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. 2022 Feb 16;298(3):101740. doi: 10.1016/j.jbc.2022.101740

Figure 1.

Figure 1

The IP35-phosphatase activity of INPP5A controls cellular late endosome and lysosome content.A and B, representative confocal images of fixed HeLa cells treated with control (SCR) or INPP5A siRNA (5AKD) and stained with specific antibodies for CD63 (green, A) or LAMP1 (green, B), respectively. Blue, DAPI-stained nuclei. The scale bar represents 25 μm. C, representative confocal images of HeLa cells treated with control (SCR) or INPP5A siRNA (5AKD) and incubated with LysoTracker (red) for 30 min before fixation with 4% PFA. Blue, DAPI-stained nuclei. The scale bar represents 25 μm. D–F, quantification of representative data shown in (A–C), respectively. Data for SCR-control siRNA-treated cells were set to 1. One-sample Student's t test for each pair. CD63: p = 0.0464, t = 3.282, and df = 3. LAMP1: p = 0.416, t = 3.427, and df = 3. LysoTracker: p = 0.031, t = 3.261, and df = 4. G, immunoblot analysis of lysates from HeLa cells treated with control (SCR) or INPP5A siRNA (5AKD). Blots were decorated with specific antibodies for LAMP1 and GAPDH as a loading control. H, representative confocal images of fixed HeLa cells treated with control (SCR) or INPP5A siRNA (5AKD) transfected alone or together with plasmids encoding siRNA-resistant active WT or phosphatase-defective inactive mutant (Mut) INPP5A and stained for CD63 (green) or LAMP1 (red), respectively. Blue, DAPI-stained nuclei. The scale bar represents 10 μm. I and J, quantification of representative data shown in H. Data for SCR-control siRNA-treated cells were set to 1. One-sample Student's t test followed by Benjamini–Hochberg correction. CD63: -/5AKD: p = 0.014, t = 8.352, and df = 2; Mut/5AKD: p = 0.0103, t = 9.777, and df = 2; WT/5AKD: p = 0.0550, t = 4.085, and df = 2. LAMP1: -/5AKD: p = 0.012, t = 9.031, and df = 2; Mut/5AKD: p = 0.031, t = 5.548, and df = 2; WT/5AKD: p = 0.6208, t = 0.5795, and df = 2. Data represent n = 4 independent experiments for CD63 and LAMP1 and five independent experiments for LysoTracker and n = 3 for rescue experiments with WT or Mut INPP5A. DAPI, 4′,6-diamidino-2-phenylindole; INPP5A, inositol polyphosphate-5-phosphatase A; IP3, inositol triphosphate; LAMP1, lysosome-associated membrane protein 1; PFA, paraformaldehyde.