Journal: Molecular Metabolism

Article Title: Picalm coordinates clathrin-mediated endocytosis and actin remodeling during myogenesis

doi: 10.1016/j.molmet.2026.102351

Figure Lengend Snippet: Picalm knockdown impairs clathrin-mediated endocytosis in C2C12 cells. (A) Co-localization of Picalm with the endosomal marker EEA1 and the AP2 adaptor complex in C2C12 cells (day 4); nuclei were stained using DAPI. Scale bar: 10 μm. (B) Pearson's correlation coefficient between fluorescent signal of Picalm (red) and EEA1 or AP2 (green) per field of view. (C) Fluorescence-labelled epidermal growth factor (Alexa555-EGF) was applied to serum-starved myoblasts (day 1) after treatment with the inhibitor Dyngo-4a for 24h. Surface binding was allowed at 0°C for 50 min, followed by washing to remove excess Alexa555-EGF and internalization was then permitted at 37°C for up to 30 min. After fixation, cells were subjected to immunofluorescent microscopy using an anti-Picalm antibody. (D) Quantification of internalized Alexa555-EGF intensity after 0, 5 and 30 min of incubation at 37°C. Intracellular signals were quantified by selecting regions of interest (ROI) in perinuclear regions (n = 3 independent experiments, 10–12 images per experiment, 12 ROI per image). (E) Representative images for EGF uptake assay. Scale bar: 10 μm. (F) The clathrin-mediated endocytosis (CME)-inhibitor Dyngo-4a was added to differentiation medium of C2C12 cells 24h after start of differentiation and incubated for 24h. (G) Expression profiles of Myog and Myh3 in si Picalm vs. siNT cells following Dyngo-4a treatment analyzed by qRT-PCR (n = 2 independent experiments, each performed in dublicates). ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.

Article Snippet: EEA1 , Monoclonal , Mouse , Santa Cruz , sc-137130.

Techniques: Knockdown, Marker, Staining, Fluorescence, Binding Assay, Microscopy, Incubation, Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Enhanced recruitment of Rab5 to lipid droplets (LDs) upon lipophagy stimulation . A , Western blot analysis of Rab5 and EEA1 GTP-pulldown in regular medium (CT) or 4 h HBSS starvation in Hep3B cells. B , quantification of the fold change of GTP-bound/total Rab5 as well as EEA1 from n = 4 independent experiments. C , quantification of total Rab5–β-actin confirming that starvation does not change overall Rab5 expression. D , Western blot analysis of LDs isolated from Hep3B cells by density gradient centrifugation showing abundant levels of Rab5 in isolated LDs following 4 h of HBSS starvation compared with the regular medium (CT). E , quantification of Rab5–Plin2 in the biochemically isolated LDs. F , fluorescence micrograph showing immunofluorescence staining of Hep3B cells expressing Myc-tagged Rab5 ( green ). Cells were treated with oleic acid for 16 h and then either maintained in regular medium (control) or subjected to 4 h of HBSS starvation before being fixed and stained with Oil Red O (ORO; red ) to label LDs. Note the marked increase in Rab5 staining around LDs in HBSS-starved cells versus control ( yellow arrowheads ). Graphs represent mean ± SD from n = 3 to 4 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CT, control; EEA1, early endosome antigen 1; HBSS, Hank’s balanced salt solution; Plin2, perilipin-2.

Article Snippet: Antibodies against Rab5A (2143S), PLIN2 (95109), and EEA1 (2411S) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Isolation, Gradient Centrifugation, Fluorescence, Immunofluorescence, Staining, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Inhibition of Rab5 GTPase activity reduces lipid droplet (LD) catabolism via its GTPase activity . A , Western blot analysis of Rab5 and EEA1 GTP-pulldown in Hep3B cells treated with DMSO (CT) or NAP (100 μM, 48 h). B , quantification of GTP/total Rab5 as well as EEA1 from n = 3 independent experiments. C , confocal micrograph showing ORO-stained LDs ( red ) and DAPI-stained nuclei from Hep3B cells treated with DMSO (control) and NAP (100 μM). D , bar graphs depict quantification of LD number/cell, total LD area/cell, and LD size. E , confocal micrograph showing ORO-stained LDs (red ) and DAPI-stained nucleus from Hep3B cells treated with DMSO (control) and NAP (100 μM) with oleic acid (OA, 150 μM). F , bar graphs depict quantification of LD number/cell, total LD area/cell, and LD size. G , cartoon depicting NAP alteration of Rab5 GTP binding. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CT, control; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EEA1, early endosome antigen 1; NAP, neoandrographolide; ORO, Oil Red O.

Article Snippet: Antibodies against Rab5A (2143S), PLIN2 (95109), and EEA1 (2411S) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Activity Assay, Western Blot, Staining, Control, Binding Assay, Two Tailed Test