Figure S5. PIEZO1 regulates Ca 2 levels... | Rockefeller University Press

Figure S5.

PIEZO1 regulates Ca2+ levels near focal adhesions.(A) Left: Representative flow cytometry results of cell-surface LAMP1 level in PIEZO1 knock-down U2OS cells. Right: Corresponding RT-qPCR assessment of PIEZO1 mRNA level in PIEZO1 knock-down U2OS cells. The expression of PIEZO1 was normalized with HPRT (n = 3 technical replicates; One-way ANOVA with Dunnett correction; ****P ≤ 0.0001). Related to Fig. 7. (B) Top: Representative CLSM images of the local Ca2+ level at FAs in WT and PIEZO1-KO HeLa cells detected by RCaMP6-FAT. Bar, 10 µm. Images are inverted to get a better contrast. Bottom: Quantification results of RCaMP6-FAT punctate size and fluorescent intensity in WT and PIEZO1-KO HeLa cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; **P ≤ 0.01; ***P ≤ 0.001). Related to Fig. 7. (C) Dual color live image of lysotracker and LAMP1-jGCaMP7b in TRPML1-depleted HeLa cells. Kymographs are generated along the dotted line in the original cell image on the left by multi-kymograph plugin from ImageJ. Related to Fig. 7.

PIEZO1 regulates Ca²⁺ levels near focal adhesions. (A) Left: Representative flow cytometry results of cell-surface LAMP1 level in PIEZO1 knock-down U2OS cells. Right: Corresponding RT-qPCR assessment of PIEZO1 mRNA level in PIEZO1 knock-down U2OS cells. The expression of PIEZO1 was normalized with HPRT (n = 3 technical replicates; One-way ANOVA with Dunnett correction; ****P ≤ 0.0001). Related to Fig. 7. (B) Top: Representative CLSM images of the local Ca²⁺ level at FAs in WT and PIEZO1-KO HeLa cells detected by RCaMP6-FAT. Bar, 10 µm. Images are inverted to get a better contrast. Bottom: Quantification results of RCaMP6-FAT punctate size and fluorescent intensity in WT and PIEZO1-KO HeLa cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; **P ≤ 0.01; ***P ≤ 0.001). Related to Fig. 7. (C) Dual color live image of lysotracker and LAMP1-jGCaMP7b in TRPML1-depleted HeLa cells. Kymographs are generated along the dotted line in the original cell image on the left by multi-kymograph plugin from ImageJ. Related to Fig. 7.