ¹⁴C incorporation assay was performed on SEB-1 cells treated with IGF-1 (20 ng/ml) and/or 50 µM of a pharmacological inhibitor of PI3-K (LY294002) or MAPK/ERK (PD98059). IGF-1 induces a robust increase in lipogenesis (black striped bars vs. solid black bars). Interestingly, addition of 50 µM PD98059 to cells treated with IGF-1 has no effect on the IGF-1 induced lipogenesis (striped red bars vs. solid red bars). Most importantly, the addition of 50 µM PI3-K inhibitor LY294002 blocks any induction of lipogenesis when cells are stimulated with IGF-1 (blue striped bars vs. blue solid bars). All samples within an experiment were done in triplicate, and each experiment was performed at least three separate times. Data were analyzed by ANOVA and considered significant if a p-value of <0.05 was observed compared to the DMSO vehicle treated control group. Abbreviations used in this figure are: C=cholesterol; CO=cholesterol oleate; FOH=fatty alcohols; OA=oleic acid; SQ=squalene; TAG=triglycerides; WE=wax esters.

a. Western blot reveals that p44/p42 (ERK1/2) is phosphorylated by IGF-1 in SEB-1 cells and that this stimulation can be blocked by 50 µM PD98059. The phospho p44/p42 blot was stripped and re-probed with an antibody that recognizes total p44/p42 regardless of phosphorylation status to serve as a loading control. Data indicate that IGF-1 activates the MAPK/ERK pathway and that this activation is blocked in the presence of a MEK inhibitor.
b. SEB-1 cells were treated with IGF-1 (20 ng/ml) and/or MEK inhibitor PD98059 (50 µM) and QPCR was performed to quantify SREBP-1a and —1c mRNA. Data are representative of 6 samples per treatment group as analyzed by the REST —XL program. These data indicate that inhibition of the MAPK/ERK pathway does not abrogate the induction of SREBP-1 mRNA by IGF-1.
c. Western blot was used to confirm that the MEK inhibitor PD98059 did not abrogate the induction of SREBP-1 protein by IGF-1 in SEB-1 cells. After being probed for SREBP-1, the membrane was stained with Simply Blue to ensure equal protein loading.

a. Western blot reveals that Akt is phosphorylated by IGF-1 at 24 hours and that this induction is blocked when cells are treated with the PI3-K inhibitor LY294002 for 30 minutes prior to the addition of IGF-1.
b. SEB-1 cells were treated with 50 µM of the PI3-K inhibitor LY294002 and/or 20 ng/mL IGF-1 for 24 hours and QPCR was performed to look for changes in the mRNA for SREBP-1a and —1c. Data are representative of 6 samples per treatment group as analyzed by the REST—XL program. These data indicate that inhibition of the PI3-K pathway with LY294002 abrogates the IGF-1 induced increase in expression of mRNA for both SREBP-1a and –1c.
c. Western blot probing for SREBP-1 was performed in SEB-1 cells in the presence or absence of IGF-1 (20 ng/ml) or the PI3-K inhibitor LY294002. β-actin was used as the loading control. Data indicate that the precursor and mature forms of the SREBP-1 protein are not induced by IGF-1 in the presence of the PI-3K inhibitor, which supports the QPCR data above.
d. Previous studies indicate that 1µM insulin (which can act through the IGF-1 receptor) increases SREBP-1 expression whereas lower doses of insulin (100 nM) did not. Western blots were performed with 2 doses of insulin in the presence and absence of the PI3-K inhibitor to test the hypothesis that Akt is activated by high dose insulin, but not low dose insulin. Data indicate that 1µM insulin induces a robust increase in pAkt that is blocked in the presence of the PI3-K inhibitor. In contrast, negligible activation of Akt is noted with 100 nM insulin. These data suggest that although lipogenesis can be stimulated by activation of the insulin receptor or the IGF-1 receptor in SEB-1 sebocytes, different intracellular signaling pathways are involved.

SEB-1 cells were treated with IGF-1 (20ng/ml) in the presence or absence of 50 µM of the PI3-K inhibitor LY294002 and QPCR was performed. IGF-1 induces a robust increase in the expression of SREBP target genes that is blocked in the presence of the PI3-K inhibitor (which blocks SREBP induction). FAS, ACS, and SCD are traditional targets of SREBP-1, as they are involved in fatty acid biosynthesis. SQE and HMGCR are involved in cholesterol biosynthesis. Data are representative of 5 samples per treatment group as analyzed by the REST program. The following abbreviations are used: FAS=fatty acid synthase; ACS=Acyl-CoA synthetase; SCD=stearoyl-CoA desaturase; SQE=squalene epoxidase; HMGCR=HMG-CoA reductase.