eea1 2411s  (Cell Signaling Technology Inc)

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

Journal: bioRxiv

Article Title: Membrane Tension Integrates Physical and Signaling Cues to Gate Cell Fate Transitions

doi: 10.64898/2026.03.04.708749

Figure Lengend Snippet: (A) Immunostains of E15.5 and E17.5 mouse distal lung epithelium wherein early endosomes (EEA1) are detected in DPs and nAT2s. AT1s (1) and AT2s (2) are identifiable at E17.5 by morphology and podoplanin (PDPN, red). Scale bars correspond to 50µm and 10µm, respectively. (B) Quantification of EEA1 in late DPs as well as nAT2s. Error bars show 95% confidence intervals; n ≥ 58 cells (p-values ***p < 0.001 calculated via one-way ANOVA). (C) Immunostain of EEA1 in alveolospheres 4 days in differentiation media treated with DMSO (Control) or raised CMT (MβCD, 1mM), scale bar represents 10µm. (D) Quantification of c showing a reduction of EEA1 when CMT is raised. Expressed as mean ± SD from three independent experiments (Control: n = 208 cells; MβCD: n = 155 cells). Statistical significance was assessed using a two-tailed Mann-Whitney test, with results indicating ***p < 0.001. (E) Immunostains of alveolospheres at day 4 following treatment with DMSO (Control) or 10 µM Dynasore for markers of AT1 (RAGE, red) and AT2 (SPC, green) fate. Scale bars indicate 10µm. (F) Quantification of e detects a significant reduction in differentiation with Dynasore treatment. Data are presented as mean ± SD (p-value ***p < 0.001 determined by two-tailed Mann-Whitney test). (G) FGFR2 immunostains of E17.5 (left) and adult (right) mouse lungs, wherein the cell surface can be detected by either E-Cadherin or membrane GFP. Lower breakouts highlight FGFR2 cell surface enrichment at E17.5 (i-iii) that transitions to intracellular (lower right, surface depicted by dashes) in adult AT2s. (H) Quantification of g finds a significant shift in localization over time. Data are mean ± SD; n = 20 cells per group (p < 0.0001 determined using a two-tailed Mann-Whitney test). (I) PROGENy score of ERK pathway activity in mouse DPs, nAT2s and AT1s sampled in a prior scRNAseq time course study. (J) Alveolospheres at day 4 under differentiation conditions treated with DMSO (Control) or ERK inhibitor (FR180204, 10µM) stained for AT markers, scale bar 100μm. (K) Quantification of j, demonstrating a significant reduction in AT2 differentiation. Data shown as mean ± SD; n = 10 spheroids per condition conducted in triplicate (p-values p< 0.0001 were determined using a 2-way ANOVA). (L) Differentiating alveolospheres in Control or raised CMT conditions (1.5% PEG) stained for DP marker (SOX9) and phosphorylated ERK (nuclei in red, pERK positivity/negativity indicated as +/-), scale 10μm. (M) Quantification of l nuclear pERK intensity. Data expressed as mean ± SD; n = 50 cells per group (p-value p < 0.0001 determined by two-tailed Mann-Whitney test). (N) Alveolospheres wherein either GFP or MEK1 was mosaically expressed then immunostained for AT markers, scale bar indicates 100μm. (O) Quantification of GFP+ cells from (N) finds a significant upregulation of AT2 fate in MEK1 expressing cells versus control. Data presented as mean ± SD. p-value p < 0.0001 was determined by two-tailed Mann-Whitney test.

Article Snippet: The following primary antibodies were used: rabbit anti pro–Sftpc (Sigma-Aldrich), rabbit anti SPC (Invitrogen), rat anti RAGE (R&D Systems), rat anti E-cadherin (Life Technologies, ECCD-2), hamster anti podoplanin (DSHB, 8.1.1), hamster anti Mucin1 (Thermo Fisher Scientific, HM1630), mouse anti FGFR2 (Santa Cruz Biotechnology), rabbit anti EEA1 (Cell Signaling Technology), rabbit anti β-catenin (Cell Signaling Technology), anti-mouse phalloidin-647 conjugated (Abcam), mouse anti YAP (Santa Cruz Biotechnology), mouse anti α-smooth muscle actin IgG2a (Invitrogen), rabbit anti RELMα (PeproTech), mouse anti SOX9 (Invitrogen), rabbit anti SOX9 (Millipore), rat anti SOX2 (Invitrogen), Sheep anti podoplanin (R&D Systems), and mouse anti TGFBR2 (Proteintech).

Techniques: Control, Two Tailed Test, MANN-WHITNEY, Membrane, Activity Assay, Staining, Marker, Expressing