rab11a  (Cell Signaling Technology Inc)

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Identification and validation of RAB11A as an FGFR3-related gene in NMIBC. ( A ) Differential gene expression analysis of TCGA bladder cancer dataset comparing Ta (non-invasive) versus T1–T4 (invasive) stages. RAB11A was identified as one of the significantly upregulated genes in Ta tumors. ( B ) Kaplan–Meier survival analysis of patients in the TCGA BLCA cohort grouped by high and low RAB11A expression ( n = 406). Higher RAB11A expression is associated with poorer overall survival. ( C , D ) Correlation analysis of RAB11A and RAB11B expression with FGFR3 expression in TCGA BLCA dataset. RAB11A showed a stronger positive correlation with FGFR3 than other Rab GTPases. ( E , F ) Immunohistochemical staining of RAB11A in tumor samples from NMIBC patients collected at Renmin Hospital of Wuhan University ( n = 36), showing elevated expression of RAB11A in Ta-stage tumors. Scale bar = 100 μm. ( G , H ) mRNA and protein levels of RAB11A detected in clinical bladder cancer tissues by qRT-PCR and Western blotting, confirming its upregulation in NMIBC compared to MIBC samples. Statistical analysis was performed using unpaired Student’s t-test. Data are shown as mean ± SD from at least three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: Biomarker Discovery, Gene Expression, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: RAB11A promotes cell proliferation and survival in FGFR3-high RT4 bladder cancer cells. ( A – B ) Western blotting and qRT-PCR were performed to evaluate the knockdown efficiency of RAB11A in RT4 cells following lentiviral transduction with sh RAB11A or non-targeting control (shCTL). ( C ) Cell proliferation was measured using CCK-8 assay at the indicated time points. ( D , E ) Colony-formation assays were performed in RT4 cells expressing control shRNA (shCTL) or sh RAB11A . Quantification includes colony number (colonies containing ≥ 50 cells), mean colony diameter, and total crystal-violet staining intensity (OD 590 ) measured after dye solubilization. Cells were seeded at 500 cells/well in 6-well plates and cultured for 12 days before fixation and crystal-violet staining. ( F – H ) Apoptosis and necrosis were assessed using Annexin V-FITC/PI double staining followed by flow cytometry. Early and late apoptotic cells were quantified in parallel with necrotic cell populations. Statistical analysis was performed using unpaired Student’s t -test. Data are shown as mean ± SD from at least three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: Western Blot, Quantitative RT-PCR, Knockdown, Transduction, Control, CCK-8 Assay, Expressing, shRNA, Staining, Cell Culture, Double Staining, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: SREBF2 directly regulates RAB11A transcription in bladder cancer. ( A ) In-silico transcription-factor screening: the RAB11A (-2000–0 bp) promoter was queried against JASPAR, PROMO, LASAGNA-Search and hTFtarget; overlapping hits are shown as a Venn diagram. ( B ) Overall-survival plot obtained from GEPIA2 using TCGA-BLCA data set stratified by median SREBF2 mRNA. ( C ) QRT-PCR validation of SREBF2 mRNA in RT4 cells stably transduced with two independent shRNAs versus non-target control, GAPDH reference. ( D , E ) Evaluation of RAB11A after SREBF2 knock-down. QRT-PCR for transcript levels ( D ) and immunoblot for protein levels ( E ), GAPDH loading control. ( F ) Dual-luciferase reporter assay in parental RT4 cells co-transfected with full-length or truncated RAB11A -promoter firefly constructs plus SREBF2 or empty vector; Renilla used for normalisation. ( G ) ChIP-qPCR of endogenous SREBF2 at the RAB11A promoter, IgG and H3K4me3 as negative and positive controls. ( H ) Immunofluorescence assay to detect the expression of RAB11A after knockdown of SREBF2 . Scale bar = 20 μm. Data represent mean ± SD of three biological replicates; statistics were performed by unpaired two-tailed t-test ( C , D ) or one-way ANOVA followed by Tukey’s test ( F , G ). A p -value < 0.05 was considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: In Silico, Quantitative RT-PCR, Biomarker Discovery, Stable Transfection, Transduction, Control, Knockdown, Western Blot, Luciferase, Reporter Assay, Transfection, Construct, Plasmid Preparation, ChIP-qPCR, Immunofluorescence, Expressing, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Phenylalanine 178 of SREBF2 is indispensable for activating RAB11A transcription and for nucleating nuclear condensates that sequester nascent RAB11A transcripts. ( A ) Immunoblot analysis of SREBF2 -knockdown (KD) RT4 cells re-expressing empty vector (pcDNA), wild-type SREBF2 (pcSREBF2), or the phenylalanine-to-alanine mutant (pcSREBF2-F178A). GAPDH serves as a loading control. ( B ) Dual-luciferase reporter assay using a − 2 kb RAB11A promoter construct (pGRAB-Luc). Co-transfection of wild-type SREBF2 markedly elevates promoter activity, whereas F178A produces only a modest increase; pGL3-Basic without an insert provides the basal reference. Data are mean ± SEM of three biological replicates; one-way ANOVA with Tukey post-hoc test. ( C ) ChIP-qPCR in SREBF2 -KD cells transfected with Flag-tagged constructs (pCSRE, pCSRE-F178A, pCVector) Enrichment of the RAB11A promoter is expressed as percentage of input DNA. IgG serves as the negative IP control. ( D ) RNA fluorescence in situ hybridisation (FISH) combined with confocal imaging. Cy3-labelled probes targeting intron 1 of RAB11A (red) were hybridised to SREBF2 -KD cells expressing RFP-tagged SREBF2 constructs (magenta). Controls include scrambled probe, RNase A pretreatment, empty pTagRFP657 vector, and probe-only conditions to validate specificity. Nuclei are counter-stained with DAPI (blue). Scale bar = 10 μm. Data represent mean ± SEM of three independent biological replicates ( n = 3). One-way ANOVA followed by Tukey’s multiple-comparison test was used for ( B ) and ( C ); unpaired two-tailed Student’s t -tests were used for Manders’ co-localisation coefficients (see Results). A p -value < 0.05 was considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: Western Blot, Knockdown, Expressing, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, Construct, Cotransfection, Activity Assay, ChIP-qPCR, Transfection, Fluorescence, In Situ, Hybridization, Imaging, Staining, Comparison, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Transcriptome and protein analyses reveal that RAB11A modulates FGFR3 membrane localization but not total expression. ( A ) Differential gene expression was analyzed by RNA sequencing (RNA-seq) of RT4 cells transduced with sh RAB11A or shCTL. Volcano plot was generated to visualize significantly upregulated and downregulated genes (adjusted p < 0.05, |log2FC| > 1). ( B ) Heatmap displaying the top 100 differentially expressed genes between the two groups based on Z-score normalized expression values. ( C ) Gene Ontology (GO) enrichment analysis was performed on downregulated genes using the DAVID database, and significant pathways were plotted based on –log10 adjusted p-values. ( D , E ) Quantitative real-time PCR ( D ) and Western blot ( E ) were conducted to detect FGFR3 mRNA and total protein expression in shCTL and sh RAB11A cells. GAPDH served as internal control. ( F ) Flow cytometry was performed to evaluate surface and total levels of FGFR3 in RT4 cells. Cells were stained with anti-FGFR3 antibody with or without permeabilization. Fluorescence intensity was quantified, and the ratio of membrane-to-total FGFR3 signal was calculated. Statistical analysis was performed using unpaired two-tailed Student’s t -test. Data are presented as mean ± SD from at least three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: Membrane, Expressing, Gene Expression, RNA Sequencing, Transduction, Generated, Real-time Polymerase Chain Reaction, Western Blot, Control, Flow Cytometry, Staining, Fluorescence, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: RAB11A regulates FGFR3 palmitoylation and protein stability through interaction with ZDHHC20. ( A – C ) RT4 cells transduced with sh RAB11A or shCTL were treated with 200 ng/mL cycloheximide (CHX) for the indicated times. Whole-cell lysates were collected and analyzed by western blot using antibodies against phospho-FGFR3 (p-FGFR3), total FGFR3, and GAPDH. Band intensities were quantified using ImageJ. ( D – E ) Acyl–biotin exchange (ABE)–like assay using hydroxylamine (HAM) treatment was performed to detect palmitoylated FGFR3. Calnexin served as a positive control. Palmitoylation ratios were calculated based on densitometric comparison of the HAM (+) and HAM (–) lanes. ( F – G ) Co-immunoprecipitation assays were performed in HEK293T cells co-transfected with HA-ZDHHC20 and either Flag-tagged FGFR3 or Myc-tagged RAB11A. Protein complexes were immunoprecipitated with anti-Flag or anti-HA antibodies and analyzed by western blot. ( H ) RT4 cells stably expressing shRNA targeting ZDHHC20 (sh ZDHHC20 ) or control shRNA (shCTL) were subjected to western blot analysis with antibodies against FGFR3, ZDHHC20, and GAPDH. Knockdown of ZDHHC20 markedly reduced FGFR3 protein levels. ( I ) Palmitoylation of FGFR3 was assessed by HAM-dependent ABE assay in ZDHHC20 -knockdown (sh ZDHHC20 ) or control (shCTL) RT4 cells. Band intensities were quantified, showing reduced FGFR3 palmitoylation upon ZDHHC20 depletion. ( J ) Immunofluorescence confocal microscopy of RT4 cells transduced with shCTL, sh RAB11A , or sh ZDHHC20 . Cells were stained for endogenous RAB11A (green), ZDHHC20 (red), and FGFR3 (magenta). Nuclei were counterstained with DAPI (blue). Scale bar = 10 μm. Statistical analysis was performed using unpaired two-tailed Student’s t -test. Data are shown as mean ± SD of three independent experiments. A p -value < 0.05 was considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: Transduction, Western Blot, Positive Control, Comparison, Immunoprecipitation, Transfection, Stable Transfection, Expressing, shRNA, Control, Knockdown, Immunofluorescence, Confocal Microscopy, Staining, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: Cholesterol-rich lipid rafts are required for proper membrane localisation of FGFR3. ( A ) Detergent-resistant membrane (DRM) fractionation. RT4 shCTL, sh RAB11A and sh ZDHHC20 cells Lysates were adjusted to 40% sucrose, over-layered with 35%, 30%, 25%, 15% and 5% sucrose steps and centrifuged at 200 000 g for 18 h (SW-41 rotor, 4 °C). Fractions F1–F4 and the detergent-soluble pellet were collected, equalised for protein and analysed by SDS-PAGE with antibodies against FGFR3, Caveolin-1and Transferrin-R. ( B ) Confocal co-localisation of FGFR3 with GM1. Cells were fixed and surface-labelled with Alexa Fluor 555–cholera-toxin B, briefly permeabilized and stained with anti-FGFR3 (1: 200) plus Alexa 488 secondary antibody. Nuclei were counter-stained with DAPI. Scale bar = 10 μm. ( C , D ) Flow-cytometric assessment of surface FGFR3 after acute modulation of plasma-membrane cholesterol. RT4 cells were treated at 37 °C for 30 min with vehicle or 10 mM methyl-β-cyclodextrin (MβCD); a subset of MβCD-treated cells was subsequently incubated with 2 mM MβCD–cholesterol complex for 1 h, and an additional group received 2 mM MβCD–cholesterol alone. The cells were stained with APC-conjugated anti-FGFR3 extracellular antibody together with an APC-matched isotype control; propidium iodide was used to exclude dead cells. ( C ) Representative histogram overlays; ( D ) geometric mean fluorescence intensity (MFI) normalized to the isotype control from three independent experiments

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: Membrane, Fractionation, SDS Page, Staining, Clinical Proteomics, Incubation, Control, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: SREBP2-RAB11A-ZDHHC20 axis orchestrates FGFR3 palmitoylation and membrane retention to drive bladder cancer progression

doi: 10.1007/s13402-026-01163-z

Figure Lengend Snippet: In vivo validation of the RAB11A–ZDHHC20 axis in regulating bladder tumor growth. ( A ) RT4 cells stably expressing shCTL, sh RAB11A , or sh ZDHHC20 were subcutaneously injected into BALB/c nude mice (5 mice/group). Tumor volume was measured every other day using calipers, and calculated as (length × width²)/2. Tumors were harvested on Day 28 and weighed. ( B ) Tumor tissues were fixed in 4% paraformaldehyde and photographed. ( C ) Formalin-fixed paraffin-embedded tumor sections were subjected to multiplex immunofluorescence. ( D ) Multiplex immunohistochemistry (mIHC) of FFPE xenograft sections using Opal™ TSA reagents (Akoya Biosciences) detected RAB11A (Opal 520, red), ZDHHC20 (Opal 570, magenta) and FGFR3 (Opal 650, green); nuclei were counter-stained with DAPI (blue). Images were captured on a Vectra Polaris multispectral scanner and spectrally unmixed with inForm 2.4. Scale bar = 100 μm. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. Data are shown as mean ± SD. p -values < 0.05 were considered statistically significant

Article Snippet: Primary antibodies against RAB11A (Cell Signaling Technology, #2413), ZDHHC20 (Novus Biologicals, NBP1-91813) or FGFR3(Abcam, ab137084) were incubated overnight at 4 °C.

Techniques: In Vivo, Biomarker Discovery, Stable Transfection, Expressing, Injection, Formalin-fixed Paraffin-Embedded, Multiplex Assay, Immunofluorescence, Immunohistochemistry, Staining