Figure 5. mTORC1 signaling activates SREBP1 to induce de novo lipid biosynthesis and growth factor-independent proliferation.

A) Levels of full length (fl) and processed (p) SREBP1 are shown from littermate-derived pairs of wild-type and either Tsc1 or Tsc2 null MEFs grown in serum-free media and subjected to a time course of rapamycin (20 nM). (Below) The ratio of processed to full length SREBP1 at the indicated time points was quantified and plotted relative to untreated wild-type cells.

B) MEFs were transfected with control non-targeting siRNAs (ctl) or siRNAs targeting Raptor or Rictor, and 48 h post-transfection, cells were serum starved for 24 hours.

C, D, E) The mTORC1-induced expression of genes involved in fatty acid (C) and sterol (D) biosynthesis and the pentose phosphate pathway (E) is dependent on SREBP. MEFs were transfected with control non-targeting siRNAs (ctl) or siRNAs targeting Raptor, Srebp1 (Sr1), Srebp2 (Sr2), or both (Sr1/2). 32 h post-transfection, cells were serum starved for 16 h in the presence or absence of rapamycin (Rap, 20 nM). The expression levels of representative genes were measured by qRT-PCR and are presented as the mean±SD relative to levels in Tsc2^+/+ cells over three independent experiments. (See also Figures S5A and B)

F) Overexpression of Rheb or the processed form of SREBP1a induces G6PD expression. HEK-293 cells were transiently transfected with empty vector (Vec), FLAG-RHEB, or FLAG-processed SREBP1a. 24 h post-transfection, cells were serum starved for 16 h in the presence of vehicle or rapamycin (20 nM). G6PD mRNA levels, measured by qRT-PCR, are presented as the mean±SD relative to levels in vector-transfected cells over three independent experiments. (See also Figures S5C and D)

G) The stimulation of de novo lipid biosynthesis downstream of mTORC1 is dependent on SREBP. MEFs were transfected with siRNAs targeting the indicated transcripts or control non-targeting siRNAs (ctl). 24 h post-transfection, cells were serum starved for 24 h in the presence of vehicle or rapamycin (20 nM) and were incubated with D-[6-¹⁴C]-glucose for the final 4 h. ¹⁴C incorporation into the lipid fraction was measured and is presented as mean±SD relative to Tsc2^+/+ (Ctl) cells. These data are representative of three independent experiments. *p<0.004 for Tsc2^+/+ versus Tsc2^−/− and Tsc2^−/− versus rapamycin-treated Tsc2^−/−; **p<0.008 versus Tsc2^−/− (ctl); ***p<0.002 versus Tsc2^−/− (ctl). A control immunoblot is provided with numbering matching the samples from the graph. (See also Figure S5E)

H) The mTORC1-driven proliferation of Tsc2 null cells in the absence of growth factors is dependent on SREBP. Tsc2^+/+ (T2+/+) and Tsc2^−/− (T2−/−) MEFs were transfected with siRNAs targeting indicated genes or non-targeting control siRNAs (siCtl). 24 h post-transfection, 1×10⁵ cells per well were re-seeded and grown in serum-free medium in the presence of vehicle or rapamycin (20 nM, Rap), with daily medium changes. Triplicate samples were counted every 24 h, and the mean cell numbers ±SD for a representative experiment are shown. (See also Figure S5F)