Deletion of both STIM isoforms rescues cell migration. (A) Representative images of WT, S1KO, S2KO, or dKO migrating cells, as indicated. (B) Scale bar = 350 μm (B)Number of migrating cells from each group was normalized to that of control (WT) cells. Bars show the mean ± SEM (WT, n = 18; S1KO, n = 13; S2KO, n = 15; dKO-C4, n = 8; dKO-B6, n = 7). (C and D) EYFP-STIM1 or EYFP-STIM2 was re-expressed in dKO cells. (C) Representative images of migrating cells from the indicated cells. Scale bar = 350 μm. (D) Number of migrating cells from STIM1 or STIM2 rescue cells was normalized to that of control (dKO) cells. Bars show the mean ± SEM (control, n = 7; STIM1, n = 6; STIM2, n = 7). (E and F) WB analysis of lysates from WT, S1KO, S2KO, and dKO cells using two different antibodies against pMLC (ab2480 or cs3675) and GAPDH as a loading control. (E) Representative images from two independent experiments. (F) Quantification of pMLC signal intensity relative to that of GAPDH and normalized to control (WT) cells. (G and H) Bars shows the mean ± SEM of normalized pMLC (n = 4) (G and H). Representative confocal images (G) and quantification (H) of anti-phosphorylated MLC (red) and phalloidin (F-actin, green) fluorescence (see the Materials and methods section). Bars show the mean ± SEM of pMLC staining at actin bundles per cell in the indicated cell type (WT, n = 30; S1KO, n = 28; S2KO, n = 32; dKO, n = 30). (I and J) Representative images (I) and quantification (J) of anti-paxillin (red) and phalloidin (F-actin, green) fluorescence (see the Materials and methods section). Bars show the mean ± SEM of FAs per cell in the indicated cell type (WT, n = 30; S1KO, n = 34; S2KO, n = 31; dKO, n = 36). Scale bar = 10 μm. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F2.
Figure 2.

Deletion of both STIM isoforms rescues cell migration. (A) Representative images of WT, S1KO, S2KO, or dKO migrating cells, as indicated. (B) Scale bar = 350 μm (B)Number of migrating cells from each group was normalized to that of control (WT) cells. Bars show the mean ± SEM (WT, n = 18; S1KO, n = 13; S2KO, n = 15; dKO-C4, n = 8; dKO-B6, n = 7). (C and D) EYFP-STIM1 or EYFP-STIM2 was re-expressed in dKO cells. (C) Representative images of migrating cells from the indicated cells. Scale bar = 350 μm. (D) Number of migrating cells from STIM1 or STIM2 rescue cells was normalized to that of control (dKO) cells. Bars show the mean ± SEM (control, n = 7; STIM1, n = 6; STIM2, n = 7). (E and F) WB analysis of lysates from WT, S1KO, S2KO, and dKO cells using two different antibodies against pMLC (ab2480 or cs3675) and GAPDH as a loading control. (E) Representative images from two independent experiments. (F) Quantification of pMLC signal intensity relative to that of GAPDH and normalized to control (WT) cells. (G and H) Bars shows the mean ± SEM of normalized pMLC (n = 4) (G and H). Representative confocal images (G) and quantification (H) of anti-phosphorylated MLC (red) and phalloidin (F-actin, green) fluorescence (see the Materials and methods section). Bars show the mean ± SEM of pMLC staining at actin bundles per cell in the indicated cell type (WT, n = 30; S1KO, n = 28; S2KO, n = 32; dKO, n = 30). (I and J) Representative images (I) and quantification (J) of anti-paxillin (red) and phalloidin (F-actin, green) fluorescence (see the Materials and methods section). Bars show the mean ± SEM of FAs per cell in the indicated cell type (WT, n = 30; S1KO, n = 34; S2KO, n = 31; dKO, n = 36). Scale bar = 10 μm. Statistics: one-way ANOVA with Tukey’s post hoc test; *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F2.

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