Fig. 2: Loss of the FA pathway results in deficiencies in lysosomal health.

a Lysosomal exocytosis was quantified using antibody binding surface LAMP-1. FaDu WT and FANCD2-null cells with and without SNAP23 repression and FA-deficient (FANCA-null/FANCA-null+Tg) cell lines were tested. LAMP-1 scars on the plasma membrane were quantified by flow analysis (n = 3 as biological replicates; unpaired t-test was used to determine statistical significance). b Graphical depiction of LysoTracker assay for assessing lysosomal membrane permeabilization. LysoTracker dye stains acidic compartments such as the lysosome. Depending on cell line, LMP damaged lysosomes can either be enlarged and have a higher fluorescent intensity or become damaged, leak, and/or dissolve which would be observed as lower fluorescence intensity using LysoTracker dyes. Using an LMP inducing agent (LLoMe or GPN) allows one to see the predicted dye retention of each cell line when a cell’s lysosomes are undergoing LMP. GPN was used for early experiments but due to the short shelf life, powdered LLoMe reconstituted fresh was later used. (All experiments in this figure were performed in triplicate and reported as geometric mean ± STD, Unpaired two-tailed t-test was used to determine statistical significance). c A graph representing the lysosomal health as measured by LysoTracker dye of FaDu WT and FANCD2-null cells expressing control non-targeting control (NTC-KD) or SNAP23 (SNAP23-KD) sgRNA in the presence or absence of LMP inducing agent (GPN). d A graph representing the lysosomal health as measured by LysoTracker dye of FaDu FANCA-null and FANCA-null rescued with overexpression of a WT FANCA transgene. LLoMe was used was used as LMP inducing agent. e The functional connections between FA pathway genes and lysosomal health were assayed in FaDu cells using LysoTracker. FANCA/B/F/L knockdowns are nonessential in FaDu and were assayed for effect in lysosomal and endosomal health for the FaDu cell line. FANCA/B/F/L knockdowns had statistically significant decreases in LysoTracker dye retention. f FITC-dextran was used to monitor early changes in lysosomal pH during the LMP process in FANCA/D2-null FaDu cells. The fluorescence intensity of FITC-dextran is reduced in normal lysosomes (pH 4–5) and upon LMP, neutralization of lysosomes leads to increase in fluorescence intensity. FITC-Dextran is overloaded into lysosomes then chased with fresh media and the fluorescence was measured using flow cytometry. LLoMe treatment was used as a positive control. g Lysosomal marker, LAMP-1 (green), was used to visualize lysosome number and morphology in FaDu WT and FANCD2-null with and without NTC or SNAP23 knockdown. Representative images are shown in (g) and quantification is shown in (h). h (Left) A plot of the distribution of the number of LAMP-1 foci per cell for FaDu WT and FANCD2-null with and without SNAP23-KD. (Right) A plot of the distribution of the average intensity of LAMP-1 foci for FaDu WT and FANCD2-null with and without SNAP23-KD. The open circles represent outliers defined by R base boxplot function as values > 1.5× outside the interquartile range (two-sided Mann–Whitney test was used to assess statistical significance).