Extended Data Fig. 6: PIKfyve inhibition activates an SREBP-dependent lipogenic transcriptional signature.
From: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer
a. Scatter plot of log2 fold change in gene expression upon 8-hour treatment with apilimod (100 nM) vs DMSO (x-axis) and ESK981 (1000 nM) vs DMSO (y-axis). A linear regression was calculated with r- and p-values displayed. b. GSEA plots of cholesterol homeostasis and fatty acid metabolism using the fold change rank-ordered gene signature from the 7940B cells treated with apilimod or ESK981 for 8 hours. (GSEA enrichment test.). c-d. Volcano plot using RNA-seq analysis on 7940B cells treated with apilimod (25 nM, c.) or ESK981 (250 nM, d.) for eight hours highlighting SREBP-1 target genes. Vertical dashed lines indicate log2 fold change = +/-0.5). Horizontal dashed line indicates FDR = 10−6. e. Immunoblot showing PIKfyve, premature SREBP1 (SREBP1 (P)), and mature SREBP1 (SREBP1 (M)) in MIA PaCa-2, PANC-1, and 7940B cells upon treatment with PIKfyve inhibitors or degrader PIK5-33d (33 d) for 8 hours. Vinculin or histone H3 were used as loading controls. The drug doses used were as follows: MIA-PaCa-2 and PANC-1: apilimod=300 nM, ESK981 = 1000 nM, PIK5-33d = 1000 nM; 7940B: apilimod=100 nM, ESK981 = 1000 nM, PIK5-33d = 1000 nM. f. qPCR of MIA PaCa-2 and PANC1 showing changes in RNA levels of FASN upon CRISPRi-mediated knockdown of PIKFYVE. Bars are +/-SD. (One-way ANOVA with Dunnett’s.). g. Immunoblot analysis of MIA PaCa-2 and PANC1 showing changes in protein levels of FASN upon CRISPRi-mediated knockdown of PIKFYVE using two independent sgRNAs targeting PIKFYVE relative to control. Vinculin was used as a loading control. h. qPCR of MIA PaCa-2, PANC-1, and 7940B showing changes in RNA levels of labeled genes upon treatment with PIKfyve inhibitors for 8 hours. The drug doses used were as follows: MIA PaCa-2 and PANC-1: apilimod = 300 nM, ESK981 = 1000 nM. 7940B: apilimod = 100 nM, ESK981 = 1000 nM. Bars are +/-SD. (One-way ANOVA with Dunnett’s.). i-j. Immunoblot analysis of MIA PaCa-2, PANC1 (i.), and 7940B (j.) cells showing changes in protein levels of labeled genes upon treatment with PIKfyve inhibitors for 24 hours. Vinculin was used as a loading control. The drug doses used are indicated on the figure or as follows for PANC-1: apilimod = 300 nM, ESK981 = 1000 nM, PIK5-33d = 1000 nM. k. Immunoblot of MIA PaCa-2, PANC1, and 7940B cells treated with apilimod (100 nM for 7940B, 300 nM for MIA PaCa-2 and PANC1) or ESK981 (1000 nM) for 24 hours showing changes in LDLR. Vinculin was used as a loading control. l. qPCR showing changes in RNA levels of SQLE, FDFT1, HMGCR, or LDLR upon treatment with apilimod (100 nM for 7940B, 300 nM for MIA PaCa-2 and PANC1), or ESK981 (1000 nM) for 8 hours. Bars are +/-SD. (One-way ANOVA with Dunnett’s.). m. Immunoblot analysis of 7940B, PANC1, and MIA PaCa-2 cells upon treatment with apilimod (100 nM for 7940B, 300 nM for PANC1, and MIA PaCa-2), ESK981 (1000 nM), fatostatin (20 μM), apilimod + fatostatin, or ESK981 + fatostatin as indicated for eight hours showing changes in premature SREBP1 (P) and mature SREBP1 (M). Vinculin was used as a loading control for all blots. n. qPCR showing changes in FASN (top) or ACACA (bottom) upon treatment with apilimod (100 nM for 7940B, 300 nM for PANC1 and MIA PaCa-2), ESK981 (1000 nM), fatostatin (20 μM), apilimod + fatostatin, or ESK981 + fatostatin as indicated for eight hours. Bars are +/-SD. (One way ANOVA with Šídák’s multiple comparisons test.). o. qPCR of HPNE, MIA PaCa-2, and PANC1 cells showing differential baseline expressions of SREBF1 (left), FASN (middle), and ACACA (right). Ct values were normalized to β-actin first and then to HPNE. Bars are +/-SD. (One-way ANOVA with Dunnett’s.). Statistics and reproducibility: e. These experiments were performed twice with similar results. f. n = 3 technical triplicates. P-value: PANC1 FASN: sgNC vs sgPIKFYVE-1:5.5e-5. This experiment was performed thrice in MIA PaCa-2 cells with similar results and once in PANC1 cells. g. These experiments were performed once each. h. n = 3 technical triplicates. P-values: MIA PaCa-2 FASN: DMSO vs apilimod:2.0e-5; ACACA: DMSO vs ESK981:2.3e-5; PANC1 FASN DMSO vs apilimod:3.8e-7; DMSO vs ESK981:4.5e-6; 7940B Fasn DMSO vs apilimod:9.3e-7; DMSO vs ESK981:7.3e-5. These experiments were performed three independent times each with similar results. i. This experiment was performed once in each cell line. j. This experiment was performed twice with similar results. k. These experiments were performed once each. l. n = 3 technical triplicates per group. P-values: MIA PaCa-2: HMGCR: DMSO vs apilimod:5.3e-7; LDLR: DMSO vs apilimod:8.9e-7; PANC1: SQLE: DMSO vs apilimod:3.2e-8; DMSO vs ESK981:1.0e-6; HMGCR: DMSO vs apilimod:3.2e-8; DMSO vs ESK981:2.0e-5; 7940B: Fdft1: DMSO vs apilimod:1.2e-7; DMSO vs ESK981:2.0e-5; Hmgcr: DMSO vs apilmod:2.0e-7; DMSO vs ESK981:7.4e-6; Ldlr: DMSO vs apilimod:4.3e-5; DMSO vs ESK981:7.7e-5. m. These experiments were performed twice in each cell line. n. n = 3 technical triplicates per group. P-values: 7940B: Fasn: apilimod vs apilimod + fatostatin:2.4e-7; PANC1: FASN: ESK981 vs ESK981 + fatostatin:2.5e-6; ACACA: apilimod vs apilimod + fatostatin:3.2e-5; ESK981 vs ESK981 + fatostatin:1.8e-5; MIA PaCa-2: FASN: apilimod vs apilimod + fatostatin:1.1e-7; ACACA: apilimod vs apilimod + fatostatin:3.4e-5; ESK981 vs ESK981 + fatostatin:1.7e-5. HPNE: FASN: DMSO vs apilimod:2.8e-11; DMSO vs ESK981:2.6e-7; apilimod vs apilimod + fatostatin:6.9e-10; ESK981 vs ESK981 + fatostatin:6.8e-9; ACACA: DMSO vs apilimod:3.0e-5; apilimod vs apilimod + fatostatin:8.5e-5; ESK981 vs ESK981 + fatostatin:1.0e-7. o. n = 3 technical triplicates per group. P-values: SREBF1: HPNE vs MIA PaCa-2:1.5e-5; HPNE vs PANC1:1.5e-8; FASN: HPNE vs MIA PaCa-2:1.1e-6; HPNE vs PANC1:1.9e-9.
