INPP4B expression regulates lysosomal localization in PDAC cells. (a) Top 20 KEGG gene sets enriched in INPP4B^high TCGA-PAAD patients as determined using GSEA. (b) KEGG lysosome, autophagy, and endocytosis enrichment plots from analysis of INPP4B^high GSEA analysis. NES, normalized enrichment score. (c) Micrographs of indicated PDAC cell lines immunostained for LAMP1 (green) and DAPI nuclear stain (blue). Scale bar: 10 µm. (d) Whole-cell lysates from indicated PDAC cell lines were subjected to IB for INPP4B and GAPDH. Quantification of INPP4B expression levels normalized to GAPDH is shown in red. (e) Illustration of image analysis technique used to segment cell areas for nucleus, inner shell (perinuclear), and outer shell (peripheral). (f) Quantitation of LAMP1 intensity distribution for images from c as measured by peripheral (outer shell)/perinuclear (inner shell) LAMP1 intensity ratio. (g) Scatterplot correlation of INPP4B protein expression in various PDAC cell lines versus outer/inner shell LAMP1 intensity. (h and j) LAMP1 immunostaining and DAPI nuclear staining of (h) PANC-1 and (j) PK-1 stably expressing pCW-INPP4B with and without 500 nM Dox. Scale bar: 20 µm for (h) PANC-1 cells and 15 µm for (j) PK-1 cells. (i and k) Quantitation of LAMP1 intensity distribution as measured by peripheral (outer shell)/perinuclear (inner shell) LAMP1 intensity ratio for (i) PANC-1 and (k) PK-1 cells. (l) HPAC cells stably expressing CRISPR-Cas9 and indicated sgRNAs for sgCDY1B, sgINPP4B-1, or sgINPP4B-2 immunostained for LAMP1 and nuclear stained with DAPI. Scale bar: 10 µm. (m) Quantitation of LAMP1 intensity distribution for l HPAC cells. Data represent ± SD from 100 to 120 cells from three independent experiments assessed per treatment per condition, with one-way ANOVA Tukey’s post hoc test performed for f and m, and unpaired two-tailed parametric t test for i and k. ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F2.