Journal: EMBO Molecular Medicine

Article Title: Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

doi: 10.1002/emmm.201100193

Figure Lengend Snippet: A. Rab25 expression decreases nutrient withdrawal (24 h) induced cell death in ovarian A2780 and IOSE29 cells measured using the Cell death ELISA plus assay (left); cells were cultured in complete media (containing 5% FBS), serum free media (SF), complete media plus 2DG, serum and glucose free media (SF-glu), or in amino acid, glucose and SF Earls buffer salt solution (EBSS). (a) p < 0.01 versus control and (b) p < 0.05 versus pcDNA. (Right) Percentage of apoptotic cells (sub- G 0 population) by flow cytometry in A2780 cells following glucose and serum withdrawal (Glu/SF). (a) p < 0.001 versus A2780 pcDNA control and (b) p < 0.05 A2780 Rab25 versus A2780 pcDNA control. B. Decreasing Rab25 expression increases sensitivity to nutrient stress induced cell death. Expression of Rab25 was down-regulated by siRNA or shRNA specific to Rab25. C. Rab25 regulates autophagy activity in ovarian cancer cells under serum and glucose deprivation conditions. Western blot for the 16kD LC3-II fragment, indicative of autophagy activity, in A2780 cells after 4 and 6 h of serum and glucose withdrawal. D. Electron microscopic analysis of autophagy in ovarian A2780 cells after 4 and 24 h of serum and glucose withdrawal (upper panel, low magnification). High magnification images of boxed areas with arrowheads depicting autophagic vacuoles (lower panel, inset; N: nucleus). Average number of autophagosomes per cell was calculated by counting the number of autophagosomes in 16 individual cells from two-independent experiments. E-H. Expression of Rab25 decreases glucose and serum deprivation induced signalling activation. Protein expression was measured by RPPA or WB analysis. E. RPPA detection of time-dependent activation of AMPK after nutrient withdrawal. F. WB analysis of AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in HEY ovarian cancer cells (upper panel). WB of phospho-ACC levels in A2780, IOSE80ht and SKOV3 ovarian cells after 1 h of nutrient withdrawal (lower panel). G. RPPA detection of phosphorylation of ACC after withdrawal of serum and glucose. H. Effect of Rab25 down-regulation on AMPK (left panel) and ACC (right panel) phosphorylation. Total cellular protein, isolated from A2780 pcDNA and Rab25 expressing cells 72 h post-transfection with either non-target (NT) RNAi or Rab25 specific RNAi, was separated by polyacrylamide gel electrophoresis (PAGE) followed by WB analysis. (a) p < 0.001 versus NT RNAi control and (b) p < 0.001, pcDNA versus Rab25.

Article Snippet: Rab25 shRNA was purchased from Origene (Rockville, MD).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Flow Cytometry, shRNA, Activity Assay, Western Blot, Activation Assay, Phospho-proteomics, Isolation, Transfection, Polyacrylamide Gel Electrophoresis

Journal: EMBO Molecular Medicine

Article Title: Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

doi: 10.1002/emmm.201100193

Figure Lengend Snippet: Expression of Rab25 increases endogenous ATP level in ovarian cancer cells. * p < 0.001 Rab25 versus pcDNA. The mean cellular concentration of ATP per mg of protein (based on the standard curve generated using a known amount of ATP) is 2.36 −10 moles/mg for IOSE29ht pcDNA cells, 6.21 −10 moles/mg for IOSE29ht Rab25 cells, 8.14 −11 moles/mg for IOSE80ht pcDNA cells, 1.05 −10 moles/mg for IOSE80ht Rab25 cells, 2.85 −10 moles/mg for A2780 pcDNA cells, 3.81 −10 moles/mg for A2780 Rab25 cells, 7.23 −10 moles/mg for HEY pcDNA cells and 9.82 −10 moles/mg for HEY Rab25 cells. Rab25 expression increases ATP synthesis and delays fall in total cellular ATP levels after glucose and serum withdrawal, p < 0.001 Rab25 versus control pcDNA. Down-regulation of Rab25 expression decreases ability to maintain ATP after glucose and serum withdrawal (a) p < 0.05 SF-Glu versus FBS and (b) p < 0.05 versus control. Expression of Rab25 increases total cellular glycogen content while down-regulation of Rab25 expression decreases total cellular glycogen content (µg of glycogen/mg protein). Cells were cultured in complete media for 24 h before glycogen measurement. (a) p < 0.01 Rab25 versus pcDNA, (b) p < 0.05 Rab25 RNAi versus NT RNAi and (c) p < 0.05 shRAb25 versus shRNA control. Decrease in cellular glycogen content after glucose and serum withdrawal * p < 0.001 Rab25 versus pcDNA. Effect of GPi (Bay U6571) and oligomycin on ATP production. A2780 pcDNA transfected and Rab25 expressing cells were pretreated with vehicle (control), 30 µM of Bay U6571 (GPi) or 25 µg/ml of oligomycin for 2 h. Cells were then cultured in serum- and glucose free RPMI 1640 in the presence of absence of Bay U6571 or oligomycin for the indicated times. (a) p < 0.01 oligomycin time 0 versus control time 0 and (b) p < 0.01 versus time 0 at each treatment group. Effect of Bay U6571 on cellular glycogen (left panel) and ATP (right panel) levels in HEY cells. (a) p < 0.01 versus pcDNA control time 0 at 5% FBS and (b) p < 0.01 GPi versus control at each corresponding treatment. Down regulation of Rab25 expression and inhibition of glycogen breakdown by GPi administration increases 2DG induced cell death * p < 0.05 versus shRNA control.

Article Snippet: Rab25 shRNA was purchased from Origene (Rockville, MD).

Techniques: Expressing, Concentration Assay, Generated, Control, Cell Culture, shRNA, Transfection, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

doi: 10.1002/emmm.201100193

Figure Lengend Snippet: A. Rab25 expression increases phospho but not total AKT and GSK3 levels in A2780 cells leading to decreased phosphorylation of GS (pGS). B. WB analysis of AKT and GSK3 phosphorylation confirming the inhibitory effect of PI3K pathways inhibitors PI103, GPi and synthase kinase 3 inhibitor (GSK3i) in HEY cells. C. Inhibition of GSK3 activity increases total cellular glycogen levels. pcDNA transfected and Rab25 expressing ovarian cancer cells were cultured in complete media in the presence of 10 µM GSK3i for the indicated times. Total cellular glycogen content was measured and normalized with total protein content. (a) p < 0.001 versus no GSK3i treated pcDNA cells. D-E. Effect of PI3K inhibitor PI103 on cellular glycogen content and ATP production. Ovarian cancer cells were pretreated with PI103 for at least 2 h in complete media. Cells were then subjected to 2 h glucose and FBS withdrawal to deplete endogenous glycogen followed by culturing cells in RPMI-glucose media in the presence of PI103 for 2 h (2 h SF). After 2 h of nutrient stress, complete media containing PI103 was added to the cells for 30 min (5% FBS) for recovery. A second glucose and FBS withdrawal (15 min SF) followed immediately to examine the effect on glycogen and ATP levels. a, p < 0.001 5% FBS versus 2 h SF, b, p < 0.01 15 min SF versus 5% FBS. D. There is an increase in glycogen content during recovery phase (5% FBS) and a decrease in glycogen when cells are subject to a second nutrient stress (15 min SF). E. Utilization of glycogen to produce ATP. F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF. G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. * p < 0.001 shRNA Rab25 versus shRNA control.

Article Snippet: Rab25 shRNA was purchased from Origene (Rockville, MD).

Techniques: Expressing, Phospho-proteomics, Inhibition, Activity Assay, Transfection, Cell Culture, shRNA, Control, Viability Assay

Journal: EMBO Molecular Medicine

Article Title: Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

doi: 10.1002/emmm.201100193

Figure Lengend Snippet: A. Rab25 over-expression increases H 3 -labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment. B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT. C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25. D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescent staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 ( i.e. yellow) is indicated by arrows. Single scale bar, 10 µM. E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206. E. AKT inhibition increases sensitivity to 2DG induced cell death. * p < 0.001 versus HEY pcDNA cells. F. Down-regulation of AKT by siRNA specific to AKT. G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. * p < 0.001 versus RNAi control cells.

Article Snippet: Rab25 shRNA was purchased from Origene (Rockville, MD).

Techniques: Over Expression, Expressing, Inhibition, Activity Assay, Control, shRNA, Staining

Journal: EMBO Molecular Medicine

Article Title: Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

doi: 10.1002/emmm.201100193

Figure Lengend Snippet: Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow. Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody. Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel. Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.

Article Snippet: Rab25 shRNA was purchased from Origene (Rockville, MD).

Techniques: Transfection, Fluorescence, Stable Transfection, Expressing, Control, Immunoprecipitation, Nucleic Acid Electrophoresis, Immunofluorescence, Staining, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

doi: 10.1002/emmm.201100193

Figure Lengend Snippet: A. Rab25 expression correlates with glycogen levels in patient tumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using QPCR and glycogen assay, respectively. B–C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curves for patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease). B. Tothill et al, 2008. C. Dressman et al, 2007. D. Prediction of breast cancer overall survival in two independent published breast datasets (upper panel) Pawitan et al, 2005 and (lower panel) Chin et al, 2006, based on Rab25-associated gene signature. Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25-associated gene expression signature (Rab25-signature) or without (non-Rab25 signature). All patients from the datasets were included in class prediction. E. Proposed model for the mechanism by which Rab25 regulates cellular bioenergetics.

Article Snippet: Rab25 shRNA was purchased from Origene (Rockville, MD).

Techniques: Expressing, Isolation, Gene Expression

Journal: Virulence

Article Title: MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells

doi: 10.1080/21505594.2020.1735051

Figure Lengend Snippet: Sequences of primers, miRNAs, and siRNAs used in this study.

Article Snippet: The membrane was blocked with the polyclonal anti-Rab25 antibody (CSB-PA019175LA01HU, cusabio), and monoclonal anti-β-actin antibody (AB0035, Abways).

Techniques: Sequencing, Over Expression, Knockdown

Journal: British Journal of Cancer

Article Title: Androgen-related expression of G-proteins in ovarian cancer

doi: 10.1038/sj.bjc.6605153

Figure Lengend Snippet: Changes in expression of G-proteins after 8-h stimulation by 10 n M DHT

Article Snippet: Monoclonal mouse anti-human Rab25 antibody was used at a concentration of 1 : 500 (clone 3F12F3, ProMab Biotechnologies, Richmond, CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Androgen-related expression of G-proteins in ovarian cancer

doi: 10.1038/sj.bjc.6605153

Figure Lengend Snippet: Rab25, Rab35 and AR expression in clinical samples. Rab25, Rab35 and AR protein expression levels were determined using immunohistochemical techniques for 154 epithelial cancer samples arrayed on a TMA. Representative sections are shown demonstrating predominantly cytoplasmic staining for Rab25 and Rab35, and nuclear staining for AR. Breast, thyroid and prostate sections were used as positive controls for Rab25, Rab35 and AR, respectively.

Article Snippet: Monoclonal mouse anti-human Rab25 antibody was used at a concentration of 1 : 500 (clone 3F12F3, ProMab Biotechnologies, Richmond, CA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: British Journal of Cancer

Article Title: Androgen-related expression of G-proteins in ovarian cancer

doi: 10.1038/sj.bjc.6605153

Figure Lengend Snippet: Correlation of expression of Rab25 and Rab35 with clinical parameters

Article Snippet: Monoclonal mouse anti-human Rab25 antibody was used at a concentration of 1 : 500 (clone 3F12F3, ProMab Biotechnologies, Richmond, CA, USA).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Androgen-related expression of G-proteins in ovarian cancer

doi: 10.1038/sj.bjc.6605153

Figure Lengend Snippet: High expression of Rab25 predicts survival by histological type. ( A ) Kaplan–Meier survival curves of all patients included in this study show differential expression by histological type. ( B ) Kaplan–Meier curves of only those tumours showing low expression of Rab25 shows no difference in survival ( C ) Kaplan–Meier curves of only those tumours showing high expression of Rab25 attenuates this finding with improved survival in mucinous and endometrioid tumours compared with serous and clear cell cancers ( P =0.043).

Article Snippet: Monoclonal mouse anti-human Rab25 antibody was used at a concentration of 1 : 500 (clone 3F12F3, ProMab Biotechnologies, Richmond, CA, USA).

Techniques: Expressing, Quantitative Proteomics

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: RAB25 is upregulated in activated HSCs induced by HBV-associated hepatocellular carcinoma cells. A: ELISA assay was applied to detect TGF-β1 level in HepG2 and HepG2.2.15 cells. ** P < 0.01, vs. the HepG2 group. B-D: LX-2 cells were subjected to 24-h co-culture with HepG2.2.15 and HepG2 cells in Transwell chambers, respectively, and then collected for CCK-8 (B), cell colony formation assay (C) and scratch assay (D) were carried out respectively to examine the changes in HSCs viability, proliferation, and migration. E: The expression levels of fibrosis markers (α-SMA and Collagen I), matrix remodeling factor MMP2, and proliferation-associated protein PCNA in HSCs were detected using Western blot. F: AQP1, CXCL10, CCL20, SOX9, and RAB25 mRNA levels in HSCs were detected using qRT-PCR. F: RAB25 protein level in HSCs was detected using Western blot. N=3 (biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. the HepG2+ HSC group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, CCK-8 Assay, Colony Assay, Wound Healing Assay, Migration, Expressing, Western Blot, Quantitative RT-PCR

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: Silencing RAB25 inhibits TGF-β1-induced activation of HSCs. A-B: si-RAB25 knockdown efficiency was detected using qRT-PCR (A) and Western blot (B), ** P < 0.01, vs. the si-NC group. C-F: The si-RAB25 and its control si-NC were transfected into cells and cultured for 48 h. HSCs were then subjected to 24-h stimulation with 4 ng/ml TGF-β1. CCK-8 (C), cell colony formation assay (D), and scratch assay (E) were carried out, respectively, to detect changes in viability, proliferation, and migration ability of HSCs in different treatment groups. F: Using Western blot, the expressions of fibrosis markers (α-SMA and Collagen I), matrix remodeling factor MMP2, and proliferation-associated protein PCNA in HSCs from treatment groups were detected. N=3 (biological replicates). ** P < 0.01, vs. Blank group; ## P < 0.01, vs. the TGF-β1+si-NC group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Activation Assay, Knockdown, Quantitative RT-PCR, Western Blot, Control, Transfection, Cell Culture, CCK-8 Assay, Colony Assay, Wound Healing Assay, Migration

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: Overexpression of RAB25 activates TGF-β1-induced activation of HSCs. A-B: The transfection efficiency of RAB25 overexpression to HSCs was detected using qRT-PCR (A) and Western blot (B), ** P < 0.01, vs. the NC group. C-F: The RAB25 overexpression and its negative control were transfected into cells and cultured for 48 h. HSCs were subsequently subjected to 24-h stimulation with 4 ng/ml TGF-β1. CCK-8 (C), cell colony formation assay (D), and scratch assay (E) were implemented, respectively, to detect changes in viability, proliferation, and migration ability of HSCs in different treatment groups. F: The expressions of α-SMA and Collagen I, MMP2, and PCNA in HSCs from treatment groups were detected by Western blot. N=3 (biological replicates). ** P < 0.01, vs. Blank group; ## P < 0.01, vs. the TGF-β1+NC group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Over Expression, Activation Assay, Transfection, Quantitative RT-PCR, Western Blot, Negative Control, Cell Culture, CCK-8 Assay, Colony Assay, Wound Healing Assay, Migration

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: Silencing RAB25 inhibits HSC activation induced by HBV-associated hepatocellular carcinoma cells. The RAB25 knockdown vector or the control vector was transfected into LX-2 and cultured for 48 h. Next, the transfected LX-2 was subjected to 24-h co-culture with HepG2.2.15 and HepG2, respectively, and the LX-2 cells were harvested for the subsequent experiments. A-C: The changes in viability, proliferation, and migration ability of HSCs in different treatment groups were detected using CCK-8 (A), cell colony formation assay (B), and scratch assay (C), respectively. D: Using Western blot, the expression levels of fibrosis markers (α-SMA and Collagen I), matrix remodeling factor MMP2, and proliferation-associated protein PCNA in HSCs in different treatment groups were detected. N=3 (biological replicates). ** P < 0.01, vs. HepG2+HSC group; ## P < 0.01, vs. HepG2.2.15+HSC+si-NC group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Activation Assay, Knockdown, Plasmid Preparation, Control, Transfection, Cell Culture, Co-Culture Assay, Migration, CCK-8 Assay, Colony Assay, Wound Healing Assay, Western Blot, Expressing

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: Effects of RAB25 overexpression on HSCs activation induced by HBV-associated hepatocellular carcinoma cells. The RAB25 overexpression vector or the control vector was transfected into LX-2 and cultured for 48 h. Next, the transfected LX-2 was subjected to 24-h co-culture with HepG2.2.15 and HepG2, respectively, and the LX-2 cells were harvested for the subsequent experiments. A-C: The changes in viability, proliferation, and migration ability of HSCs in different treatment groups were explored using CCK-8 (A), cell colony formation assay (B), and scratch assay (C), respectively. D: Using Western blot, the expression levels of α-SMA, Collagen I, MMP2 and PCNA in HSCs in different treatment groups were detected. N=3 (biological replicates). ** P < 0.01, vs. HepG2+HSC group; ## P < 0.01, vs. HepG2.2.15+HSC+NC group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Over Expression, Activation Assay, Plasmid Preparation, Control, Transfection, Cell Culture, Co-Culture Assay, Migration, CCK-8 Assay, Colony Assay, Wound Healing Assay, Western Blot, Expressing

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: RAB25 promotes PI3K-AKT signaling activation in HBV-associated hepatocellular carcinoma cell-induced HSCs activation. A: si-RAB25#1 and its control product were transfected into LX-2 and cultured for 48 h. Next, the transfected LX-2 was subjected to 24-h co-culture with HepG2.2.15 and HepG2, respectively. The total and phosphorylated PI3K and AKT level in HSCs from all groups was then detected using Western blot. ** P < 0.01, vs. HepG2+HSC group; ## P < 0.01, vs. the HepG2.2.15+HSC+si-NC group. B: RAB25 overexpression vector or the control vector was transfected into LX-2 and cultured for 48 h. Next, the transfected LX-2 was subjected to 24-h co-culture with HepG2.2.15 and HepG2, respectively. Then, the total and phosphorylated PI3K and AKT levels in HSCs from all groups were detected using Western blot. N=3 (biological replicates). ** P < 0.01, vs. HepG2+HSC group; ## P < 0.01, vs. the HepG2.2.15+HSC+NC group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Activation Assay, Control, Transfection, Cell Culture, Co-Culture Assay, Western Blot, Over Expression, Plasmid Preparation

Journal: Virus Research

Article Title: RAB25 promotes hepatitis B virus-induced liver fibrosis progression through activation of the PI3K/AKT signaling pathway

doi: 10.1016/j.virusres.2026.199698

Figure Lengend Snippet: Rescue of RAB25-mediated HSCs activation by PI3K/AKT inhibition. HepG2.2.15 cells were co‑cultured with LX‑2 hepatic stellate cells transiently transfected with a RAB25 overexpression vector; cells were treated with either dimethyl sulfoxide (DMSO) as vehicle control or the selective PI3K inhibitor LY294002 at a concentration of 10 μM for 24 h. Then, treated HSCs were examined for A: the total and phosphorylated PI3K and AKT level using Western blot; B: cell viability using CCK-8; C: cell proliferation by colony formation assay; D: cell migration through scratch assay; E: the expression levels of α-SMA, Collagen I, MMP2, and PCNA using Western blot. N=3 (biological replicates). ** P < 0.01, vs. HepG2.2.15+HSC-RAB25+DMSO group.

Article Snippet: Serum samples were collected from all participants after overnight fasting and stored at −80°C for subsequent serum RAB25 expression analysis by ELISA (Human RAB25 ELISA Kit, SLD4057Hu, Sunlong Biotech, Hangzhou, China).

Techniques: Activation Assay, Inhibition, Transfection, Over Expression, Plasmid Preparation, Control, Concentration Assay, Western Blot, CCK-8 Assay, Colony Assay, Migration, Wound Healing Assay, Expressing

Journal: Communications Biology

Article Title: Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits

doi: 10.1038/s42003-021-02934-0

Figure Lengend Snippet: List of Antibodies used in this paper.

Article Snippet: Purified anti-human RAB25 , Cell Signaling , 13048 T , 1:500.

Techniques: Flow Cytometry, Microscopy, Western Blot, Purification, Enzyme-linked Immunosorbent Assay

Journal: Angiogenesis

Article Title: Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking

doi: 10.1007/s10456-021-09791-9

Figure Lengend Snippet: Rab10 and Rab25 work in combination to traffic LH3 to CIVC vesicles. ( a ) Representative images of ECs expressing RFP-Rab10 wild-type (WT) and stained for collagen IV (Col IV) (green). Green arrowheads indicate collagen IV-containing (CIVC) vesicles only and yellow arrowheads indicate Rab10 puncta only. ( b ) Representative images of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs and stained for Col IV (red), actin (green), and DNA (blue). Arrowheads denote extracellular Col IV secretion. ( c ) Graph of extracellular Col IV ratio of scramble, Rab10, Rab25, or LH3 siRNA-treated ECs cultured in serum-starve (SS) media. ( d ) Representative images of fibrin-bead sprouts between indicated siRNA treatment groups. Sprouts were stained for actin (grey) to delineate sprout morphology. ( e – g ) Graphs of sprouting parameters for scramble, Rab10, Rab25, or LH3 siRNA-treated sprouts. ( h ) Representative images of ECs cultured in VEGF-containing or SS media and stained for Col IV (green), LH3 (red), and DNA (blue). Green arrowheads indicate CIVC vesicles only and yellow arrowheads indicate co-localized puncta. ( i ) Graph of percent CIVC vesicles co-localized with LH3 in ECs cultured in VEGF-containing or SS media. ( j ) Representative images of scramble, Rab10, Rab25, or both Rab10/25 siRNA-treated ECs and stained for Col IV (green), LH3 (red), actin (grey), and DNA (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV only puncta, and red arrowheads indicate LH3 puncta only. ( k ) Graph of percent CIVC vesicles co-localized with LH3 in scramble, Rab10, Rab25, or LH3 siRNA-treated conditions cultured in SS media or SS media with CLQ (10 µM). ( l ) Representative images of ECs expressing RFP-Rab10 WT, RFP-Rab10 constitutively active (CA), or RFP-Rab10 dominant negative (DN), stained for Col IV (green) and LH3 (blue). Yellow arrowheads indicate co-localized puncta only, green arrowheads indicate Col IV puncta, and red arrowheads indicate LH3 puncta. ( m ) Graph of percent CIVC vesicles co-localized with LH3 in ECs expressing RFP-Rab10 WT/CA/DN. ( n ) Graph of percent Rab10 wild-type (WT) puncta co-localized with Rab25 in either VEGF-containing or SS media. ( o ) Graph of percent Rab10 puncta co-localized with Rab25 in ECs transfected with indicated constructs. ( p ) Schematic diagram showing how Rab10 and Rab25 function to coordinate delivery of LH3 to CIVC vesicles for proper secretion of Col IV. For all graphs n number of cells unless otherwise indicated. For all experiments, data represented as mean ± 95% confidence intervals. Black bars indicate comparison groups with indicated p -values. All p -values are from two-tailed Student’s t -test from at least three experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns not significant

Article Snippet: Full-length human Rab25 cDNA was purchased from Origene (RC203413, ORIgene) and cloned into pME-MCS as described supplementary table 1.

Techniques: Expressing, Staining, Cell Culture, Dominant Negative Mutation, Transfection, Construct, Comparison, Two Tailed Test

Journal: Angiogenesis

Article Title: Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking

doi: 10.1007/s10456-021-09791-9

Figure Lengend Snippet: Notch1 deficient mouse retinas have reduced collagen IV. ( a ) Representative images of wild-type (WT; Cdh5-PAC-CreER +/− ; Notch +/+ ), Notch EC knockout (ecko)/− (HET, Cdh5-PAC-CreER +/− ; Notch flox/+ ), or Notch ecko/ecko (NULL, Cdh5-PAC-CreER +/− ; Notch flox/flox ) retinas harvested at P5 and stained for isolectin B4 (IB4). ( b ) Graph of vasculature area for vessels between indicate genotypes. ( c ) Graph of number of branch points at vascular front. ( d ) Top- schematic of Col IV measurements taken on sprouts. Graph of collagen IV (Col IV) intensity starting at the vascular front and measured back every 50 μm between indicated groups. Intensities are normalized to Col IV levels at 200 μm for each group. ( e ) Representative images of retinas harvested at P5 and stained for Col IV (red), DNA (blue), and IB4 (green) to identify blood vessels between indicated genotypes. ( f ) Graph of Col IV fluorescence intensity across indicated groups. ( g ) Model- VEGF-binding at the tip cells decreases Notch activation. In stalk cells, Notch activation promotes expression of DENNd4c which activates Rab10. Active Rab10 works with Rab25 to traffic Lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles, allowing for secretion of Col IV into the extracellular space. For all experiments, data represented as mean ± 95% confidence intervals. N number of measurements. In all conditions, 6 or more mice were used per group. Black bars indicate comparison groups with indicated p -values. All p -values are from two-tailed Student’s t-test from duplicate experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns, not significant

Article Snippet: Full-length human Rab25 cDNA was purchased from Origene (RC203413, ORIgene) and cloned into pME-MCS as described supplementary table 1.

Techniques: Knock-Out, Staining, Fluorescence, Binding Assay, Activation Assay, Expressing, Comparison, Two Tailed Test