Fig 2: RPS6KB1 drives NCOR1 nuclear localization upon loss of ULK1 activity. (A) Representative immunoblot shows the level of NCOR1 in nuclear extracts in control, Ulk1 KD and Ulk1 + Rps6kb1 double knockdown AML-12cells. Whole cell lysates were used to assess the knockdown efficiency. (B, C) Representative immunofluorescence image and quantification of NCOR1-FLAG-transfected cells using anti-FLAG antibody (red) and DAPI (green pseudo color) in cells treated with Ulk1 alone or ULK1 + RPS6KB1 siRNA for 48 h (scale bar: 200 µm). Bars represent the mean of the respective individual ratios ±SD (n = 5, #p < 0 .05 denotes significant between Ulk1 KD vs.Control and, *p < 0 .05 denotes significant difference between Ulk1 KD vs. Ulk1 KD + Rps6kb1 KD cells.

Fig 3: PIP4K2A negatively regulates cellular proliferation, clonogenic growth, and the AKT signaling pathway in patient-derived primary GBMs. (A) Effects of PIP4K2A on in vitro proliferation of GBM cells that were transduced with either control (non-target) or PIP4K2A-expressing virus. Values are presented as mean ± SD (n = 5). (B) PIP4K2A suppresses tumorsphere formation. Bar, 100 µm. (C and D) LDA for in vitro tumor sphere formation. LDA clonogenic significance was measured by linear regression analysis. (E) A schematic representation of dual-color competition assay of GBM4 cells in vivo. A total of 100,000 cells from a 1:1 mixture of RFP-labeled PIP4K2A WT cells (red) and GFP-labeled control (NT) cells (green) was injected into the mouse brains. (F) Immunoblot analysis of PIP4K2A in patient-derived GBM4 cells transduced with NT control or PIP4K2A-WT vector. (G) FACS analysis of post–in vivo dual-color competition assay. (H) Bar graph represents the relative percentage of GFP- and RFP-positive cells from the FACS analysis. (I) Representative bar graph of the number of GFP- and RFP-positive cells that were counted from different regions that were selected randomly from the post–in vivo competition assay. Values are presented as mean ± SD (n = 4). (J) Immunofluorescence images of cryosectioned mouse brains from the in vivo dual-color competition assay. Bars, 1,000 (left) and 100 µm (right). (K) Representative confocal images of immunofluorescence staining of pAKT in GBM cells that were transduced with NT virus or PIP4K2A-expressing virus. Bar, 20 µm. (L) Representative immunoblot analysis of phosphorylated-AKT (pAKT), total AKT, phosphorylated-S6K (pS6K), total S6K, and PIP4K2A expression in patient-derived tumor cells and NPCs after they were transduced with either PIP4K2A or control. Actin was used as a loading control. (M) Densitometric analysis of the blots in L. Values are presented as mean ± SEM. Data shown in A–D, F, and K–M are representative of three independent and reproducible experiments. P values: A and M, two-tailed Student’s t test. *, P = 0.05; ***, P = 0.001. Endo., endogenous; Exo., exogenous.

Fig 4: Starvation induces ER-phagy in RCS and HeLa cells through TFEB and TFE3 Western blot analysis of phospho-P70S6K (T389), P70S6K, and phospho-TFEB (S142) in RCS cultured in complete medium or HBSS for 16 h. ß-actin was used as a loading control. Bar graph shows quantification of phospho-TFEB (S142) normalized to ß-actin. Mean ± standard error of the mean (SEM) of N = 3 biological replicates. Student's paired t-test *P < 0.05.Subcellular localization analysis of TFE3 (green) in RCS chondrocytes starved with HBSS for 8 h. Torin 1 was used at 1 µM for 2 h as positive control. Nuclei were stained with DAPI (blue). Quantification analysis showed % of cells with nuclear TFEB and TFE3 in RCS with indicated treatments. Mean ± standard error of the mean (SEM) of N = 3 biological replicates/treatment. Scale bar 10 µm. n = 62 cells (control), 83 cells (HBSS), 47 cells (Torin 1). One-way analysis of variance (ANOVA) P < 0.0001; Sidak's multiple comparison test ***P < 0.0005.qRT–PCR analysis of Fam134b isoforms in wild type (ctrl) and TFEB;3KO RCS. Cells were starved overnight with HBSS where indicated. Values were normalized to Cyclophilin gene and expressed as fold change relative to control (complete medium). Mean ± standard error of the mean (SEM) of N = 3 biological replicates. One-way analysis of variance (ANOVA) P < 0.0001; Sidak's multiple comparison test ***P < 0.0005; NS, not significant.EATR assay in chondrocytes with indicated genotypes (ctrl = wild type) showing % of cells with acidified ER measured by FACS analysis. HBSS starvation was for 16 h. Mean ± standard error of the mean (SEM) of N = 3 biological replicates/treatment/genotype. One-way analysis of variance (ANOVA) P < 0.0001; Sidak's multiple comparison test ***P < 0.0005; *P < 0.05.Co-staining of CLIMP63 (green) and TMEM192-HA (lysosomes, red) in chondrocytes with indicated genotypes (ctrl = wild type) and starved with HBSS for 16 h where indicated. BaFA1 was used at 100 nM for 4 h. Scale bar 10 and 2 µm (higher magnification boxes). Bar graph shows quantification of relative CLIMP63 fluorescence in TMEM192-HA decorated lysosomes. Mean ± standard error of the mean (SEM) of N = 3 biological replicates. N = 10 cells/experiment were analyzed. One-way analysis of variance (ANOVA) P < 0.0001; Sidak's multiple comparison test ***P < 0.0005.Co-staining of CLIMP63 (green) and TMEM192-HA (lysosomes, red) in wild type (ctrl) and TFEB;3KO HeLa cells with indicated genotypes (ctrl = wild type) and starved with HBSS for 16 h where indicated. BaFA1 was used at 100 nM for 4 h. Scale bar 15 and 2 µm (higher magnification boxes). Quantification showed CLIMP63 relative fluorescence in TMEM192-HA decorated lysosomes. Mean ± standard error of the mean (SEM) of N = 3 biological replicates. N = 26 (HeLa full media), N = 29 (HeLa HBSS), N = 22 (HeLa TFEB;3KO full media), N = 25 (HeLa TFEB;3KO HBSS) cells were analyzed. Student's paired t-test *P < 0.05; NS, not significant.EATR assay in HeLa cells with indicated genotypes (ctrl = wild type) showing % of cells with acidified ER measured by FACS analysis. HBSS starvation was for 16 h. Mean ± standard error of the mean (SEM) of N = 5 biological replicates/treatment/genotype. One-way analysis of variance (ANOVA) P = 0.0002; Sidak's multiple comparison test **P < 0.005; NS, not significant. Source data are available online for this figure.

Fig 5: Activation of mTOR signaling and promotion of NAFLD in E. coli NF73-1–fed mice. (A) Increasing nuclear translocation of SREBP-1(N) and expression of SREBP-1 (P), as well as its targets, such as ACC1 and FASN, after E. coli NF73-1 treatment and decreasing PPAR-a expression after E. coli NF73-1 treatment. (B) Positive immunostaining for phosphorylated S6 (brown color), the predominant location in lipid-rich hepatocytes (red arrows) of NAFLD mice. (C) Representative immunoblots of phosphorylation of mTOR, S6K1, and S6 in livers from three mice in each group. Total liver lysates of the indicated genotypes were subjected to western blot analysis and densitometry analysis. Data are means ± SEM (n = 3). NAFLD, non-alcoholic fatty liver disease; mTOR, mammalian target of rapamycin; N, cleaved nuclear (68 kDa) form of SREBP-1; P, precursor (125 kDa) form of SREBP-1; S6K1, S6 kinase 1; SREBP-1, sterol-regulatory element binding protein-1; ACC1, acetyl-coenzyme A carboxylase; FASN, fatty acid synthase; PPAR-a, peroxisome proliferator-activated receptor alpha.