Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Functional characterization of H3C14 in GCB resistance in bladder cancer cells . (A) Western blot analysis of histone H3.2 (H3C14) protein expression in GCB‐sensitive bladder cancer cells (T24 and 5637) versus their GCB‐resistant counterparts (T24GCB and 5637GCB) and J82 cells. (B) Western blot analysis of histone H3.2 (H3C14) protein levels in EVs isolated from 5637, 5637GCB, T24, T24GCB and J82 cells. (C) Western blot analysis of histone H3.2 (H3C14) in T24 and 5637 cells after 24 h treatment with conditioned media (ConMed) or EVs derived from T24, T24GCB and 5637, 5637GCB cells. (D) RT‐qPCR analysis was used to evaluate the knockdown efficiency of H3C14 RNA in T24 and 5637 cells transfected with H3C14 siRNA (siH3C14#1 and siH3C14#2). (E) MTT assay was used to evaluate cell viability in T24 and 5637 cells transfected with scramble siRNA (Vector) or siH3C14 following treatment with a series of GCB concentrations (0–3 µM) for 48 h ( n = 6 per group). (F and I) Colony formation assays were used to evaluate measuring clonogenic survival clonogenicity of T24 and 5637 cells transfected with Vector or siH3C14 over 14 days ( n = 3 per group). (G and J) Migration assays were used to evaluate the migratory capacity of T24 and 5637 cells transfected with Vector or siH3C14 ( n = 3 per group). (H and K) Invasion assays were used to evaluate the invasive ability of T24 and 5637 cells transfected with Vector or siH3C14 ( n = 3 per group). (M and N) Flow cytometry analysis of apoptosis in T24 and 5637 cells transfected with Vector or siH3C14 following treatment with 0.01 µM gemcitabine. Apoptosis was assessed using PI and Annexin V staining ( n = 3 per group). (L) Western blot analysis of histone H3.2 (H3C14) and GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) in T24 and 5637 cells transfected with Vector, siH3C14#1, or siH3C14#2. For all panels, data are presented as mean ± SEM. An unpaired two‐tailed Student's t ‐test was used for comparisons between groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. All experiments were independently repeated at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; PI, propidium iodide.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Functional Assay, Western Blot, Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Knockdown, Transfection, MTT Assay, Plasmid Preparation, Migration, Flow Cytometry, Staining, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Functional characterization of H3C14 gain‐of‐function in GCB‐resistant bladder cancer cells and its role in GCB sensitivity . (A) RT‐qPCR analysis was conducted to confirm H3C14 overexpression in T24GCB, 5637GCB and J82 cells transfected with H3C14‐GFP plasmid. (B) Representative fluorescence microscopy images show subcellular localization of H3C14‐GFP in T24GCB, 5637GCB, and J82 cells. Scale bar: 20 µm. (C) MTT assays were used to evaluate cell viability in T24GCB‐H3C14‐GFP, 5637GCB‐H3C14‐GFP and J82‐H3C14‐GFP cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (D) Western blot analysis of histone H3.2 (H3C14) and GCB‐metabolizing enzymes and transporters (CNT1, ENT1, NT5C2 and CNT3) in T24GCB, 5637GCB and J82 cells transfected with either vector or H3C14‐GFP, with or without 0.01 µM GCB treatment. (E and F) Western blot analysis of histone H3.2 (H3C14) protein stability in T24 and T24GCB cells treated with cycloheximide (CHX, 50 µg/mL) for 0, 2, 4 or 6 h, and with MG132 (10 µM) for 6 h. (G) Schematic diagram of xenograft tumour model in nude mice—T24GCB‐Vector or T24GCB‐H3C14 cells were subcutaneously injected, followed by GCB treatment (1 mg/kg every 2 days for 28 days). (H) Representative tumour images from the four treatment groups: Vector, Vector + GCB, H3C14 and H3C14 + GCB ( n = 3 mice per group). (I) Tumour weight comparison among the four groups ( n = 3 mice per group). (J) Tumour growth curves present changes in tumour volume over time across treatment groups ( n = 3 mice per group). (K) Histogram of final tumour weights from each treatment group ( n = 3 mice per group). (L–O) IHC staining of H3C14, CNT3 and TK1 in xenograft tumours from the four groups. H‐scores were calculated to quantify expression levels ( n = 3 mice per group). (P–R) Kaplan–Meier survival analysis of patients with bladder cancer from TCGA datasets based on high versus low expression of H3C14, CNT3 and TK1. (S) TCGA survival analysis based on high H3C14 and low CNT3 expression revealed the highest survival probability in this subgroup. Data are presented as mean ± SEM. Statistical comparisons were made using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were independently repeated at least three times. GCB, gemcitabine; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; IHC, Immunohistochemical; TCGA, The Cancer Genome Atlas.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Functional Assay, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Injection, Comparison, Immunohistochemistry, Expressing, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemical staining

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Rab27A regulates EV release and histone H3.2 (H3C14) protein excretion in GCB‐resistant bladder cancer cells . (A) RT‐qPCR analysis was used to evaluate Rab27A knockdown efficiency in T24GCB and 5637GCB cells following siRab27A transfection. (B) Representative fluorescence microscopy images of T24GCB‐Vector and T24GCB‐siRab27A cells stained with H3C14‐FITC, Rab27A‐APC and DAPI. Scale bar: 20 µm. (C) Nanoparticle tracking analysis (NS300) of EVs released from T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells revealed particle size distribution and concentration. (D) MTT assay was used to evaluate cell viability of T24GCB‐Vector, T24GCB‐siRab27A and 5637GCB‐Vector, 5637GCB‐siRab27A cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (E) Representative electron microscopy images show MVBs and ILVs in T24GCB‐Vector, T24GCB‐siRab27A and 5637‐Vector, 5637GCB‐siRab27A cells. (F, G) Quantification of MVBs per image (F) and ILVs per MVB (G) from panel L. (H, I) Colony formation assays were used to evaluate clonogenicity of T24GCB and 5637GCB cells transfected with Vector or siRab27A over 10 days ( n = 3 per group). (J) Western blot analysis of Rab27A, H3C14, CNT3 and TK1, as well as EV‐associated proteins (CD9, CD63, CD81, TSG101 and Alix) in T24GCB and 5637GCB cells transfected with Vector or siRab27A. (K) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB and 5637GCB cells treated with DMSO, neticonazole (1 µM), or ketoconazole (1 µM) for 24 h. (L, M) Colony formation assays in T24GCB and 5637GCB cells treated with DMSO, neticonazole, or ketoconazole (1 µM each) ± GCB (0.001 µM) for 7 days ( n = 3 per group). (N) Western blot analysis of Rab27A, H3C14, Alix, p‐ERK1/2, total ERK1/2 and BCL‐2 in T24GCB and 5637GCB cells after 24 h treatment with DMSO, neticonazole, or ketoconazole. (O) RT‐qPCR analysis was used to evaluate CNT3 knockdown efficiency in T24GCB and 5637GCB cells after siCNT3 transfection. (P) Nanoparticle tracking analysis (NS300) of EVs derived from T24GCB‐Vector, T24GCB‐siCNT3, and 5637GCB‐Vector, 5637GCB‐siCNT3 cells. (Q) MTT assay was used to evaluate viability of T24GCB‐Vector, T24GCB‐siCNT3 and 5637GCB‐Vector, 5637GCB‐siCNT3 cells treated with GCB (0–3 µM) for 48 h ( n = 6 per group). (R) Western blot analysis of CNT3, H3C14, Rab27A, EV markers (CD9, CD63, CD81, Alix and TSG101) and anti‐apoptotic proteins (BCL‐2 and BCL‐XL) in T24GCB and 5637GCB cells transfected with Vector or siCNT3. All data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Knockdown, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Staining, Concentration Assay, MTT Assay, Electron Microscopy, Western Blot, Derivative Assay, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization of H3.2 (H3C14)‐carrying EVs in GCB Resistance . (A) Schematic illustration of the isolation of EV subpopulations from T24GCB cells using MicroBeads Pan, targeting CD9, CD63 and CD81. (B) MTT assay was used to assess the viability of T24 and 5637 cells treated with PBS, ultracentrifugation‐derived T24GCB‐EVs (or 5637GCB‐EVs), or MicroBeads Pan‐isolated EVs (+MicroBeads Pan), or EVs not captured by MicroBeads Pan (–MicroBeads Pan). Cells were subsequently treated with a series of GCB concentrations (0–3 µM) for 48 h ( n = 6 per group). (C) Mass spectrometry‐based comparative proteomic profiling of Transport‐EVs and Excretion‐EVs. The heatmap illustrates differentially expressed proteins, with red indicating higher expression and blue indicating lower expression levels. (D) Western blot analysis of EV‐associated markers (CD9 and CD81), histone H3.2 (H3C14), LAMB1 and CD147 in EVs derived from ultracentrifugation, MicroBeads Pan‐enriched (+MicroBeads Pan), or non‐captured (–MicroBeads Pan) fractions from T24GCB‐EVs and 5637GCB‐EVs. EVs may be broadly classified into Transport‐EVs (either CD9+, CD63+, or CD81+) and Excretion‐EVs (lacking CD9, CD63 and CD81). (E) Western blot analysis of histone H3.2 (H3C14), Alix, LAMB1 and CD147 in EVs isolated via ultracentrifugation or immunoprecipitation using magnetic beads conjugated with IgG, anti‐CD147, or anti‐LAMB1 antibodies from T24GCB‐EVs. (F–I) Imaging flow cytometry analysis of EVs derived from T24‐EVs, T24GCB‐EVs and T24GCB‐H3C14‐EVs (TG‐H3C14 EVs). EVs were stained with a lipid bilayer dye and labelled with CD9‐APC, CD63‐FITC, CD81‐PE, LAMB1‐AF568 and CD147‐Cy7 antibodies. Percentage gated values represent the proportion of EVs co‐expressing the indicated surface markers: (F) CD63⁺CD9⁺, CD63⁺CD81⁺, CD63⁺LAMB1⁺ and CD63⁺CD147⁺ (G) CD9⁺CD81⁺, CD9⁺LAMB1⁺ and CD9⁺CD147⁺ (H) CD81⁺LAMB1⁺ and CD81⁺CD147⁺ (I) CD147⁺LAMB1⁺ For all panels, data are presented as mean ± SEM. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; PBS, paraformaldehyde.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Isolation, MTT Assay, Derivative Assay, Mass Spectrometry, Expressing, Western Blot, Immunoprecipitation, Magnetic Beads, Imaging, Flow Cytometry, Staining, Two Tailed Test, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Schematic diagram depicting the H3C14‐mediated regulation of GCB metabolism and dynamics of EV subtypes in bladder cancer . Gemcitabine (GCB, dFdC) enters the bladder cancer cells via the equilibrative nucleoside transporter ENT1. After internalization, it undergoes sequential phosphorylation by DCK, CMPK1 and NME2 to form its active metabolite dFdCTP (dFdC → dFdCMP → dFdCDP → dFdCTP), which inhibits DNA synthesis in the nucleus. Alternatively, GCB can be phosphorylated through TK1 in the cytosol or TK2 in the mitochondrion. In GCB‐resistant cells, CNT1 and CNT3—localized at the plasma membrane and endoplasmic reticulum, respectively—are upregulated to facilitate drug efflux. NT5C2 further contributes to resistance by dephosphorylating GCB metabolites. Under normal conditions, the histone variant H3.2 (H3C14) suppresses CNT3 expression. However, in resistant cells, H3.2 (H3C14) is selectively excreted via the ESCRT machinery into MVBs and secreted extracellularly through EVs. These EVs can be classified into two major subtypes: (1) Transport‐EVs, marked by CD9, CD63 and/or CD81, which propagate GCB‐resistant phenotypes to recipient cells by shaping a pro‐tumorigenic microenvironment; (2) Excretion‐EVs, which lack CD9, CD63 and CD81 but are enriched in CD147 and LAMB1, thereby promoting GCB resistance by excreting tumour‐suppressive regulators from the GCB‐resistant cells. GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies.

Article Snippet: After centrifugation, the cells were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal H3C14 monoclonal (1:500, LSBio, Beijing, China) and rabbit polyclonal Rab27A (1:800, Cell Signalling Technology, Danvers, MA, USA).

Techniques: Phospho-proteomics, DNA Synthesis, Clinical Proteomics, Membrane, Variant Assay, Expressing