Figure 5

PAT4 regulates a rapamycin-resistant form of mTORC1 in HCT116 cells and increases rapamycin resistance in HEK-293 cells. (a and b) Clones of HCT116 cells carrying the IPTG-inducible PAT4 shRNA construct, shPAT4(4.8), and the IPTG-inducible non-targeting control construct, shNT, were cultured for 5 days in the absence or presence of IPTG and, if required, treated with rapamycin for the last 24 h. Rapamycin (3 nM; see Supplementary Figures S2A and B) strongly reduces phospho-S6 levels (p-S240/244-S6), and partially affects the Ser65-phosphorylated 4E-BP1 (p-S65-4E-BP1) γ-band (arrow). This rapamycin-resistant phospho-4E-BP1 γ-band is almost completely lost after PAT4 knockdown (b). (c) shPAT4(4.8) cells and shNT controls were cultured for 8 days in the absence or presence of IPTG and treated with rapamycin for 3 days. Cells were then counted revealing reduced proliferation for both cell lines in the presence of rapamycin, and also for shPAT4(4.8) in the presence of IPTG (n=3). The combination of both rapamycin and IPTG leads to further reduction in the cell number of shPAT4(4.8). (d and e) Normal HEK-293 cells or cells stably transfected with a constitutively expressed GFP-PAT4 construct were cultured for 24 h in the presence or absence of 100 nM rapamycin (see Supplementary Figure S2F), and then cell lysates analysed by western analysis. GFP-PAT4 expression reduces the effect of rapamycin on the γ-phosphorylated 4E-BP1 band (e), but not phospho-S6 (p-S240/244-S6), phospho-Akt (p-S473-AKT) or phospho-ERK (p-T202/Y204-ERK). All blots were probed with an anti-tubulin antibody as a loading control. (*P<0.05; n=3). The cell proliferation experiment was repeated three times. NS, not significant.