nb100 1770ss rrid ab 10128178  (Novus Biologicals)

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) Structure of the THRAP3 protein with putative CIT phosphosites derived from our previous mass spectrometry data indicated in red (top). In vitro ADP-Glo assays were performed using recombinant CIT kinase domain (rec CIT) and the indicated peptides. The green dashed line indicates values for CIT in the absence of substrate peptide. Data are represented as means ± SEM. Biological triplicates were analyzed. Peptide sequences are listed below the graph (bottom). aa, amino acids. ( B ) LNCaP cells were transfected with a construct encoding THRAP3, THRAP3 S243A, THRAP3 S379A, or empty vector (EV). At 96 hours later, cells were lysed and analyzed via Western blotting. ( C ) LNCaP cells were transfected as under (A). Ninety-six hours later, cell viability was assessed using an MTS assay read at 490 nm in biological quintuplicate. ( D ) Cells representing diverse CaP stages were transfected, and cell viability was assessed as under (B). VCaP, treatment-naïve CaP cells; C4-2, CRPC cells; 42F ENZR , enzalutamide-resistant, neuroendocrine CaP (NEPC) cells. ( E ) LNCaP and C4-2 cells were transfected with a construct encoding THRAP3, THRAP3 S243A, THRAP S379A, THRAP3 S243A or THRAP3 S379A, or EV. At 96 hours later, an MTS assay was performed as above. ( F ) LNCaP cells were transfected as under (D). Ki67 immunofluorescence staining was performed 96 hours later, and the percentage of Ki67-postive cells was recorded. ( G ) LNCaP cells were transfected with expression constructs encoding wild-type (WT) THRAP3, THRAP3 S243A and THRAP3 S379A, or EV. Ninety-six hours later, immunofluorescence using an antibody targeting FLAG was performed. Representative images are shown (right). Percentage of cells that show nuclear and cytoplasmic THRAP3 expression was determined (left). At least 100 cells per transfection group were evaluated for THRAP3 expression. Data are represented as means ± SEM. H3, histone 3 peptide. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant. Two-way t tests.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Derivative Assay, Mass Spectrometry, In Vitro, Recombinant, Transfection, Construct, Plasmid Preparation, Western Blot, MTS Assay, Immunofluorescence, Staining, Expressing

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) Top: LNCaP cells expressing DOX-inducible shRNA targeting CIT were treated with DOX (1 μg/ml) or vehicle (DMSO). CLIP-seq was performed using an antibody targeting THRAP3. Bottom: First, THRAP3 peaks that are significantly (FDR < 0.05) and ≥1.5-fold enriched under DMSO treatment compared to input samples were defined (purple dots). Second, fraction of 3396 peaks whose binding was altered ≥1.5 FC at FDR < 0.05 by DOX treatment was determined (circle plot, inset). Red: peaks whose height is decreased; blue: peaks whose height increases after CIT silencing (circle plot, inset). Created in BioRender. H. Heemers (2026) https://BioRender.com/ckag7u7 . ( B ) Cells were treated as under (A). After 96 hours, RIP was performed on technical triplicates using an antibody targeting THRAP3 or nontargeting immunoglobulin G (IgG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) used primers targeting pre-mRNA sequences for CPT1A , AMACR , PRUNE2 , and FN1 . Data are expressed as relative expression (%) to input values. ( C ) Tracks for THRAP3 binding at CPT1A and AMACR pre-mRNAs. ( D ) LNCaP and C4-2 cells were transfected with small interfering RNAs (siRNAs) targeting CIT or THRAP3 or nontargeting siRNA (ctrl). Ninety-six hours later, qRT-PCR was performed for CPT1A and AMACR alternate transcripts. Transcript levels were normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) levels and represented as relative expression using the value obtained from one of the biological triplicates from the vehicle-treated condition as 1. ( E ) LNCaP cells were transfected with expression constructs encoding FLAG-tagged WT THRAP3 (WT), THRAP3 S243A (S243A), or EV. Ninety-six hours later, RIP was performed. qRT-PCR used primers targeting pre-mRNA sequences for CPT1A and AMACR . Data are expressed as the percentage values from input. ( F ) LNCaP cells were transfected as under (E). Ninety-six hours later, qRT-PCR was performed for CPT1A and AMACR transcripts as in (F). si, siRNA. * P < 0.05; ** P < 0.01; *** P < 0.001. Data are represented as means ± SEM.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Expressing, shRNA, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Construct

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) LNCaP cells were transfected with expression constructs encoding FLAG-tagged WT THRAP3 (WT), THRAP3 S243A (S243A), or EV. At 96 hours later, qRT-PCR was performed using primers recognizing THRAP3 mRNA expression. THRAP3 mRNA levels were normalized to GAPDH levels and are represented as relative expression using the value obtained from one of the biological triplicates from the vehicle-treated condition as 1. Columns : means of values from biological triplicates; bars: SEM. ( B ) Overview of alternative splicing patterns. A3′SS (alternative 3′ splice sites), A5′SS (alternative 5′ splice sites), MXE (mutually exclusive exons), RI (retained introns), and SE (skipped exons). ( C ) RNA-seq data were analyzed for THRAP3-dependent splicing events using rMATS. Number and type of basal CIT-dependent splicing events are shown for comparison EV versus WT-THRAP3 (top), EV versus THRAP3 S243A (middle), and WT THRAP3 versus THRAP3 S243A. Red: down-regulated; blue: up-regulated after THRAP3 silencing. ( D ) Overlap in splicing events from rMATS analyses of EV versus WT-THRAP3 and EV versus THRAP3 S243A transfected cells. Data were summarized on the basis of gene symbols. * P < 0.05; ** P < 0.01. Two-way t tests. ( E ) GO:BP gene sets that are significantly (FDR < 0.25) associated with genes whose alternative splicing patterns are affected by WT THRAP3 (left) or THRAP3 S243A (right).

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Transfection, Expressing, Construct, Quantitative RT-PCR, Alternative Splicing, RNA Sequencing, Comparison

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) GO BP enrichment analysis (FDR < 0.25, P < 0.05) on 311 CIT-dependent THRAP3-bound genes (top) and other THRAP3-bound genes (bottom). Top 10 results for both gene sets are shown. The complete list of returned GO BP results is shown in tables S3 and S4. ( B ) Heatmap summarizing results of hallmark enrichment analysis on 311 CIT-dependent THRAP3-bound genes (left) and other THRAP3-bound genes (right). Results with FDR < 0.25 and P < 0.05 are listed. FDR is marked by color coding, and gene ratios are indicated by the size of the dots.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Analysis

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) LNCaP cells were transfected with siRNA targeting THRAP3 or control (ctrl) siRNA. Ninety-six hours later, THRAP3 mRNA levels were determined by qRT-PCR on biological triplicates, normalized to GAPDH expression, and represented as relative expression using the value from one biological triplicate from the vehicle-treated condition as 1. Columns: means of values from biological triplicates; bars: SEM. ( B ) RNA isolated under (A) was subjected to RNA-seq. Genes whose expression was ≥2-fold increased (up) or decreased (down) after THRAP3 silencing were determined. ( C and D ) GSEA analyses define MSigDB hallmark (C) or GO:BP gene sets (D) that are significantly (FDR < 0.25) associated with THRAP3-dependent genes. ( E ) Overview of alternative splicing patterns: A3′SS, A5′SS, MXE, RI, and SE (left). RNA-seq data were analyzed for THRAP3-dependent splicing events using rMATS (right). Red: down-regulated; blue: up-regulated after THRAP3 silencing. ( F ) LNCaP cells were transfected with siRNA targeting all CPT1A transcripts, CPT1A transcript variant 1 (v1), CPT1A v2, or control (ctrl) siRNA. Ninety-six hours later, CPT1A transcript variant mRNA levels were determined by qRT-PCR as under (A). ( G ) RNA isolated from biological triplicates under (F) was subjected to RNA-seq. Genes whose expression was ≥2-fold increased (up) or decreased (down) after CPT1A (transcript variant) silencing were determined. ( H and I ) GSEA analyses define MSigDB hallmark gene sets (H) and GO:BP gene sets (I) that are significantly (FDR < 0.25) associated with CPT1A (transcript variant)–dependent genes. Up to top 10 of gene sets is shown. If more than 10 significant results were obtained, then they are shown in tables S5 to S7. ( J ) Overlap in THRAP3- and CPT1A ( transcript variant)–dependent genes for up-regulated (top) and down-regulated genes (bottom). * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Isolation, RNA Sequencing, Alternative Splicing, Variant Assay

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) Charts: number of alternatively spliced transcripts at different CaP stages [left to right: primary CaP (pri-PCa), CRPC, and NEPC]. Overlap with THRAP3-bound genes (red; pie charts); number of alternative splicing events (ASEs) within THRAP3-bound genes (bar charts). Right: Fisher’s exact tests determined significance of overlaps. A3′SS, A5′SS, MXE, SE, and RI. ( B ) Overview of CIT-(in)dependent CPT1A and ESYT2 transcripts. Numbers: exons. ( C ) LNCaP and C4-2 cells were transfected with constructs encoding ESYT2 v1, ESYT2 v2, CPT1A v1, CPT1A v2, or EV. Ninety-six hours later, MTS assays were read in biological quintuplicate. ( D ) Cells were transfected as under (C). Ninety-six hours later, percentage of Ki67-positive cells was determined. ( E ) LNCaP cells that DOX-inducibly express CPT1A v1 or CPT1A v2 were treated with DOX (1 μg/ml) or vehicle (DMSO) for 96 hours. CPT1A transcript levels were determined by qRT-PCR, normalized to GAPDH expression, and represented as relative expression using value from a biological vehicle-treated triplicate as 1. Cells were grafted subcutaneously in nude mice. Once xenografts reached 100 mm 3 , animals were randomized to DOX (60 μg/ml; red) or DMSO (blue). CaP volumes were measured every other day for 28 days. Two-way ANOVA. ( F ) PDOs generated from fresh CaP tissues were transduced with vectors encoding CPT1A v1, CPT1A v2, or EV (left). At day 15, qRT-PCR was done for CPT1A v1 or v2 as under [(E); left)]. Surface area of 100 PDOs per transduction group was determined at day 0 and days 3, 7, 10, and 15 posttransduction (right). ( G ) PDXOs were generated from the LuCaP 86.2 PDX, transduced and analyzed as under (E). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001; n.s., not significant. t tests. Data are represented as means ± SEM.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Alternative Splicing, Transfection, Construct, Quantitative RT-PCR, Expressing, Generated, Transduction

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) Logos obtained for CIT-dependent THRAP3 RNA binding motifs (top) and other THRAP3 RNA binding motifs (bottom) via MEME motif analysis on THRAP CLIP-seq data. E value represents statistical significance of the motif. Percentage of enrichment and percentage of THRAP3 binding peaks in which the motif is present. ( B ) RNA pull-down experiment on nuclear extracts from LNCaP cells using different concentrations of a biotinylated RNA decoy molecule that consists of three copies of the THRAP3 RNA binding motif from the CPT1A pre-mRNA or a control (ctrl) RNA decoy. ( C ) LNCaP cells were transfected with the same RNA decoy or scrambled (scr) control. At 96 hours later, CPT1A transcript variant levels were determined by qRT-PCR and were normalized to GAPDH expression. Data are represented as relative expression using the value obtained from one of the biological triplicates from the vehicle-treated condition as 1. Columns: means of values obtained from independent biological triplicates; bars: SEM. ( D ) Cells representing different CaP stages and treatment resistance were transfected with two concentrations of the same RNA decoy and its scr control. At 96 hours later, an MTS assay was read in biological quintuplicate. Data are expressed as means ± SEM. ( E ) RWPE-1 cells were transfected and analyzed as in (D). *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001; n.s., not significant. t tests.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: RNA Binding Assay, Analysis, Binding Assay, Pull Down Assay, Control, Transfection, Variant Assay, Quantitative RT-PCR, Expressing, MTS Assay

Journal: Science Advances

Article Title: Citron kinase activity controls alternative splicing events that drive prostate cancer growth

doi: 10.1126/sciadv.aee5257

Figure Lengend Snippet: ( A ) Scheme for ASO spanning the THRAP3 recruitment motif in CPT1A pre-mRNA (top). Overexpression of this ASO does not affect THRAP3 expression in LNCaP cells (bottom). ( B ) Cells representing different CaP stages and treatment resistance were transfected with increasing concentrations of the CPT1A pre-mRNA–derived ASO or a scrambled control ASO. At 96 hours later, an MTS assay was read in biological quintuplicate. ( C ) RWPE-1 cells were transfected and processed as under (B). ( D ) PDXOs were generated from LuCaP 86.2 PDX tissues. PDXOs were treated with the same specific or scr ASO from (B) to (D). Top left: At day 15, qRT-PCR assays were done using primers targeting CPT1A v1 or v2. mRNA levels were normalized to GAPDH expression. Top right: The surface area of 100 PDOs per transduction group was determined at day 0 and at days 3, 7, 10 and 15 posttransduction. Bottom: Representative images at day 7. Scale bars, 400 μm. ( E ) LNCaP cells were treated with the same specific or scr ASOs as above. At 96 hours later, CPT1A transcript variant levels were determined by qRT-PCR as above. ( F ) LNCaP cells were treated as under (F). At 96 hours later, an RIP assay was performed on technical replicates using antibody targeting THRAP3 and primers detecting the CPT1A pre-mRNA. ( G ) CIT-mediated phosphorylation regulates pre-mRNA splicing and drives cell proliferation in CaP. CIT phosphorylates the RNA processing factor THRAP3 in CaP cells, facilitating its nuclear localization and binding to substrate RNA transcripts. THRAP3-RNA binding promotes generation of oncogenic splice variants of the substrate RNA transcripts resulting in increased cell proliferation and cancer growth. Created in BioRender. H. Heemers (2026) https://app.biorender.com/citation/6a26ea5b6a65c95e84d182ea . Columns: means; bars: SEM. *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.001; n.s., not significant. t tests.

Article Snippet: Antibodies were purchased from BD [CIT/CRIK (611377, RRID: AB_398899)]; Abcam [Ki67 (ab15580, RRID: AB_443209), Lamin A + C (ab108595, RRID: AB_10866185)]; Cell Signaling Technology [β-actin (4967, RRID: AB_330288), SRF (5147S, RRID: AB_10694554), cleaved poly(ADP-ribose) polymerase (PARP; 9541, RRID: AB_331426), PARP (9542, RRID: AB_2160739), normal rabbit immunoglobulin G (IgG; 2729, RRID: AB_1031062), anti-mouse IgG Alexa Fluor 594 (8890, RRID: AB_2714182), and anti-rabbit IgG Alexa Fluor 488 (4412, RRID: AB_1904025)]; Millipore [anti-mouse IgG (12-371, RRID: AB_145840)]; Novus [THRAP3 (NB100-40848, RRID: AB_792260), FAM62B (NBP1-85627, RRID: AB_11016669)]; Sigma-Aldrich [FLAG (F3165, RRID: AB_259529]; and LICORbio [IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (926-32211, RRID: AB_621843), IRDye 800CW Goat anti-Mouse IgG Secondary Antibody (926-32210, LICORbio catalog #926-32210, RRID: AB_621842)].

Techniques: Over Expression, Expressing, Transfection, Derivative Assay, Control, MTS Assay, Generated, Quantitative RT-PCR, Transduction, Variant Assay, Phospho-proteomics, Binding Assay, RNA Binding Assay

Journal: Advanced Science

Article Title: Alkylating Agent–Induced FANCL Impairment Damages Pulmonary Arterial, Venous, and Microvascular Endothelial Cells and Predisposes to PAH/PVOD

doi: 10.1002/advs.78081

Figure Lengend Snippet: MMC or CDDP Treatment Induces Progressive DNA Damage Accumulation in Pulmonary Vascular Endothelium. (A–H) Immunoblots and analyzed graphs showing the γ‐H2AX protein expression in the lung tissue of rats treated with MMC (3 mg/kg, once weekly) for 2 or 4 weeks (A–D), and CDDP (4 mg/kg, once weekly) for 2 or 4 weeks (E–H). n = 6–12 rats per group. Protein expression levels were calculated as the ratio of the target protein band intensity to that of β‑actin and were normalized to the mean values of the respective 2‑week or 4‑week NC groups, expressed as fold change. (I, J) Immunohistochemical staining for the FA core factors (FANCL and FANCD2) and the DNA damage marker γ‐H2AX in lung sections from rats treated with MMC (3 mg/kg, once weekly) or CDDP (4 mg/kg, once weekly) for 2 or 4 weeks, followed by quantification of the percentage of positive cells within the pulmonary vasculature. n = 6 rats per group. (K, L) Immunofluorescence staining for the endothelial cell marker vWF and the DNA damage marker γ‐H2AX in lung sections from rats treated with MMC (3 mg/kg, once weekly) or CDDP (4 mg/kg, once weekly) for 4 weeks, followed by correlation analysis between γ‐H2AX fluorescence intensity within vWF‑positive regions and vessel wall area percentage. n = 6 rats per group, with 10–25 vessels analyzed per group. Data are presented as mean ± SD. Differences between two groups were analyzed by t ‐test for C (FANCD2, FANCL), D (all panels), G (FANCD2, γ‐H2AX), H (all panels), and L (CDDP), Mann‐Whitney U‐test for C (γ‐H2AX), D (PCNA), G (FANCL), and L (MMC), or one‐way ANOVA with Tukey's honestly significant difference (Tukey's HSD) post hoc test for J. A value of P < 0.05 is considered statistically significant, n.s. indicates no statistical significance. Scale bar = 50 µm in I and 25 µm in K. NC: normal control.

Article Snippet: Primary antibodies for fibronectin (FN, #15613‐I‐AP, Proteintech, Rosemont, IL, USA), Twist (ab50887, Abcam, Cambridge, MA, USA), Vimentin (#5741, Cell Signaling Technology, Danvers, MA, USA), Snail (#ab53519, Abcam, Cambridge, MA, USA), ZO‐1 (21773‐1‐AP, proteintech, Wuhan, China), VEGFR2 (9698, CST, Boston, USA), Slug (YM8357, Immunoway, California, USA), proliferating cell nuclear antigen (PCNA, ab29, Abcam, Cambridge, MA, USA), CD31 (AF3628, R&D Systems, Minneapolis, MN, USA), Cleaved caspase3 (#9664, Cell Signaling Technology, Danvers, MA, USA), Bax (#2772S, Cell Signaling Technology, Danvers, MA, USA), B‐cell lymphoma/leukemia 2 (Bcl‐2, 13–8800, Thermo Fisher Scientific, Rockford, IL, USA), γ‐H2AX (#05‐635, EMD Millipore/Merck, USA), α‐smooth muscle actin (α‐SMA, ab124964 and ab7817, Abcam, Cambridge, MA, USA), FANCL (LS‐C807939, LifeSpan BioSciences, Seattle, WA, USA), FANCD2 (NB100‐182, Novus Biologicals, USA), general control nonderepressible 2 (GCN2, #46041, Cell Signaling Technology, Danvers, MA, USA), and β‐actin (66009‐1‐Ig, Proteintech, Rosemont, IL, USA) were used for immunoblotting, immunofluorescence (IF), and immunohistochemistry (IHC) staining.

Techniques: Western Blot, Expressing, Tissue, Immunohistochemical staining, Staining, Marker, Immunofluorescence, Analysis, Fluorescence, MANN-WHITNEY, Control