Journal: Acta Pharmacologica Sinica

Article Title: Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

doi: 10.1038/aps.2014.106

Figure Lengend Snippet: Nutlin-3 causes DNA DSB damage in SMMC-7721 cells. (A) Nutlin-3 increased γH2AX expression level. β-Actin served as the loading control. (B) The relative expression levels of γH2AX. The value represents the mean±SD derived from triplicate tests. bP<0.05 vs NC group. (C) Representative images of γH2AX foci formation (1000×). SMMC-7721 cells were treated with 0.1% DMSO, Etoposide (40 μmol/L), or Nutlin-3 (10 μmol/L) for 48 h and analyzed for γH2AX (red). Nuclei were counter-stained with DAPI (blue). The images were merged using Image-Pro plus 6.0. (D) Statistical analysis of the number of γH2AX foci. Data are shown as the mean±SD (n=3). bP<0.05 vs NC group.

Article Snippet: The primary antibodies were rabbit anti-γH2AX polyclonal antibody (bs-3185R, Bioss, diluted 1:200), mouse anti-IFI16 monoclonal antibody (ab50004, Abcam, diluted 1:1000), rabbit anti-H2b polyclonal antibody (BS1657, Bioworld, diluted 1:500), and β-actin antibody (BA2305, Boster, diluted 1:500).

Techniques: Expressing, Derivative Assay, Staining

Journal: Acta Pharmacologica Sinica

Article Title: Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

doi: 10.1038/aps.2014.106

Figure Lengend Snippet: Nutlin-3 induces the chromatin-bound protein IFI16 to partially localize to the cytoplasm of SMMC-7721 cells and increases the expression level of IFNB1 mRNA. (A) Nutlin-3 increased the expression level of IFI16 mRNA. The left panel shows representative gels of IFI16 and IFNB1. The TP53 and ACTB gene were used as the positive and internal control, respectively. The right graph shows the relative expression levels of IFI16 and IFNB1 mRNA. The value represents the mean±SD (n=3). bP<0.05 vs NC group. (B) Chromatin fractions were analyzed via Western blot in SMMC-7721 cells. H2b and β-actin served as the quality control for the nuclear fraction and the cytoplasmic fraction, respectively. S1, cytoplasmic proteins; S2, soluble nuclear proteins; P2, chromatin-enriched sediment; WCE, whole-cell extracts.

Article Snippet: The primary antibodies were rabbit anti-γH2AX polyclonal antibody (bs-3185R, Bioss, diluted 1:200), mouse anti-IFI16 monoclonal antibody (ab50004, Abcam, diluted 1:1000), rabbit anti-H2b polyclonal antibody (BS1657, Bioworld, diluted 1:500), and β-actin antibody (BA2305, Boster, diluted 1:500).

Techniques: Expressing, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

doi: 10.1038/aps.2014.106

Figure Lengend Snippet: IFI16 subcellular localization regulated by Nutlin-3 in L02 cells. (A) IFI16 subcellular localization was detected in L02 cells using fluorescence microscopy (1000×). Cells were treated as described above and stained for IFI16 (green). Nuclei were counter-stained with DAPI (blue). Images were merged using Image-Pro plus 6.0. (B) Chromatin fractions were analyzed via Western blot in L02 cells. H2b and β-actin served as the quality control for the nuclear fraction and the cytoplasmic fraction, respectively. S1, cytoplasmic proteins; S2, soluble nuclear proteins; P2, chromatin-enriched sediment; WCE, whole-cell extracts.

Article Snippet: The primary antibodies were rabbit anti-γH2AX polyclonal antibody (bs-3185R, Bioss, diluted 1:200), mouse anti-IFI16 monoclonal antibody (ab50004, Abcam, diluted 1:1000), rabbit anti-H2b polyclonal antibody (BS1657, Bioworld, diluted 1:500), and β-actin antibody (BA2305, Boster, diluted 1:500).

Techniques: Fluorescence, Microscopy, Staining, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: HnRNP-L-regulated circCSPP1/miR-520h/ EGR1 axis modulates autophagy and promotes progression in prostate cancer

doi: 10.1016/j.omtn.2021.10.006

Figure Lengend Snippet: circCSPP1 promotes prostate cancer autophagy in vitro (A) The overexpression of circCSPP1 promotes autophagy in prostate cancer cell lines, and the conversions of P62, LC3-II, and β-actin under BAF or CQ treatment was detected by western blotting. (B) The knockdown of circCSPP1 attenuates autophagy in prostate cancer cell lines. (C) The overexpression or knockdown of circCSPP1 increases or attenuates the accumulation of LC3-II puncta (green and red overlap). ∗∗p < 0.01, ∗p < 0.05. (D) Autophagosomes (arrow) observed by transmission electron microscopy (TEM) in circCSPP1 overexpression and knockdown prostate cancer cell lines.

Article Snippet: For detecting relative autophagy markers LC3 and P62, cancer cells were treated with EBSS containing 10 μM BAF or 50 nM CQ for 8 h before being lysed by RIPA buffer with PMSF on ice for 15 min. Then the cell lysis was mixed with 5× protein loading buffer and subsequently denaturalized at 100°C for 10 min. Total protein denaturants were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), and blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 detergent for 1 h. The membranes were incubated with primary antibodies against β-actin (BA2305, Boster), LC3A (NB100-2331, Novus), SQSTM1/P62 (sc-28359, Santa Cruz), HnRNP-L (ab6106, Abcam), and EGR1 (#4154 CST) at 4°C overnight.

Techniques: In Vitro, Over Expression, Western Blot, Transmission Assay, Electron Microscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: HnRNP-L-regulated circCSPP1/miR-520h/ EGR1 axis modulates autophagy and promotes progression in prostate cancer

doi: 10.1016/j.omtn.2021.10.006

Figure Lengend Snippet: circCSPP1 sponges miR-520h to promote prostate cancer migration, invasion, proliferation, and autophagy in vitro and in vivo (A and B) The migration and invasion capabilities of DU145 transfected with lentivirus-circCSPP1 and/or miR-520h mimics were determined with the wound-healing and Transwell assays. (C and D) The proliferative ability of DU145 transfected with lentivirus-circCSPP1 and/or miR-520h mimics was determined through the colony-formation and CCK-8 assays. (E–G) Image of subcutaneous tumors derived from DU145 cells transfected with lentivirus-circCSPP1 and/or miR-520h mimics in the xenograft model. Tumor volumes were measured every 6 days up to 30 days, and the final tumor weight was calculated. (H) The conversion of P62, LC3-II normalized to β-actin in DU145, and PC3 transfected with lentivirus-circCSPP1 and/or miR-520h mimics under BAF treatment were detected by western blotting. (I) The accumulation of LC3-II puncta (yellow, green, and red overlap) was detected in DU145 and PC3 transfected with circCSPP1-overexpressing lentivirus or miR-520h mimics or co-transfected with both circCSPP1-overexpressing lentivirus and miR-520h mimics. (J) Autophagosomes (arrow) were observed by TEM in DU145 and PC3 transfected with lentivirus-circCSPP1 and/or miR-520h mimics. Data are shown as means ± SD. Student’s t test, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Article Snippet: For detecting relative autophagy markers LC3 and P62, cancer cells were treated with EBSS containing 10 μM BAF or 50 nM CQ for 8 h before being lysed by RIPA buffer with PMSF on ice for 15 min. Then the cell lysis was mixed with 5× protein loading buffer and subsequently denaturalized at 100°C for 10 min. Total protein denaturants were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), and blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 detergent for 1 h. The membranes were incubated with primary antibodies against β-actin (BA2305, Boster), LC3A (NB100-2331, Novus), SQSTM1/P62 (sc-28359, Santa Cruz), HnRNP-L (ab6106, Abcam), and EGR1 (#4154 CST) at 4°C overnight.

Techniques: Migration, In Vitro, In Vivo, Transfection, CCK-8 Assay, Derivative Assay, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: HnRNP-L-regulated circCSPP1/miR-520h/ EGR1 axis modulates autophagy and promotes progression in prostate cancer

doi: 10.1016/j.omtn.2021.10.006

Figure Lengend Snippet: EGR1 is direct target of miR-520h in prostate cancer cells (A) Venn diagram shows EGR1 is the only gene in the overlap. (B) The mRNA expression level of EGR1 was downregulated or upregulated in DU145 and PC3 transfected with miR-520h mimics or inhibitors. (C) The protein expression level of EGR1 was downregulated or upregulated in DU145 and PC3 transfected with miR-520h mimics or inhibitors. (D) RNA in situ hybridization detected the co-localization between EGR1 (green) and miR-520h (red) in PC3 cells. (E) The interaction of miR-520 with EGR1 gene sequences was predicted by bioinformatics, and dual-luciferase reporter assays were performed to confirm their direct target sites. ∗∗p < 0.01. (F and G) Pattern of the expression of circCSPP1/miR-520h/ EGR1 normalized to that of β-actin and the Pearson correlation analysis between any two of three above in 10 pairs of human prostate cancer and benign tissues or 6 pairs of xenograft tumors with vector or circCSPP1 overexpression. Data are shown as means ± SD. Student’s t test, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Article Snippet: For detecting relative autophagy markers LC3 and P62, cancer cells were treated with EBSS containing 10 μM BAF or 50 nM CQ for 8 h before being lysed by RIPA buffer with PMSF on ice for 15 min. Then the cell lysis was mixed with 5× protein loading buffer and subsequently denaturalized at 100°C for 10 min. Total protein denaturants were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), and blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 detergent for 1 h. The membranes were incubated with primary antibodies against β-actin (BA2305, Boster), LC3A (NB100-2331, Novus), SQSTM1/P62 (sc-28359, Santa Cruz), HnRNP-L (ab6106, Abcam), and EGR1 (#4154 CST) at 4°C overnight.

Techniques: Expressing, Transfection, RNA In Situ Hybridization, Luciferase, Plasmid Preparation, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: HnRNP-L-regulated circCSPP1/miR-520h/ EGR1 axis modulates autophagy and promotes progression in prostate cancer

doi: 10.1016/j.omtn.2021.10.006

Figure Lengend Snippet: miR-520h silences EGR1 to inhibit prostate cancer migration, invasion, proliferation, and autophagy in vitro (A and B) The migration and invasion capabilities of DU145 and PC3 transfected with pcDNA3.1- EGR1 and/or miR-520h mimics were determined with the wound-healing and Transwell assays. (C and D) The proliferative ability of DU145 and PC3 transfected with pcDNA3.1- EGR1 and/or miR-520h mimics was determined through the colony-formation and CCK-8 assays. (E) EGR1 expression and the conversion of P62, LC3-II normalized to β-actin in DU145 and PC3 transfected with pcDNA3.1- EGR1 and/or miR-520h mimics under BAF treatment were detected by western blotting. (F) The accumulation of LC3-II puncta (yellow, green, and red overlap) was detected in DU145 and PC3 transfected with pcDNA3.1- EGR1 and/or miR-520h mimics. (G) Autophagosomes (arrow) were observed by TEM in DU145 and PC3 transfected with pcDNA3.1- EGR1 and/or miR-520h mimics. Data are shown as means ± SD. Student’s t test, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

Article Snippet: For detecting relative autophagy markers LC3 and P62, cancer cells were treated with EBSS containing 10 μM BAF or 50 nM CQ for 8 h before being lysed by RIPA buffer with PMSF on ice for 15 min. Then the cell lysis was mixed with 5× protein loading buffer and subsequently denaturalized at 100°C for 10 min. Total protein denaturants were separated by SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), and blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 detergent for 1 h. The membranes were incubated with primary antibodies against β-actin (BA2305, Boster), LC3A (NB100-2331, Novus), SQSTM1/P62 (sc-28359, Santa Cruz), HnRNP-L (ab6106, Abcam), and EGR1 (#4154 CST) at 4°C overnight.

Techniques: Migration, In Vitro, Transfection, CCK-8 Assay, Expressing, Western Blot