Journal: The Journal of Cell Biology

Article Title: PI(3,5)P 2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

doi: 10.1083/jcb.201412127

Figure Lengend Snippet: KD of PIKfyve expression leads to accumulation of cortactin and actin at LE membrane. (A) Representative images of endogenous cortactin (red) localized at discrete subdomains of hVac14-EGFP + (green) vesicular structures in SCC61 (left) and HeLa (right) cells. Bars: (main panels) 20 µm; (enlarged views) 3 µm. n = 3 independent experiments for each cell line. (B) Representative images of mCherry-ML1N*2 (red) and endogenous cortactin (blue) localization to EGFP-Rab7 + (green) endosomes. Small white boxes are enlarged on the right. Bars, 20 µm (3 µm for magnifications). (C) Magnifications of boxed areas labeled with asterisks in B show colocalization of mCherry-ML1N*2 (red) with EGFP-Rab7 (green). Bars, 5 µm. (D) Immunoblot of PIKfyve expression in PIKfyve-KD MDA-MB-231 cells. NTC, nontargeting control. (E) Immunofluorescence of PIKfyve siRNA-treated cells using antibodies recognizing cortactin (green), actin (red), and Rab7 (blue). Bars, 20 µm (3 µm for magnifications). (F) Percentage of colocalization of cortactin or actin with Rab7. 3 independent experiments, n ≥ 61 cells in each condition. ****, P < 0.0001.

Article Snippet: Antibodies were as follows: mouse anti-cortactin (clone 4F11; Millipore), rabbit anti-Rab7 (#9367; BD), rabbit anti-GST antibody (#2622; Cell Signaling Technology), rhodamine-phalloidin (Molecular Probes), mouse anti-PIK5KIII (sc-100408; Santa Cruz Biotechnology, Inc.), rabbit anti-cofilin/phospho-cofilin (#5175/#3313; Cell Signaling Technology).

Techniques: Expressing, Labeling, Western Blot, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: PI(3,5)P 2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

doi: 10.1083/jcb.201412127

Figure Lengend Snippet: Inhibition of PI(3,5)P 2 production leads to accumulation of cortactin and actin at LE membrane. (A) Representative images showing cortactin (green) and actin (red) localization on Rab7 + endosomes (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control in control and cortactin–KD MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (B) Immunoblot of cortactin expression and β-actin loading control in MDA-MB-231 cells. (C–E) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7 or Rab7 area per cell. 3 independent experiments, n ≥ 55 cells in each condition. (F and G) Number and intensity of actin dots on Rab7 + endosomes. More than 170 vesicles from 12–19 cells analyzed for each condition. Data shown as box and whiskers plots, with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. ****, P < 0.0001 .

Article Snippet: Antibodies were as follows: mouse anti-cortactin (clone 4F11; Millipore), rabbit anti-Rab7 (#9367; BD), rabbit anti-GST antibody (#2622; Cell Signaling Technology), rhodamine-phalloidin (Molecular Probes), mouse anti-PIK5KIII (sc-100408; Santa Cruz Biotechnology, Inc.), rabbit anti-cofilin/phospho-cofilin (#5175/#3313; Cell Signaling Technology).

Techniques: Inhibition, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: PI(3,5)P 2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

doi: 10.1083/jcb.201412127

Figure Lengend Snippet: Recruitment of cortactin to late endosomes depends on Arp2/3 complex activity. (A) Representative images show cortactin (green), actin (red), and Rab7 (blue) localization after 2 h treatment of MDA-MB-231 cells with 800 nM YM201636 ± 200 µM CK-666. Bars, 20 µm (5 µm for magnifications). (B) Images from cells treated with YM201636 and 100 or 200 µM CK-666 were analyzed for percentage of colocalization of cortactin or actin with Rab7. The DMSO and YM201636 datasets (no CK-666) are the same data as that shown in ( C and D). Data shown as box and whiskers plots with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. 3 independent experiments, n ≥ 50 cells for each condition. ****, P < 0.0001.

Article Snippet: Antibodies were as follows: mouse anti-cortactin (clone 4F11; Millipore), rabbit anti-Rab7 (#9367; BD), rabbit anti-GST antibody (#2622; Cell Signaling Technology), rhodamine-phalloidin (Molecular Probes), mouse anti-PIK5KIII (sc-100408; Santa Cruz Biotechnology, Inc.), rabbit anti-cofilin/phospho-cofilin (#5175/#3313; Cell Signaling Technology).

Techniques: Activity Assay

Journal: The Journal of Cell Biology

Article Title: PI(3,5)P 2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

doi: 10.1083/jcb.201412127

Figure Lengend Snippet: WASH localization is controlled by PI(3,5)P 2 levels. (A) Representative images from MDA-MB-231 (top) and SCC61 (bottom) showing localization of WASH (red), cortactin (green), and actin (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control. Bars, 20 µm. (B) Representative images of MDA-MB-231 cells stably expressing mRFP-Rab7 (pseudocolored blue) immunostained by WASH (red) and cortactin (green) after 2-h treatment with 800 nM YM201636 or DMS0 diluent control. Bars, 20 µm (5 µm for magnifications). (C and D) Images were analyzed for percentage of colocalization of WASH with Rab7 (C) and Rab7 area/cell (D). 2 independent experiments, n ≥ 62 cells in each condition. ****, P < 0.0001.

Article Snippet: Antibodies were as follows: mouse anti-cortactin (clone 4F11; Millipore), rabbit anti-Rab7 (#9367; BD), rabbit anti-GST antibody (#2622; Cell Signaling Technology), rhodamine-phalloidin (Molecular Probes), mouse anti-PIK5KIII (sc-100408; Santa Cruz Biotechnology, Inc.), rabbit anti-cofilin/phospho-cofilin (#5175/#3313; Cell Signaling Technology).

Techniques: Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Encephalomyocarditis virus non-structural protein 2C degrades NDP52 autophagy protein to promote its own survival

doi: 10.1101/2024.12.13.628388

Figure Lengend Snippet: (A)A549 cells were infected or uninfected with EMCV for 2 h (MOI=1) and then treated for 7 h with dimethyl sulfoxide (DMSO), MG132 (10 µM), CQ (50 µM), or Z-VAD-FMK (20 µM), respectively. Western blotting was used to detect the expression of NDP52 protein and VP1. GAPDH was used as a loading control. (B) A549 cells were infected or uninfected with EMCV for 2 h (MOI=1) and then treated for 7 h with CQ (25 µM, 50 μM, 100 μM), respectively, and western blotting was used to detect the expression of NDP52 protein and VP1. GAPDH was used as a loading control. (C, D) A549 cells were infected or uninfected with EMCV for 2 h (MOI=1) and then treated for 7 h with PBS, 3-MA (20 mM), Bafilomycin A1 (1 mM), or NH 4 Cl (20 mM), respectively. Western blotting was used to detect the expression of NDP52 protein and VP1. GAPDH was used as a loading control. (E) A549 cells were infected or uninfected with EMCV for 2 h (MOI=1), then treated for 0, 6, 9, 12 h with cycloheximide (CHX) (100 µg/ml), dimethyl sulfoxide (DMSO), CQ (50 µM), respectively. Western blotting was used to detect the expression of NDP52 protein and VP1. GAPDH was used as a loading control. (F)A549 cells were transfected with 1.0 µg of mCherrry-NDP52 plasmid for 24 h, then infected or uninfected with EMCV for 3 h (MOI=3). The cells were stained with Lyso-Tracker Green for 1h and Hoechst 33342 for 10min, followed by fluorescence analysis. Scale bar = 10 μm. (G, H, I) A549 cells were transfected with 20mM of siNC or siRNA (siRab7, siRab9) for 36 h. And then infected or uninfected with EMCV for 9 h (MOI=1). Western blotting was used to detect the expression of NDP52 protein, Rab7/rab9 and VP1. GAPDH was used as a loading control.

Article Snippet: Rab7 Rabbit mAb and Rab9A Rabbit mAb were purchased from Zenbio (Chengdu, China).

Techniques: Infection, Western Blot, Expressing, Control, Transfection, Plasmid Preparation, Staining, Fluorescence