ar v7  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A ) Immunofluorescence staining and fluorescence intensity quantification of A − CAFs and A + CAFs showing the expression of APCDD1 (green) and COL1A1 (red). Scale bars: 50 μm. ( B and C ) Schematic of the Boyden chamber coculture system and colony formation assays used to indirectly coculture C4-2 and LNCaP cells with A + CAFs or A − CAFs with or without ENZ treatment. Quantification of colonies is shown on the right ( n = 3). ( D ) Images of PDOs cocultured with A + CAFs or A − CAFs, with and without ENZ treatment. Quantification of organoid size is shown on the right ( n = 30). Scale bars: 100 μm. ( E ) Schematic of in vivo experiments. C4-2 cells were co-injected with A + CAFs or A − CAFs into castrated NSG mice, followed by ENZ treatment. Tumor growth analysis confirms that A + CAFs significantly enhanced tumor growth under ENZ treatment ( n = 5 for each group). ( F ) CCK-8 assay showing increased cell viability in C4-2 cells treated with A + CTM, but not A − CTM, under ENZ treatment. ( G and H ) Gene ontology enrichment and gene set enrichment analysis of differentially expressed genes in C4-2 cells treated with A + CTM versus A − CTM, highlighting RNA splicing–related pathways ( n = 5 for each group). ( I ) RT-PCR analysis of AR and AR-V7 expression in C4-2 cells treated with A + CTM, showing an increase in AR-V7 levels. ( J ) CCK-8 assay showing the reversal of A + CTM-induced ENZ resistance in C4-2 cells treated with an AR-V7 PROTAC. Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test [(B) to (D)] and two-way ANOVA [(E), (F), and (J)] followed by a multiple comparisons test. ** P < 0.01, and *** P < 0.001. ns, not significant.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, In Vivo, Injection, CCK-8 Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A to C ) AR-V7 expression and ENZ resistance after heat inactivation, proteinase K (PK), DNase, or ribonuclease (RNase) treatment with A + CTM in C4-2 cells. ( D ) Heatmap showing untargeted metabolomic profiling of A + CTM and A − CTM ( n = 6 for each group). ( E ) KEGG pathway analysis of differentially abundant metabolites in A + CTM versus A − CTM. ( F ) Principal components analysis (PCA) of targeted metabolomics data from A + CTM and A − CTM ( n = 6 for each group). ( G ) Heatmap of key metabolites detected in A + CTM and A − CTM. ( H ) Gene set enrichment analysis in A + CAFs. ( I ) Volcano plot of metabolomic data comparing A + CAFs and A − CAFs ( n = 6 for each group). ( J ) Quantification of relative lactate levels in the conditioned medium from A + CAFs, A − CAFs, C4-2 cells, and controls. h, hours. ( K ) Colony formation assays of C4-2 cells treated with DMSO or lactate (LAC) with and without ENZ treatment ( n = 3). ( L ) AR and AR-V7 expression in C4-2 cells treated with DMSO or lactate. ( M ) Tumor growth of C4-2 cells injected into castrated NSG mice treated with PBS or lactate with or without ENZ ( n = 5 for each group). ( N ) Images of PDOs treated with lactate or DMSO with or without ENZ. Quantification of organoid size is shown on the right ( n = 30). Scale bars: 100 μm. ( O and P ) Schematic and colony formation assay of medium change experiments in the coculture system of A + CAFs and C4-2 cells with or without ENZ. The medium was changed every 24 or 72 hours to modulate lactate concentration within the medium ( n = 3). Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test and two-way ANOVA followed by a multiple comparisons test. ** P < 0.01, *** P < 0.001. All experiments were independently replicated three times in the laboratory ( n = 3) to ensure biological reproducibility.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: Expressing, Metabolomic, Injection, Colony Assay, Concentration Assay, Two Tailed Test

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A ) OCR analysis of C4-2 cells treated with 10 or 20 mM lactate and PBS, followed by mitochondrial stress tests using oligomycin, carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP), and rotenone. Quantification of maximum and basal respiration is shown on the right ( n = 5). ( B ) CCK-8 assay showing cell proliferation of C4-2 cells treated with DMSO or rotenone under continuous ENZ treatment. ( C ) RT-PCR and Western blot analysis of AR and AR-V7 expression in C4-2 cells cultured with A + CTM and treated with DMSO or rotenone. ( D ) Western blot analysis showing pan-lactylation (Pan-Lacyl), pan-ubiquitination (Pan-Ubi), pan-phosphorylation (Pan-Pho), and pan-acetylation (Pan-Ace) in C4-2 cells treated with 1640 or A + CTM. ( E ) Western blot analysis of Pan-Lac in C4-2 cells treated with NALA or DMSO. ( F ) Heatmap and KEGG pathway enrichment analysis of differentially lactylated proteins in C4-2 cells treated with A + CTM compared to A − CTM ( n = 3). ( G ) Volcano plot showing the differentially lactylated proteins in C-2 cells treated with A + CTM compared to A − CTM. ( H ) CCK-8 assay showing cell proliferation of C4-2 cells treated with siSNRPA or siNC, NALA or DMSO, and ENZ. ( I ) RT-PCR analysis of AR and AR-V7 expression in C4-2, LNCaP, and VCaP cells treated with siSNRPA and siNC. Data represent the means ± SD. Statistical significance was determined by a two-way ANOVA followed by a multiple comparisons test. * P < 0.05, ** P < 0.01, and *** P < 0.001. All experiments were independently replicated three times in the laboratory ( n = 3) to ensure biological reproducibility.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: CCK-8 Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Cell Culture, Ubiquitin Proteomics, Phospho-proteomics

Journal: Science Advances

Article Title: Lactate derived from cancer-associated fibroblasts promotes alternative splicing and castration resistance in prostate cancer

doi: 10.1126/sciadv.ady5324

Figure Lengend Snippet: ( A ) Schematic representation of SNRPA protein domains. ( B ) MS analysis of K123 as a lactylation site in SNRPA. ( C and D ) Lactylation in SNRPA K123WT and K123R C4-2 and LNCaP cells treated with NALA or DMSO. ( E ) Dot plot showing transcriptomic analysis of SNRPA K123WT or K123R C4-2 cells under NALA and ENZ treatment ( n = 3 for each group). ( F ) Gene ontology enrichment analysis of differentially expressed genes in K123R and K123WT cells. ( G ) CCK-8 assay showing that NALA treatment reverses ENZ resistance in LNCaP cells expressing K123R SNRPA but not in WT SNRPA-expressing cells. ( H ) AR and AR-V7 expression in C4-2 cells expressing WT or K123R SNRPA under NALA or DMSO treatment. ( I ) Schematic of the AR-V7 minigene construct containing ISEm and ESEm. ( J and K ) RNA pull-down and RIP assay showing increased binding of SNRPA to ESEm in NALA or DMSO-treated C4-2 cells ( n = 3). ( L ) Schematic of the human AR gene. ( M ) ChIP analysis demonstrating the enhanced recruitment of SNRPA to P1-to-P3 regions following NALA treatment ( n = 3). ( N and O ) Co-IP assays showing a direct interaction between SNRPA and AARS1. ( P ) SNRPA K123 lactylation in C4-2 cells after AARS1 knockdown. ( Q ) CCK-8 assay showing that AARS1 knockdown reverses NALA-induced ENZ resistance in C4-2 cells. ( R ) RT-PCR and Western blot analysis of AR and AR-V7 expression in C4-2 cells with AARS1 knockdown. Data represent the means ± SD. Statistical significance was determined by a two-tailed unpaired t test and two-way ANOVA followed by a multiple comparisons test. * P < 0.05, ** P < 0.01, and *** P < 0.001. All experiments were independently replicated three times in the laboratory ( n = 3) to ensure biological reproducibility.

Article Snippet: C4-2 and LNCaP cells were plated in 96-well plates at a density of 3000 cells per well and treated with A + CTM, A − CTM, NALA (L7022, Sigma-Aldrich), l -lactate (L1750, Sigma-Aldrich), PROTAC AR-V7 degrader-1 (HY-145479, MedChem Express), or ENZ.

Techniques: CCK-8 Assay, Expressing, Construct, Binding Assay, Co-Immunoprecipitation Assay, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test