Fig. 4: TMEM165 preserves lysosomal Ca2+stores. | Nature Communications

Fig. 4: TMEM165 preserves lysosomal Ca2+stores.

From: Lysosomal TMEM165 controls cellular ion homeostasis and survival by mediating lysosomal Ca2+ import and H+ efflux

Fig. 4

Relative Fluo-4-AM FLIs (a) and pH-corrected Fura-2-AM FLI ratios (b, see Supplementary Fig. 4a, b for the basis of pH corrections) in indicated HeLa and MDA-MB-468 cells treated with 300 μM GPN for 300 sec. siRNA transfection was performed 72 h before the treatment (a). Bars, SD of three independent experiments with ≥10000 (a) or ≥15000 (b) cells analyzed in each sample. Ca2+ signals in HEK293 cells transiently transfected with TRPML1-GCaMP6s and mCherry (control, n = 19), TMEM165-WT-mCherry (n = 17) or TMEM165-R126C-mCherry (n = 17) (c) or indicated siRNAs (n = 4) from a representative experiment (d). Thin and bold lines represent Ca2+ signal curves in response to 10 μM ML-SA1 in individual cells and the mean values of all cells analyzed, respectively. The bar charts represent mean values for the area under the curve corresponding to relative fluorescence unit (RFU) x seconds. Bars, SEM of 17–19 (c) and 4 (d) independent experiments with 3–10 cells in each sample. e Representative images of live HEK293 cells transfected as in d and stained with CellMaskTM green plasma membrane stain, LysoTrackerTM Deep Red and Hoechst-33342 (left) and same cells treated with 1 µM apilimod for 16 h, fixed, and stained with LAMP1 antibodies and DAPI (right). Asterisks indicate colocalization of LysoTracker/LAMP1 and TMEM165. Scale bars, 10 µm. f Schematic presentation of Lyso-GCaMP8s. g Relative Lyso-GCaMP8s FLIs in indicated HeLa (left and middle) and MDA-MB-468 (right) cells. siRNA transfection was performed 72 h before the analyses (left). Bars, SD of three independent experiments with ≥10000 cells analyzed in each sample. h Lyso-GCaMP8s (top) and Lyso-mKeima FLIs in HeLa-WT (left) and HeLa-TMEM165-KO (right) cells transfected with Lyso-GCaMP8s/Lyso-mKeima 48 h earlier and treated three times with 10 µM ML-SA1 for 5 min. Cells were allowed to recover in fresh medium for 7 min between the treatments. Mean values of 30 cells in a representative (n = 3) experiment are shown. i Mean heights of ML-SA1-induced perilysosomal Ca2+ peaks shown in (h). Bars, SD of three independent experiments with 30 randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by two-way Anova with Dunnet’s (a, b, g, i) or Tukey’s (c, d) multiple comparison.

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