Extended Data Fig. 9: Inhibition of PIKfyve activates SREBP-dependent transcriptional signatures through disrupting lysosomal lipid homeostasis pathways.
From: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer
a. Immunoblots of 7940B, PANC1, MIA PaCa-2, and HPNE cells upon treatment with apilimod (100 nM for 7940B, 300 nM for PANC1, MIA PaCa-2, and HPNE), ESK981 (1000 nM), chloroquine (30 μM), or bafilomycin A1 (30 nM) for eight hours showing changes in SREBP1 (P), SREBP1 (M), and LC3A/B. Vinculin or histone H3 were used as loading controls. b. qPCR of 7940B, PANC1, MIA PaCa-2, or KPC1361 cells upon treatment with apilimod (100 nM for 7940B, 300 nM for PANC1, MIA PaCa-2, KPC 1361), ESK981 (1000 nM), chloroquine (CQ, 30 μM), or bafilomycin A1 (BF, 30 nM) for eight hours showing changes in FASN or ACACA mRNA levels as indicated. Bars are +/-SD. (Two-way ANOVA with Dunnett’s multiple comparison test, setting DMSO as the reference for each target for each cell line.). c. Immunoblots of PANC1 and MIA PaCa-2 cells treated with apilimod (300 nM), ESK981 (1000 nM), chloroquine (30 μM), bafilomycin (30 nM), or U18666A (5 μg/mL) for 8 hours showing changes in SREBP1 (P) and SREBP1 (M). Histone H3 or vinculin are used as loading controls. d. qPCR of MIA PaCa-2, PANC1, and 7940B cells assessing changes in RNA of FASN upon treatment with apilimod (100 nM for 7940B, 300 nM for PANC1, MIA PaCa-2) or U18666A (5 μg/mL) for 8 hours. Bars are +/-SD. (One-way ANOVA with Dunnett’s, with DMSO as the baseline.). e. Immunofluorescence images of PANC1 cells treated with DMSO, apilimod (1000 nM), ESK981 (1000 nM), or U18666A (5 μg/mL) for 24 hours stained with filipin or LAMP1. White squares indicate cropped area used for Fig. 3f. Scalebars= 5 μm. Statistics and reproducibility: a. This experiment was performed twice in MIA PaCa-2 cells with similar results and once in all other cells. b. n = 3 technical replicates per group. P-values: 7940B: Fasn: DMSO vs apilimod:1.6e-5; DMSO vs ESK981:1.2e-5; DMSO vs bafilomycin:3.0e-6; Acaca: DMSO vs apilimod:2.7e-5; DMSO vs ESK981:2.0e-5; KPC 1361: Fasn: DMSO vs apilimod:5.9e-7; DMSO vs ESK981:4.3e-5; DMSO vs BAF:7.8e-5; Acaca: DMSO vs apilimod:3.8e-6; DMSO vs ESK981:1.4e-5 PANC1: FASN: DMSO vs ESK981:5.8e-6; ACACA: DMSO vs ESK981:6.3e-6 MIA PaCa-2: FASN: DMSO vs apilimod:6.3e-8; DMSO vs chloroquine:9.1e-7; DMSO vs bafilomycin:5.9e-10; ACACA: DMSO vs bafilomycin:1.5e-5. c. These experiments were performed once each. d. n = 3 technical replicates per group. P-values: FASN: MIA PaCa-2: DMSO vs apilimod:1.5e-4; PANC1: DMSO vs apilimod:4.4e-5; Fasn: 7940B: DMSO vs apilimod:4.4e-6. e. These images are representative of n = 2 images each.
