Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF is highly expressed in spermatocytes and spermatids of the testis (A) NCBI database analysis of the expression profile of Carf mRNA in mouse tissues. (B) qPCR analyses of Carf mRNA levels in multiple organs of mice. Data are presented as the mean ± SEM, n = 3. (C) Immunofluorescence staining analysis of CARF (green) in testis sections. γH2AX (red) was used as a marker for spermatocytes. The nuclei were stained with DAPI (blue). Spc indicates spermatocytes, Ser indicates Sertoli cells, Ley indicates Leydig cells, and Rspd indicates round sperm. Scale bar: 50 μm. (D) Immunofluorescence staining analysis of CARF (red) and PNA (green) in testis sections. The nuclei were stained with DAPI (blue), Scale bar: 50 μm. (E,F) The expression pattern of CARF in the mouse germline atlas was analyzed via a single-cell sequencing database (http://malehealthatlas.cn/ ).

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Expressing, Immunofluorescence, Staining, Marker, Sequencing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF interacts with PABPC1 to participate in RNA alternative splicing (A) Abnormal alternative splicing patterns, including skipped exons, alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE) and retained intron (RI) caused by Carf defects. (B) Abnormal variable splicing of the functional gene Hira, which is related to germ cell development caused by Carf defects. It belongs to the alternative 5′ splice site (A5SS) exception mode. (C) Abnormal variable splicing of the functional gene Surf1, which is related to germ cell development caused by Carf defects. It is an alternative 3′ splice site (A3SS). (D) Abnormal variable splicing of the functional gene Usf2, which is related to germ cell development caused by Carf defects. It belongs to the mutually exclusive exons (MXE). (E) Abnormal variable splicing of the functional gene Lrmp, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (F) Abnormal variable splicing of the functional gene Pkd2l1, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (G) Co-IP analysis of the interaction between CARF and PABPC1. PABPC1 expression was detected in the IP products of CARF, and IgG was used as a control. GAPDH served as a loading control. (H) Co-IP analysis of the interaction between CARF and PABPC1. CARF expression was detected in the IP products of PABPC1, and IgG was used as a control. GAPDH served as a loading control. (I) Immunostaining of PABPC1 in wild-type and Carf–/– testis sections (PABPC1: green); DAPI was used to stain the dye the nuclei, scale bar: 50 μm. (J) Western blot analysis of PABPC1 protein levels in testes from wild-type and Carf–/– mice. GAPDH served as a loading control. (K) Immunohistochemical analysis of the expression of PABPC1 in testes from wild-type and Carf–/– testis sections. Scale bar: 50 μm. (L) Quantitative results of (K). n = 3, Data are presented as the mean ± SEM. ***P < 0.001.

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Alternative Splicing, Functional Assay, Co-Immunoprecipitation Assay, Expressing, Control, Immunostaining, Staining, Western Blot, Immunohistochemical staining

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) Inhibitor of CDK p16 INKa mRNA (n=5), and (B) the relative amounts of p16 INKa protein (n=3) calculated from (C) the western blots of p16 INKa protein (using a goat anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were cultured in 25 cm 2 flasks and supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on the indicated weeks, total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) Inhibitor of CDK p16 INKa mRNA (n=3), and (B) the relative amounts of p16 INKa protein (n=4) calculated from (C) the western blots of p16 INKa protein (using a goat anti-human antibody) and against GAPDH. TF, tissue factor; Wk, week.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on week 4, gDNA was extracted from the cells (3×10 4 cells) and bisulphite conversion of the gDNA (750 ng) was carried out using the MethylDetector Bisulfite Modification Kit. DNA was also extracted from HT-29 cells (methylated control) and HDBEC (unmethylated control) and processed as aforementioned. The modified DNA (10 ng/reaction) was amplified with methylation-specific, and unmethylated-specific sets primers to the p16 gene promoter region. Each of the nested amplification steps was carried out for 35 cycles at an annealing temperature of 60°C. Aliquots (4 µl) from the outer reactions were then used as the template for the inner PCR reactions using primers specific for methylated and unmethylated DNA. Both amplicons generated bands of 149 bp. A control β-actin sample was also amplified and examined alongside. (A) The products were examined by 2% (w/v) agarose gel electrophoresis and (B) the band intensities determined and the ratios of the methylated:unmethylated DNA calculated (n=3). TF, tissue factor; gDNA, genomic DNA.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Recombinant, Modification, Methylation, Control, Amplification, Generated, Agarose Gel Electrophoresis

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Proposed model for the mechanism by which the level of TF on the cell surface may have differential outcomes on G1/S checkpoint regulation. The presence of TF on the cell surface differentially upregulates the expression of Inhibitor of CDK p16 INKa , CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 and Alternative reading frame p14 ARF , which is dependent on the concentration of TF and the ability of the cell to dissipate excess TF. Therefore, alterations in p21 CIP1/WAF1 are highly effective in the regulation of the cellular response to acute stress. The interplay between these proteins modulates the signal permitting passage through the cell cycle, or alternatively its arrest. Consequently, the concentration of TF may be an ideal gauge for determining the level of cellular damage. However, the adaptive loss of p16 INK4a function may be promoted by prolonged inflammation leading to permissive transition through the G1/S checkpoint, even in the absence of mutational loss of p16 INKa . TF, tissue factor; PAR2, protease-activated receptor 2, Rb, retinoblastoma protein; E2F, early region 2 binding factor; p21 CIP1/WAF1 , CDK interacting protein/wildtype p53-activated fragment; p16 INK4a , inhibitor of CDK; p14 ARF , alternative reading frame.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Expressing, Concentration Assay, Binding Assay

Journal: Oncology reports

Article Title: M2 macrophages reduce the radiosensitivity of head and neck cancer by releasing HB‑EGF.

doi: 10.3892/or.2020.7628

Figure Lengend Snippet: Figure 1. Detection of HPV infection and radiosensitivity in HNSCC tissues. (A) Representative images of H&E staining using magnification, x200 (T, HNSCC tissues; N, head and neck normal tissues). (B) Representative images of negative and positive HPV‑16/18 detected by DNA ISH. Negative and positive HPV16 RNA ISH detected by RNAscope. Negative and positive p16 detected by p16 IHC using magnification, x200 (HPV+, HPV‑positive tumor tissues; HPV‑ T, HPV‑negative tumor tissues; HPV+/HPV‑ N, head and neck normal tissues; P, positive control‑cervical cancer tissues; N, negative control‑glioma tissues). (C) Representative images of the γ‑H2AX staining of frozen HNSCC sections used to evaluate the extent of radiosensitivity; magnification, x200 (green, γ‑H2AX staining; blue, DAPI). (D) Number of the γ‑H2AX foci per cell; HPV‑positive cases (n=19), HPV‑negative cases (n=33). Results are presented as the mean ± SD. **P<0.01. HPV, human papilloma virus; HNSCC, head and neck squamous cell carcinoma; H&E, hematoxylin and eosin; ISH, in situ hybridization; IHC, immunohistochemistry; γ‑H2AX, γ H2A histone family member X.

Article Snippet: The expression of p16 (cat. no. TA500036; OriGene Technologies, Inc.), inducible nitric oxide synthase (iNOS; 1:100; product code ab53769; Abcam), CD163 (1:100; cat. no. ab87099; Abcam) and EGFR (1:100; product code ab52894; Abcam) in tumor and normal tissues was determined by IHC analysis.

Techniques: Infection, Staining, RNAscope, Virus, In Situ Hybridization, Immunohistochemistry

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Strategy for introducing focal knock-in of CMV-EGFP to replace exon 2 of CDKN2A.

Article Snippet: Low-passage NHMs (passage 2–5 after isolation) were electroporated as described above with 800 ng each of pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734), pEN35-CDKN2A-Ex2-L (Addgene #110736) and pEN35-CDKN2A-Ex2-R (Addgene #110737) and plated directly into media containing 5 µM L755507 for enhancement of HDR recombination ( Yu et al., 2015 ). pHDR plasmids alone were transfected as no Cas9 controls.

Techniques: Knock-In

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Schematic of experimental set-up. For each experiment, NHMs are derived from donated tissue and engineered for CDKN2A loss as in Figure 1. After isolation, CDKN2A null and wild-type sibling cells are monitored via digital holographic cytometry for 72 hr. The rate of cell division, motility, morphology, growth arrest and detachment are quantified. Representative holographic phase shift images (bottom left) show colored comet tails tracking cells.

Article Snippet: Low-passage NHMs (passage 2–5 after isolation) were electroporated as described above with 800 ng each of pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734), pEN35-CDKN2A-Ex2-L (Addgene #110736) and pEN35-CDKN2A-Ex2-R (Addgene #110737) and plated directly into media containing 5 µM L755507 for enhancement of HDR recombination ( Yu et al., 2015 ). pHDR plasmids alone were transfected as no Cas9 controls.

Techniques: Derivative Assay, Isolation, Cytometry

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Volcano plot comparing transcriptomes of three independently derived pairs of CDKN2A null NHMs and matched wild-type sibling cells. Differentially expressed transcripts with q-values < 0.05 and log2 fold change > 1.7 are highlighted in aqua. The BRN2 transcript is highlighted in red.

Article Snippet: Low-passage NHMs (passage 2–5 after isolation) were electroporated as described above with 800 ng each of pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734), pEN35-CDKN2A-Ex2-L (Addgene #110736) and pEN35-CDKN2A-Ex2-R (Addgene #110737) and plated directly into media containing 5 µM L755507 for enhancement of HDR recombination ( Yu et al., 2015 ). pHDR plasmids alone were transfected as no Cas9 controls.

Techniques: Derivative Assay

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Fraction of nevi or melanomas with mono- or bi-allelic CDKN2A disruption. Melanomas are sub-categorized as melanoma in situ (MIS), thin invasive melanoma (Stage T1), thick invasive melanoma (Stage T2+) and distal metastatic melanoma (obtained from the TCGA public database).

Article Snippet: Low-passage NHMs (passage 2–5 after isolation) were electroporated as described above with 800 ng each of pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734), pEN35-CDKN2A-Ex2-L (Addgene #110736) and pEN35-CDKN2A-Ex2-R (Addgene #110737) and plated directly into media containing 5 µM L755507 for enhancement of HDR recombination ( Yu et al., 2015 ). pHDR plasmids alone were transfected as no Cas9 controls.

Techniques: In Situ

Journal: Cancer cell

Article Title: Bi-allelic loss of CDKN2A initiates melanoma invasion via BRN2 activation

doi: 10.1016/j.ccell.2018.05.014

Figure Lengend Snippet: (A) Gene set enrichment analysis of three CDKN2A null NHM lines compared to three CDKN2A wild-type NHM lines (Engineered NHMs) or CDKN2A wild-type regions compared to CDKN2A null regions of the clinical cohort (Transition case cohort). # indicates both the NOM p value and FDR q-values for the enrichment score < 0.0005.

Article Snippet: Low-passage NHMs (passage 2–5 after isolation) were electroporated as described above with 800 ng each of pHDR-CDKN2A-Ex2-CMV-EGFP (Addgene #110734), pEN35-CDKN2A-Ex2-L (Addgene #110736) and pEN35-CDKN2A-Ex2-R (Addgene #110737) and plated directly into media containing 5 µM L755507 for enhancement of HDR recombination ( Yu et al., 2015 ). pHDR plasmids alone were transfected as no Cas9 controls.

Techniques:

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: Senescence markers in primary human and mouse VSMCs undergoing senescence. ( A ) % EdU + in cultured human VSMCs (Control), after 24h treatment with 500 nM doxorubicin (Dox 1d), after an additional 21 days recovery in control conditions (control 21d) or after doxorubicin (Dox 1d+ 21d), or at replicative senescence (RS). ( B – D ) mRNA levels of Lamin B1, p16, and p21 in cell populations described in ( A ) relative to control (1d) cells. ( E ) Western blot for Lamin B1, p16, p21, and p53 for cells treated in ( A ). n = 6–8 human VSMC isolates. ( F and G ) EdU + % ( F ) or SAβG + % ( G ) of mouse p16-3MR VSMCs treated increasing concentrations of Doxorubicin for 1 day followed by 7 days recovery vs. vehicle control. ( I – K ) qPCR for Lamin B1, IL6, p16, or p21 mRNA expression for cells treated in ( F ). ( L ) Western blot of mouse cells as treated in ( F ) for Lamin B1, p16, or p21. n = 3–8 mouse VSMC isolates. Data are means (SD), one-way ANOVA with correction for multiple comparisons ( A ) or unpaired Student’s t -test vs. Control 1d ( B – D ) or vs. Vehicle (Dox 0 nM) ( F – K ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Cell Culture, Control, Western Blot, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: p16/Cdkn2a is detected in VSMCs in mouse atherosclerotic plaques. ( A and B ) UMAP plots showing scRNA-seq profiles of unsorted aortic cells from Myh11Cre ERt2+ /Confetti + mice ( A ), or sorted Confetti + VSMCs from atherosclerotic plaque and media of fat-fed Myh11Cre ERt2 /Confetti + /ApoE −/− mice ( B ). Log-transformed expression levels of Myh11 and p16/Cdkn2a are shown alongside e-Cadherin/Cdh5 and Pdgfrα ( A ) or Cd68 and Ly6a/Sca1 ( B ) using a scale from white to dark red. Insets show high power regions of clusters 6, 8, and 9 and expression of p16 in ( B ). Feature plots show log-normalized expression levels. ( C ) % cells in each cluster with detectable expression of p16/cdkn2a after 14 weeks or 18 weeks or high-fat feeding, or combined.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Transformation Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: GCV treatment of p16-3MR mice does not affect atherosclerosis, but induces inflammation. ( A ) Aortic root plaques in ApoE→ApoE, p16→ApoE, ApoE→p16, or p16→p16 mice + GCV, or p16→p16 mice + saline, stained with Masson’s trichrome, TUNEL, or Mac3. Scale bar = 300 µm. High power inset shows apoptotic cell and nuclear debris from outlined area. ( B ) Plaque area for mice in ( A ). ( C and D ) Number of TUNEL + cells/aortic root plaque ( C ) or %Mac3 + cells ( D ) for mice in ( A ). ( E – G ) Relative mRNA expression for p16, IL18, or TNFα in experimental mice. Data are means (SD) n = 5–10 mice. One-way ANOVA with correction for multiple comparisons ( B – D ) or Kruskal–Wallis H test followed by Dunn’s multiple comparisons test ( E – G ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Saline, Staining, TUNEL Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 (Navitoclax) selectively reduces senescent VSMCs. ( A and B ) Photomicrographs ( A ) or quantification ( B ) of mouse VSMCs stained for SAβG, as replicating control cells or after dox1 + 7 days treatment, or each group ±1 µM ABT-263 treatment for 48 h. ( C ) Western blot and quantification for p16 in cells treated in ( A and B ). ( D ) Fold change in mRNA expression compared with control replicating cells for p16 and a range of SASP cytokines against the housekeeping gene HMBS. Data are means (SD), n = 4–5. Unpaired Student’s t -test. ( E ) Mouse macrophages cultured for 28 days, then treated with 1 µM ABT-263 for 48 h and stained for SAβG. Data are means (SD), n = 3. Unpaired Student’s t -test.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Staining, Control, Western Blot, Expressing, Cell Culture

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 reduces atherosclerosis, but not local SASP cytokine expression. ( A ) ORO staining of mouse descending aorta treated with control (vehicle) or ABT-263, and quantification of %ORO area ( n = 11–14). Scale bar = 3 mm. ( B ) Masson’s trichrome histochemistry of aortic root atherosclerotic plaque from mice treated in ( A ). Panels below show high power view of outlined area. Arrow shows necrotic core. Scale bar = 200 µm. ( C – E ) Aortic root plaque area/total area ( C ) Cap area ( D ), or Core area ( E ) for mice in ( A ). n = 11–13. ( F ) qPCR for relative expression of p16 or SASP cytokines in aortic arches of experimental mice against the housekeeping gene HMBS ( n = 7). Data are means (SD), n = 10. Unpaired Student’s t -test ( A, C – E ) or Mann–Whitney U test ( F ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Expressing, Staining, Control, MANN-WHITNEY

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Cell proliferation of PANC-1 cells stably expressing empty expression vector, codon-optimized CDKN2A, one of three synonymous variants (p.L32L, p.G101G, p.V126V), or one of three pathogenic variants (p.L32P, p.G101W, p.V126D) over 14 days in culture. Cell proliferation values are given as mean of three repeats ± standard deviation normalized to PANC-1 cells that stably express empty vector. Statistically significant inhibition of cell proliferation inhibition in PANC-1 cells that stably express synonymous variants (*; p-value<0.001; Students t-test). ( B ) PANC-1 cells stably expressing codon-optimized CDKN2A transduced with a CellTag lentiviral library of 20 nonfunctional barcodes were cultured and representation (percent of reads supporting each barcode) before (day 9) and after a period of in vitro cell proliferation (day 45) was determined using next-generation sequencing. Percent values are given as the mean of three repeats ± standard deviation. Figure 1—figure supplement 1—source data 1. Raw data in . Figure 1—figure supplement 1—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Standard Deviation, Inhibition, Transduction, Cell Culture, In Vitro, Next-Generation Sequencing

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: PANC-1 cell stably expressing 1 of 20 CDKN2A variants, 19 missense variants, and 1 synonymous variant, at residue p.V126 or p.R144 were cultured. Variant representation, as the percent of reads supporting the variant sequence, before and after a period in vitro cell proliferation determined by next-generation sequencing for the two residues, p.V126 ( A ) or p.R144 ( B ). CDKN2A variant p.V126D (*) was previously reported as pathogenic and increased representation during in vitro proliferation. CDKN2A variant p.R144C (**) was previously reported as benign variant and maintained representation during in vitro proliferation. Figure 1—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Stable Transfection, Expressing, Variant Assay, Residue, Cell Culture, Sequencing, In Vitro, Next-Generation Sequencing

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Functional classifications for 3120 CDKN2A variants, including 2964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious, indeterminate function, or neutral based on p-value using gamma generalized linear model (GLM). 525 (17.7%) variants were classified as functionally deleterious. ( B ) Log 2 p-value (gamma GLM) for 32 benchmark pathogenic variants, 6 benign variants, 31 variants of uncertain significance (VUSs) previously reported to have functionally deleterious effects, and 18 VUSs previously reported to have functionally neutral effects. ( C ) Heatmap with p-values (gamma GLM) for all 3120 CDKN2A variants assayed. Figure 2—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Distribution of log 2 p-value (gamma GLM) for all possible CDKN2A missense variants. ( B ) Distribution of log 2 p-value (gamma GLM) for benchmark pathogenic variants (red box), benchmark benign variants (blue box), variants of uncertain significance (VUSs) previously reported to have functionally deleterious effects (orange box), and VUSs previously reported to have functionally neutral effects (green box). ( C ) Dot plot showing log 2 p-value (gamma GLM) of all possible CDKN2A missense variants per residue. Figure 2—figure supplement 1—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Residue

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Dot plot showing log 2 normalized fold change of all possible CDKN2A missense variants by residue. ( B ) Log 2 normalized fold change for 32 benchmark pathogenic variants, 6 benign variants, 31 variants of uncertain significance (VUSs) previously reported to have functionally deleterious effects, and 18 VUSs previously reported to have functionally neutral effects. ( C ) Functional classifications for 3120 CDKN2A variants, including 2964 missense variants and 156 synonymous variants. Variants were classified as functionally deleterious, indeterminate function, or neutral based on log 2 normalized fold change. ( D ) Comparison of functional classification of all possible CDKN2A missense variants by log 2 p-value (gamma GLM) and log normalized fold change. Figure 2—figure supplement 2—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Residue, Functional Assay, Comparison

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Dot plot showing log 2 p-value (gamma GLM) for 560 CDKN2A missense variants assayed in duplicate. ( B ) Comparison of functional classifications for 560 CDKN2A missense variants assayed in duplicate. ( C ) Dot plot showing log 2 normalized fold change for 560 CDKN2A missense variants assayed in duplicate. Figure 2—figure supplement 3—source data 1. Raw data in . Figure 2—figure supplement 3—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Comparison, Functional Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Proportion of all possible 2964 CDKN2A missense variants in the day 9 cell pool (replicate 1 if duplicated). ( B ) Percent of functionally deleterious variants (black box), variants of indeterminate function, and functionally neutral variants (white box) by variant proportion in the day 9 cell pool (replicate 1 if duplicated). Left graph variants grouped as <2% and ≥2% in day 9 cell pool. Right graph, variants grouped as <2%, 1% intervals from 2% to 8%, ≥8% in the day 9 cell pool. Figure 2—figure supplement 4—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Variant Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: Variant effect predictions for CDKN2A missense variants using CADD, PolyPhen-2, SIFT, VEST, AlphaMissense, ESM1b, and PrimateAI-3D. Predicted deleterious, damaging, or pathogenic effects (black box) and predicted neutral, tolerated, benign, or ambiguous effects (white box) presented as percent of missense variants with an available prediction. Number of missense variants with an available prediction for each in silico model given in parentheses. Accuracy shown as a red line. CADD: Combined Annotation Dependent Depletion; PolyPhen-2: Polymorphism Phenotyping v2; SIFT: Sorting Intolerant From Tolerant; VEST: Variant Effect Scoring Tool score. Figure 3—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Variant Assay, In Silico

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Schematic representation of CDKN2A with ankyrin repeats 1–4 represented. ( B ) Percent of functionally deleterious (black box), indeterminate function (gray box), and functionally neutral variants (white box) within ankyrin repeats and non-ankyrin repeat regions of CDKN2A. Ank; ankyrin repeat. ( C ) Dot plot showing distribution of percent functionally deleterious missense variants per residue. Figure 2—figure supplement 5—source data 1. Raw data in . Figure 2—figure supplement 5—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Residue

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Number of algorithms predicting deleterious effect for 904 CDKN2A missense variants with predictions from seven algorithms. ( B ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect. ( C ) Number of algorithms predicting deleterious effect for 904 CDKN2A missense variants grouped by ankyrin repeats and non-ankyrin repeat regions. ( D–H ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect in Ank1 ( D ), Ank2 ( E ), Ank3 ( F ), Ank4 ( G ), and non-ankyrins repeat regions ( H ) of CDKN2A. Figure 3—figure supplement 1—source data 1. Raw data in . Figure 3—figure supplement 1—source data 2. Raw data in . Figure 3—figure supplement 1—source data 3. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques:

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Number of algorithms predicting deleterious effect for 2060 CDKN2A missense variants with predictions from five algorithms. ( B ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect. ( C ) Number of algorithms predicting deleterious effect for 2060 CDKN2A missense variants grouped by ankyrin repeats and non-ankyrin repeat regions. ( D–H ) Percent of functionally deleterious (black box) and indeterminate function or functionally neutral (white box) variants grouped by the number of algorithms predicting deleterious effect in Ank1 ( D ), Ank2 ( E ), Ank3 ( F ), Ank4 ( G ), and non-ankyrins repeat regions ( H ) of CDKN2A. Figure 3—figure supplement 2—source data 1. Raw data in . Figure 3—figure supplement 2—source data 2. Raw data in . Figure 3—figure supplement 2—source data 3. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques:

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Somatic missense variants in CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box). ( B ) Distribution of functionally deleterious missense somatic mutations CDKN2A reported in COSMIC, TCGA, JHU, or MSK-IMPACT by ankyrin (ANK) repeat. ( C ) Percent of missense somatic mutations in CDKN2A that were classified as functionally deleterious (black box), indeterminate function (gray box), or functionally neutral (white box) group by tumor type. Missense somatic mutations reported in COSMIC, TCGA, JHU, and MSK-IMPACT were combined. The number of missense somatic mutations for each tumor type given in parentheses. COSMIC; the Catalogue Of Somatic Mutations In Cancer, TCGA; The Cancer Genome Atlas, JHU; The Johns Hopkins University School of Medicine, MSK-IMPACT; Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. Figure 4—source data 1. Raw data in . Figure 4—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay, Mutagenesis

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Percent of missense somatic mutations in CDKN2A reported in either COSMIC, TCGA, JHU, or MSK-IMPACT that were classified as pathogenic or likely pathogenic (black box), variant of uncertain significance (VUS) (gray box), or benign or likely benign (white box) using American College of Medical Genetics (ACMG) interpretation guidelines. ( B ) Percent of missense somatic mutations in CDKN2A that were classified as pathogenic or likely pathogenic (black box), VUS (gray box), or benign or likely benign (white box) using ACMG interpretation guidelines grouped by mutation database. ( C ) Number of patients with a pathogenic or likely pathogenic missense somatic mutation grouped by mutation database. Patients with p.His83Tyr mutation (black box), patients with p.Asp84Asn mutations (gray box), and patients with other mutations highlighted. COSMIC: the Catalogue Of Somatic Mutations In Cancer; TCGA: The Cancer Genome Atlas; JHU: The Johns Hopkins University School of Medicine; MSK-IMPACT: Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. Figure 4—figure supplement 1—source data 1. Raw data in . Figure 4—figure supplement 1—source data 2. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Variant Assay, Mutagenesis

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: Percent of missense somatic mutations in CDKN2A reported in either COSMIC ( A ), TCGA ( B ), JHU ( C ), or MSK-IMPACT ( D ) that were classified as functionally deleterious (black box), indeterminate (gray box), or functionally neutral (white box) in our CDKN2A functional assay grouped by tumor type. The number of missense somatic mutations for each tumor type given in parentheses. COSMIC: the Catalogue Of Somatic Mutations In Cancer; TCGA: The Cancer Genome Atlas; JHU: The Johns Hopkins University School of Medicine; MSK-IMPACT: Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets. Figure 4—figure supplement 2—source data 1. Raw data in .

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay, Mutagenesis

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet: ( A ) Synonymous and missense variants in CDKN2A reported in gnomAD. ( B ) 287 CDKN2A missense variants reported in gnomAD, by American College of Medical Genetics (ACMG) guideline classification. ( C ) 264 missense variants in CDKN2A reported in gnomAD, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box). ( D ) 395 missense variants in CDKN2A reported in ClinVar, by functional classification (deleterious – black box; indeterminate – gray box; neutral – white box).

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Functional Assay

Journal: eLife

Article Title: Functional characterization of all CDKN2A missense variants and comparison to in silico models of pathogenicity

doi: 10.7554/eLife.95347

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pHAGE-CDKN2A (plasmid) , Addgene , RRID: Addgene_116726 , Lentiviral vector expressing CDKN2A.

Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Mutagenesis, Modification, Transfection, Software, Control