Fig 1: TMEM120A is broadly distributed throughout cells. HEK293 cells were transfected with tdTomato-Tmem120a, GFP-Piezo1 or GFP-Piezo2, labeled with Sir-Actin, and confocal images were obtained as described in the Materials and methods section. (A) Representative confocal images of tdTomato-TMEM120A and Sir-Actin. (B) Representative confocal images of GFP-PIEZO2 and Sir-Actin, which labels F-Actin. (C) Representative confocal images of GFP-PIEZO1 and Sir-Actin. Two independent transfections were performed and 26–30 cells per group were imaged.
Fig 2: TMEM120A inhibits PIEZO2 currents. Whole-cell patch-clamp experiments at −60 mV in cells transiently transfected with Piezo2 with or without Tmem120a were performed as described in the Materials and methods section. (A) HEK293 cells were transfected with Piezo2 and GFP with or without Tmem120a. In some cells, GFP-tagged Piezo2 was used instead of Piezo2 plus GFP. Current amplitudes are plotted (mean ± SEM) for Piezo2 expressing cells (black) and for cells expressing Piezo2 and Tmem120a (red). (B) Scatter plots and mean ± SEM for current amplitudes at 4.8 μm indentation. Statistical significance was calculated with the Mann-Whitney test. (C) Representative current traces. (D) HEK293 cells were transfected with GFP-tagged Piezo2 and with tdTomato-tagged Tmem120a or tdTomato. Current amplitudes are plotted (mean ± SEM) for Piezo2 expressing cells (black) and for cells expressing Piezo2 and Tmem120a (red). (E) Scatter plots and mean ± SEM for current amplitudes at 4.4 μm indentation. Statistical significance was calculated with the Mann-Whitney test. (F) Representative current traces. (G) Piezo1 deficient N2A cells were transfected with GFP-tagged Piezo2 and with tdTomato-tagged Tmem120a or tdTomato. Current amplitudes (mean ± SEM) are plotted for Piezo2 expressing cells (black) and for cells expressing Piezo2 and Tmem120a (red). (H) Scatter plots and mean ± SEM for current amplitudes at 6.4 μm indentation. Statistical significance was calculated with the Mann-Whitney test. (I) Representative current traces.
Fig 3: TMEM120A does not affect the actin cytoskeleton. HEK293 cells were transfected with tdTomato-Tmem120A and GFP-Piezo1 or GFP-Piezo2, labeled with Sir-Actin, and TIRF images were obtained as described in the Materials and methods section. (A) Representative TIRF images for GFP-PIEZO1 and Sir-Actin. Cell outlines displayed in white on merged images. (B) TIRF intensity of Sir-Actin with and without TMEM120A. (C) Pearson’s coefficient for colocalization of GFP-PIEZO1 and Sir-Actin with and without TMEM120A. (D) Representative TIRF images for GFP-PIEZO2 and Sir-Actin. Cell outlines displayed in white on merged images. (E) TIRF intensity of Sir-Actin with and without TMEM120A. (F) Pearson’s coefficient for colocalization of GFP-PIEZO2 and Sir-Actin with and without TMEM120A. (G) Representative TIRF images for Sir-Actin in untreated and cytochalasin D treated cells. (H) TIRF intensity of Sir-Actin with and without cytochalasin D treatment. Bar graphs show mean ± SEM and scatter plots. Individual symbols show the average value of cells for one coverslip (5–22 cells/coverslip) from two independent transfections. Statistical significance was calculated with the two-sample t test.
Fig 4: TMEM120A does not affect the tubulin cytoskeleton. HEK293 cells were transfected with tdTomato-Tmem120A and GFP-Piezo1 or GFP-Piezo2, labeled with Spy650-tubulin, and TIRF images were obtained as described in the Materials and methods section. (A) Representative TIRF images for GFP-PIEZO1 and Spy650-tubulin. Cell outlines displayed in white on merged images. (B) TIRF intensity of Spy650-tubulin with and without TMEM120A. Statistical significance was calculated using the two-sample t test. (C) Pearson’s coefficient for colocalization of GFP-PIEZO1 and Spy650-tubulin with and without TMEM120A. Statistical significance was calculated using the two-sample t test. (D) Representative TIRF images for GFP-PIEZO2 and Spy650-tubulin. Cell outlines displayed in white on merged images. (E) TIRF intensity of Spy650-tubulin. Statistical significance was calculated using the Welch’s t test. (F) Pearson’s coefficient for colocalization of GFP-PIEZO2 and Spy650-tubulin with and without TMEM120A. Statistical significance was calculated using the two-sample t test. (G) Representative TIRF images for Spy650-tubulin in untreated and colchicine treated cells. (H) TIRF intensity of Spy650-tubulin with and without colchicine treatment. Statistical significance was calculated with the Mann-Whitney test. Bar graphs show mean ± SEM and scatter plots. Individual symbols show the average value of cells for one coverslip (5–22 cells/coverslip) from two independent transfections.
Fig 5: TMEM120A negatively regulates rapidly adapting mechanically activated currents in mouse DRG neurons. Data from Fig. 4 showing further analysis of Tmem120a-siRNA in mouse DRG neurons and knockdown confirmation in N2A cells. (A) Scatter plots and mean ± SEM for the inactivation time constant (tau) for rapidly adapting (RA) currents. Statistical significance was calculated with two-sample t test. (B) Scatter plots and mean ± SEM of capacitance for neurons displaying RA currents. Statistical significance calculated with the Mann-Whitney test. (C) Piezo1 deficient Neuro2A cells were transfected with Sham-siRNA or Tmem120a-siRNA for Western blot analysis as described in the methods section. Representative Western blot image with β-tubulin antibody application (top panel) and TMEM120A antibody application (bottom panel). (D) The ratio of TMEM120A band intensity to β-tubulin for each Western blot (three independent transfections) was normalized to Sham-siRNA, scatter plots, and mean ± SEM. Statistical significance calculated with two-sample t test. Source data are available for this figure: SourceData FS7.
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