Fig. 4: Analysis of the endolysosomal pathway in the TMEM16K absence.

RUSH assay 1st row: Scheme of the RUSH construct biosynthetic pathway. 2nd and 3rd rows: Representative images of WT and TMEM16K KO cells at the indicated time points. 4th row: Quantification at indicated time point. a RUSH assay with mCherry-GPI. Quantification of surface vs. total at 60 min. Two-tailed t-test, p value = 0.53 n.s. (three biological replicates, n = 53 WT and 56 TMEM16K KO cells) b RUSH assay with mCherry-TfR. Pearson’s correlation coefficient at 120 min. Two-tailed t-test, p value = 0.21 n.s. (three biological replicates, n = 129 WT, and 111 TMEM16K KO cells) c RUSH assay with GFP-CD-MPR. Pearson’s correlation coefficient at 60 min with GM130 (three biological replicates, n = 126 WT, and 127 TMEM16K KO cells, Two-tailed t-test, p value = 0.45 n.s.), and Pearson’s correlation coefficient at 120 min with mCherry-TfR RUSH (three biological replicates, n = 144 WT and 134 TMEM16K KO cells, Two-tailed t-test, p value =  0.17 n.s.) d Fluorescence intensity of transferrin at 60 min in the WT (n = 100) and TMEM16K KO (n = 50) cells from three biological replicates, Two-tailed t-test, p value = 0.63 n.s. e EGF-Alexa647 pulse-chase experiment was quantified for colocalization with endogenous Rab7, Two-tailed t-test between WT and KO at each measured time point (three biological replicates, n = 130 WT, and 117 TMEM16K KO cells at 10 min, p value = 0.75 n.s., 91 WT, and 103 KO cells at 15 min, p value = 0.88 n.s., 77 WT and 61 KO cells at 40 min, p value = 0.60 n.s., 89 WT, and 118 KO cells at 60 min, p value = 0.043*). f Fluorescence intensity of Lysosensor Green DNP-189 in WT (n = 114) and TMEM16K KO (n = 116) cells. Single factor ANOVA p value = 8E−25*** from three biological replicates. g Representative trace from three independent experiments of protonophore FCCP at a final concentration 2 µM added at 120 s to cells loaded with Lysosensor Green DNP-189. (WT slope is −0.0445, y = −0.0445x + 128.45, R² = 0.9537; TMEM16K KO slope is −0.0305, y = −0.0305x + 109.5, R² = 0.9581) h Evaluation of wild type and mutant TMEM16K cDNA ability to rescue acidification defect. WT or TMEM16K KO cells were co-transfected with mCherry-CAAX to visualize transfected cells, and TMEM16K wild type cDNA (TMEM16K-FLAG) or TMEM16K mutant cDNA (Ca5MUT-FLAG) and evaluated for acidification with Lysosensor Green D-189. Single factor ANOVA, p value = 1.46E−39 with post-test Bonferroni-corrected two sided t-test with pairwise comparison with WT + 16K wild type (three biological replicates, n = 50 WT + 16K wild type; 50 WT + 16K mutant, p value = 1.85E−07***; 40 KO + 16K wild type; 40 KO + 16K mutant cells, p value = 3.27E−19***). In the box and whiskers plot, the box includes the first quartile and the third quartile, with the central line representing the median. Whiskers represent the minimum and maximum values of data. X inside the box represents the mean of data. Source data are provided as a Source Data file.