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: PLoS ONE

Article Title: Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling

doi: 10.1371/journal.pone.0131674

Figure Lengend Snippet: A: IHC with anti-CD34 (red) and anti-BrdU (green) staining of hair follicles following 4-day BrdU pulse-chase in the Foxp1 fl/fl (WT) and K14-Cre; Foxp1 fl/fl ( cKO ) mice (P20-P23). The upper panel showed the timing of BrdU injection and sectioning. Abbreviations: Bu, bulge; HG, hair germ; DP, dermal papillae. Scale bars: 25 μm. B: Quantification of the number of BrdU + cells in the bulges of (A). The cKO hair follicles at P24 displayed extensive BrdU + cells in the hair germ and bulge cells, whereas the WT controls had few BrdU + cells in the identical regions (n = 3,4). *, p<0.05. C: IHC for hair follicles at P55 following 28-day BrdU pulse-chase. The upper panel showed the timing of BrdU injection and sectioning. Scale bars: 75 μm. D: Quantification of the percentages of LRC in the bulges of (C). Few label-retaining cells (LRC) were detected in the bulges of Foxp1 cKO mice. n = 4; *, p<0.05. E : NAC treatment and BrdU injection once a day from P23 to P26 enhanced cell proliferation of HFSCs in WT early anagen. Scale bar, 50μm. F: Quantification of the frequency of BrdU + cells in HFSCs in (E). *, p<0.05. G-H: Western blotting demonstrated a decrease of Foxp1 (G) and p19 ARF (H) protein levels within cKO hair follicles at anagen (P23). I: Down-regulation of p19 ARF transcripts within cKO anagen (P23) hair follicles relative to the WT as determined by qRT-PCR. J: S15 phosphorylated-p53 protein level was relatively decreased within cKO anagen (P23) hair follicles.

Article Snippet: The primary antibodies used were: anti-Foxp1 (Millipore, ABE68, USA, 1:1000); anti-phospho serine/threonine/tyrosine (Abcam, ab15556, UK, 1:1000); anti-p53 (Cell Signaling, 2524, USA, 1:1000); anti-p-p53 (Cell Signaling, 9284, USA, 1:1000); anti-β-actin (Santa Cruz, sc-81178, USA, 1:1000); anti-p19 ARF (Santa Cruz, sc-32748, USA, 1:1000).

Techniques: Staining, Pulse Chase, Injection, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling

doi: 10.1371/journal.pone.0131674

Figure Lengend Snippet: A: Anti-Trx1 IHC confirming extensive expression of Trx1 in HFs at anagen (P23), catagen (P40) and telogen (P55). B: Interaction of Foxp1 and Trx-1 endogenous proteins in anagen HFs as determined by Co-IP of protein lysates. C: Interaction of Foxp1 and Trx-1 following ectopic expression of Foxp1-His and Trx1 in transfected HeLa cells. Cell lysates were immunoprecipitated by anti-Trx1 antibody and detected by anti-His antibody. D: Colocalization of Trx1-RFP and Foxp1-EGFP protein within the nuclei (blue, DAPI) of HaCat cells. E: Flow cytometry of DCFDA-stained HEK293T cells following transient transfection of the indicated constructs (2 μg Foxp1 and/or 2 μg Trx1 expressing vector) indicated that Foxp1 releases inhibition of Trx-1-mediated ROS accrual. F: Model for the mechanism by which Foxp1 regulates redox homeostasis during hair cycling. Foxp1 is located within nuclei under conditions of low oxidative stress. Foxp1 suppresses the function of the Trx1 protein in decreasing ROS levels, and then imposes cell cycle arrest through p19/p53 axis. Foxp1 is exported into the cytoplasm when the ROS levels approach a high threshold.

Article Snippet: The primary antibodies used were: anti-Foxp1 (Millipore, ABE68, USA, 1:1000); anti-phospho serine/threonine/tyrosine (Abcam, ab15556, UK, 1:1000); anti-p53 (Cell Signaling, 2524, USA, 1:1000); anti-p-p53 (Cell Signaling, 9284, USA, 1:1000); anti-β-actin (Santa Cruz, sc-81178, USA, 1:1000); anti-p19 ARF (Santa Cruz, sc-32748, USA, 1:1000).

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Flow Cytometry, Staining, Construct, Plasmid Preparation, Inhibition

Journal: Cells

Article Title: Artesunate Inhibits the Cell Growth in Colorectal Cancer by Promoting ROS-Dependent Cell Senescence and Autophagy

doi: 10.3390/cells11162472

Figure Lengend Snippet: Artesunate caused excessive mitochondrial ROS to induce cell senescence and inhibit cell proliferation. ( A ) Artesunate arrested cell cycle at G0/G1 phase in SW480 and HCT116. Cells were seeded in 6-well plates. Once attached, cells were maintained in FBS-free medium for about 16~24 h. Afterwards, cell were treated with artesunate (1, 2, and 4 μM in medium containing 10% FBS) for 72 h and then harvested for PI staining to analyze cell cycle by flow cytometry. ( B ) Artesunate induced cell senescence in SW480 and HCT116. Cells were seeded in 6-well plates and treated with artesunate (1, 2, and 4 μM) for 72 h. Cell senescence was represented via SA-β-gal activity, which was assayed using a SA-β-gal staining kit after artesunate treatment. ( C ) Artesunate treatment downregulated the protein levels of CDK 2/4/6 and upregulated the protein levels of CDKIs, p16, and p21 in SW480 and HCT116. Cells were seeded in 60 mm dishes and treated with artesunate (1, 2, and 4 μM) for 72 h. Then, cells were lysed with RIPA to extract total protein. The protein levels were measured by western blotting. The gray values of protein blots were evaluated by Image J. Relative protein expression was normalized to β-actin. ( D ) NAC attenuated the effect of artesunate on protein expression of p16 in SW480 and HCT116. NAC was used at a concentration of 2 mM and added alone or together with artesunate (4 μM) for 72 h. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Ctrl. ### p < 0.001 vs. cells treated with artesunate alone. Data were shown as mean ± SD.

Article Snippet: CDK2, CDK4, CDK6, Cyclin D1, Cyclin E1, p21, Rb, phosphorylated-Rb, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP, Bax, Bcl-2, BIP, PERK, IRE1α, CHOP, DR5, Beclin 1, LC3A/B, Atg3, Atg5, Atg7, and Atg12 primary antibodies for western blotting were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). p16 and phosphor-IRE1α primary antibodies for western blotting were purchased from Abcam (Cambridge, UK). p16, p21, and Cyclin D1 primary antibodies for immunohistochemistry and β-actin, XBP1S, and XBP1U primary antibodies for western blotting were purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Staining, Flow Cytometry, Activity Assay, Western Blot, Expressing, Concentration Assay

Journal: Cells

Article Title: Artesunate Inhibits the Cell Growth in Colorectal Cancer by Promoting ROS-Dependent Cell Senescence and Autophagy

doi: 10.3390/cells11162472

Figure Lengend Snippet: Artesunate inhibited the growth of CT26-derived tumor in vivo. ( A ) Experimental timeline of CT26-derived tumor model in balb/c mice. Balb/c mice were injected CT-26 cells (1 × 10 5 cells for each mouse) subcutaneously to establish the CT26-derived tumor model. Tumor-loaded mice were gavaged with artesunate at 30 mg/kg or 60 mg/kg for 24 days. Body weights and tumor volumes were recorded every three days. ( B ) Curve of tumor volume. ( C ) Tumor weight. Tumor tissues were collected and weighted after treatment. ( D ) Immunohistochemical images of Ki67, Cyclin D1, p16, p21, LC3B, and p-IRE1α. Immunohistochemistry assay was performed using a SABC-POD staining kit. ( E ) Curve of body weight. ( F ) Organ indexes. Lung, heart, spleen, liver, and kidney were collected and weighted to calculate the organ indexes after treatment. * p < 0.05, *** p < 0.001 vs. Model group. Data were shown as mean ± SD.

Article Snippet: CDK2, CDK4, CDK6, Cyclin D1, Cyclin E1, p21, Rb, phosphorylated-Rb, caspase 3, cleaved-caspase 3, PARP, cleaved-PARP, Bax, Bcl-2, BIP, PERK, IRE1α, CHOP, DR5, Beclin 1, LC3A/B, Atg3, Atg5, Atg7, and Atg12 primary antibodies for western blotting were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). p16 and phosphor-IRE1α primary antibodies for western blotting were purchased from Abcam (Cambridge, UK). p16, p21, and Cyclin D1 primary antibodies for immunohistochemistry and β-actin, XBP1S, and XBP1U primary antibodies for western blotting were purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Derivative Assay, In Vivo, Injection, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: The Prostate

Article Title: A CD24-p53 Axis Contributes to African-American Prostate Cancer Disparities

doi: 10.1002/pros.23973

Figure Lengend Snippet: (A) Representative IHC data showed no CD24 staining in normal prostate (left panel) or benign prostatic hyperplasia (BPH, right panel). (B) Representative IHC data showed CD24 staining of prostate cancer cells with no staining of adjacent normal prostate epithelial cells. The red arrows indicate adjacent normal prostate epithelial cells. (C) CD24, mutp53, MDM2, and ARF staining of representative UAB prostate cancer samples. Representative IHC data showed co-expression of CD24 and mutant p53 from the same case. (D) CD24 and mutp53 IHC staining of two representative PCaP TMA prostate cancer samples. mutp53, mutant p53 protein. All experiments were repeated three times.

Article Snippet: Antibodies were human CD24 (ML5, BD Biosciences), p53 (DO-1, Santa Cruz Biotechnology), MDM2 (SMP14, BD Biosciences), and ARF (also p14-ARF, E3X6D, Cell Signaling).

Techniques: Staining, Expressing, Mutagenesis, Immunohistochemistry

Journal: The Prostate

Article Title: A CD24-p53 Axis Contributes to African-American Prostate Cancer Disparities

doi: 10.1002/pros.23973

Figure Lengend Snippet: Ethnic/racial distribution comparisons in gene expressions

Article Snippet: Antibodies were human CD24 (ML5, BD Biosciences), p53 (DO-1, Santa Cruz Biotechnology), MDM2 (SMP14, BD Biosciences), and ARF (also p14-ARF, E3X6D, Cell Signaling).

Techniques: Significance Assay

Journal: The Prostate

Article Title: A CD24-p53 Axis Contributes to African-American Prostate Cancer Disparities

doi: 10.1002/pros.23973

Figure Lengend Snippet: (A-C) Pearson correlations of the H-scores of CD24 relative to mutp53, MDM2, and ARF staining in UAB prostate cancer samples. (D-G) Protein expression levels of CD24, mutp53, MDM2, and ARF for AAs and EAs in the UAB prostate cancers. mutp53, mutant p53 protein; AA, African-American; EA, European-American.

Article Snippet: Antibodies were human CD24 (ML5, BD Biosciences), p53 (DO-1, Santa Cruz Biotechnology), MDM2 (SMP14, BD Biosciences), and ARF (also p14-ARF, E3X6D, Cell Signaling).

Techniques: Staining, Expressing, Mutagenesis

Journal: Cancers

Article Title: CDKN2A -Mutated Pancreatic Ductal Organoids from Induced Pluripotent Stem Cells to Model a Cancer Predisposition Syndrome

doi: 10.3390/cancers13205139

Figure Lengend Snippet: IHC/IF staining conditions for antibodies used in this study.

Article Snippet: P14 , mouse , Cell Signaling , 2407S , ST Citrate , 1:50.

Techniques: Staining