Journal: American Journal of Translational Research

Article Title: BMI1 governs the maintenance of mouse GC-2 cells through epigenetic repression of Foxl1 transcription

doi:

Figure Lengend Snippet: BMI1 is vital for GC-2 cell survival. (A) Western blotting of BMI1 expression in GC-2 cells treated with the indicated PTC-209 concentrations for 48 h. The experiments were repeated three times. (B) Quantification of (A). (C) Viability assay in GC-2 cells treated with PTC-209 (10 μM) or DMSO (control) for the indicated times. n = 6 for each group. (D) Flow cytometric analysis of the cell cycle in GC-2 cells treated with PTC-209 (10 μM) or DMSO (control) for 48 h. n = 3 for each group. (E) Quantification of (D). (F) TUNEL assay in GC-2 cells treated with PTC-209 (10 μM) or DMSO (control) for 48 h. n = 6 for each group. Scale bar: 20 μm. (G) Quantification of (F). For (B) data were analyzed using one-way ANOVA with Dunnett’s post hoc test. For (C, E and G), data analysis was performed using the Student’s two-tailed t-test. **P < 0.01, ***P < 0.001.

Article Snippet: GC-2 cells were lysed in RIPA buffer and incubated with anti-BMI1 antibody (Proteintech) at 4°C overnight.

Techniques: Western Blot, Expressing, Viability Assay, Control, TUNEL Assay, Two Tailed Test

Journal: American Journal of Translational Research

Article Title: BMI1 governs the maintenance of mouse GC-2 cells through epigenetic repression of Foxl1 transcription

doi:

Figure Lengend Snippet: BMI1 represses Foxl1 transcription. (A) Immunofluorescence staining of BMI1 in GC-2 cells. The experiments were repeated three times. Scale bar: 20 μm. (B) Immunoprecipitation assay in GC-2 cells using an anti-BMI1 antibody. The precipitates were subjected to mass spectrometry for the identification of the interacting proteins. (C) The density of BMI1 ChIP-seq reads at Foxl1 loci. (D) Sequence analysis of the BMI1 binding peak at the promoter region of Foxl1. (E) ChIP-qPCR for BMI1 distribution in the promoter region of Foxl1 in GC-2 cells. Gapdh served as a negative control. n = 3 for each group. (F) RT-qPCR analysis of Foxl1 expression in GC-2 cells treated with PTC-209 as indicated for 48 h. n = 3 for each group. (G-I) ChIP-qPCR for H2AK119ub (G), Ring1B (H), and H3K4me3 (I) distribution in the promoter region of Foxl1 in GC-2 cells treated with PTC-209 (10 μM) or DMSO (control) for 48 h. The y-axis represents fold-enrichment relative to IgG controls. n = 3 for each group. For (E, G, H and I), data were analyzed using the Student’s two-tailed t-test. For (F), data analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: GC-2 cells were lysed in RIPA buffer and incubated with anti-BMI1 antibody (Proteintech) at 4°C overnight.

Techniques: Immunofluorescence, Staining, Immunoprecipitation, Mass Spectrometry, ChIP-sequencing, Sequencing, Binding Assay, ChIP-qPCR, Negative Control, Quantitative RT-PCR, Expressing, Control, Two Tailed Test

Journal: American Journal of Translational Research

Article Title: BMI1 governs the maintenance of mouse GC-2 cells through epigenetic repression of Foxl1 transcription

doi:

Figure Lengend Snippet: Foxl1 is involved in BMI1-mediated GC-2 cell maintenance. (A) RT-qPCR analysis of Foxl1 expression in GC-2 cells transfected with negative control (NC) siRNA (si-NC; 50 nM) or siRNAs targeting Foxl1 (si-Foxl1; 50 nM) for 48 h. n = 3 for each group. (B) Cell counting kit-8 (CCK-8) assay in GC-2 cells transfected with si-NC or si-Foxl1 at the indicated time points. n = 6 for each group. (C) Colony formation assay in GC-2 cells transfected with si-NC or si-Foxl1 for 48 h. n = 3 for each group. (D) Quantification of (C). (E) CCK-8 assay in GC-2 cells treated as indicated for 48 h. n = 6 for each group. (F) Flow cytometric analysis of the cell cycle in GC-2 cells treated as indicated for 48 h. n = 3 for each group. (G) Quantification of (F). (H) TUNEL assay in GC-2 cells treated as indicated for 48 h. n = 3 for each group. (I) Quantification of (H). (J) Immunostaining of β-catenin in GC-2 cells treated as indicated for 48 h. n = 3 for each group. (K) Percentage of nuclear β-catenin-expressing cells (white arrows) in (J). For (E-K), PTC-209 and si-Foxl1 were used at the concentrations of 10 μM and 50 nM, respectively. DMSO and si-NC were used as controls (Ctr). Data were analyzed by one-way ANOVA with Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: GC-2 cells were lysed in RIPA buffer and incubated with anti-BMI1 antibody (Proteintech) at 4°C overnight.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Negative Control, Cell Counting, CCK-8 Assay, Colony Assay, TUNEL Assay, Immunostaining

Journal: American Journal of Translational Research

Article Title: BMI1 governs the maintenance of mouse GC-2 cells through epigenetic repression of Foxl1 transcription

doi:

Figure Lengend Snippet: BMI1 epigenetically represses Foxl1 transcription in mouse testes. (A) RT-qPCR analysis of Foxl1 expression in the testes of wild-type (WT) and Bmi1-knockout (KO) mice. (B) ChIP-qPCR of BMI1 distribution in the promoter region of Foxl1 in mouse testes. (C, D) ChIP-qPCR of H2AK119ub and H3K4me3 distribution in the promoter region of Foxl1 in the testes of Bmi1-WT and Bmi1-KO mice. For (A-D), n = 3 adult mice per genotype. Data analysis was performed using the Student’s two-tailed t-test. *P < 0.05, **P < 0.01. WT, wild-type; KO, knockout.

Article Snippet: GC-2 cells were lysed in RIPA buffer and incubated with anti-BMI1 antibody (Proteintech) at 4°C overnight.

Techniques: Quantitative RT-PCR, Expressing, Knock-Out, ChIP-qPCR, Two Tailed Test

Journal: American Journal of Translational Research

Article Title: BMI1 governs the maintenance of mouse GC-2 cells through epigenetic repression of Foxl1 transcription

doi:

Figure Lengend Snippet: Schematic illustration of the working model for the role of BMI1 in GC-2 cells. BMI1 facilitates the monoubiquitination of histone H2A at K119 to repress Foxl1 expression, thereby maintaining spermatocyte development. In the absence of BMI1, PRC1 function is disrupted and Foxl1 is transcriptionally activated via H3K4me3.

Article Snippet: GC-2 cells were lysed in RIPA buffer and incubated with anti-BMI1 antibody (Proteintech) at 4°C overnight.

Techniques: Expressing

Journal: Stem Cell Research & Therapy

Article Title: Enhancing therapeutic efficacy of human adipose-derived stem cells by modulating photoreceptor expression for advanced wound healing

doi: 10.1186/s13287-022-02892-2

Figure Lengend Snippet: Increased proliferation of green OLED-treated hADSCs. a KI67 staining (blue: nuclei, green: KI67, scale bar = 50 µm) of hADSCs at 0, 24, and 48 h after green OLED irradiation for 24 h with quantification results ( n = 6). b Western Blot analysis of KI67 expression in hADSCs at 0, 24, and 48 h after green OLED irradiation for 24 h. c Gene expression levels of C-MYC in hADSCs at 0, 24, and 48 h after green OLED irradiation for 24 h as evaluated by qRT-PCR ( n = 5). d Cell proliferation of hADSCs at 0, 24, and 48 h after green OLED irradiation for 24 h as measured by CCK-8 assay ( n = 5). * p < 0.05; ** p < 0.001 compared to no treatment group with unpaired t test

Article Snippet: Cell viability was evaluated using CCK-8 assay kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan).

Techniques: Staining, Irradiation, Western Blot, Expressing, Quantitative RT-PCR, CCK-8 Assay

Journal: Viruses

Article Title: Stromal Antigen 2 Deficiency Induces Interferon Responses and Restricts Porcine Deltacoronavirus Infection

doi: 10.3390/v14081783

Figure Lengend Snippet: Establishment of STAG2-depleted IPEC-J2 cells line. ( A ) Western blot analysis for STAG2 expression in IPEC-J2 cell line; ( B ) Gene sequence alignment revealed that were heterozygous for STAG2 knockout alleles generated using CRISPR-Cas9 gene editing; ( C ) Cell viability was determined using CCK-8 detection; ( D ) Western blot analysis for STAG2 expression of WT IPEC-J2, STAG2 − / − IPEC-J2 passage 5 (P. 5), passage 10 (P. 10), passage 15 (P. 15) and passage 20 (P. 20).

Article Snippet: Cell viabilities were assessed using a cell counting kit-8 (CCK-8) (Cat NO. GK10001, GLPBIO, Montclair, CA, USA).

Techniques: Western Blot, Expressing, Sequencing, Knock-Out, Generated, CRISPR, CCK-8 Assay

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Low-dose graphene oxide promotes tumor cells proliferation by activating PI3K-AKT-mTOR signaling via cellular membrane protein integrin αV.

doi: 10.1016/j.envpol.2023.121817

Figure Lengend Snippet: Fig. 3. The effects of low-dose GO treatment on the cell cycle of PC3 cells. (a) Statistical data of cell cycle phages distribution for PC3 cells post GO nanosheet (GO-L, GO-M, and GO-S) treatment at 0, 5, 10, 15, 20, and 50 μg/mL for 24 h n = 3. (b) Western blotting of the expression levels of Cyclins and CDKs proteins within PC3 cells underwent GO nanosheet (GO-L, GO-M, and GO-S) treatment at 0, 2, 5, 10, 15, 20, and 50 μg/mL for 24 h *: P < 0.05, **: P < 0.001, relative to un treated control.

Article Snippet: The antibodies (Ab) used in this study were: anti-PI3K(p110β) Ab (1:1000 dilution, CST, Danvers, MA, USA), anti-P-PI3K(Tyr458/Tyr199) Ab (1:1000 dilution, CST), anti-AKT(pan) Ab (1:1000 dilution, CST), anti-P-AKT(Ser473) Ab (1:2000 dilution, CST), anti-mTOR Ab (1:1000 dilution, CST), anti-P-mTOR(Ser2448) Ab (1:1000 dilution, CST), anti-Cyclin A2 Ab (1:2000 dilution, Proteintech, Chicago, IL, USA), anti-Cyclin B1 Ab (1:1000 dilution, Proteintech), anti-Cyclin D1 Ab (1:5000 dilution, Proteintech), anti-CDK1 Ab (1:500 dilution, Proteintech), anti-CDK2 Ab (1:1000 dilution, Proteintech), anti-CDK4 Ab (1:2000 dilution, Proteintech), anti-integrin αV Ab (1:2000 dilution, Abcam, Cambridge, UK), anti-integrin β1 Ab (1:1000 dilution, Proteintech), and anti-β-actin Ab (1:5000 dilution, Proteintech).

Techniques: Western Blot, Expressing, Control

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Low-dose graphene oxide promotes tumor cells proliferation by activating PI3K-AKT-mTOR signaling via cellular membrane protein integrin αV.

doi: 10.1016/j.envpol.2023.121817

Figure Lengend Snippet: Fig. 5. Role of integrin subunits in the process of low- dose GO nanosheet promoting tumor cell prolifera tion. (a) Western blotting of protein expression levels of integrin αV and β1 subunits in PC3 cells post GO nanosheet (GO-L, GO-M, and GO-S) treatment at 0, 2, 5, 10, 15, 20, and 50 μg/mL for 24 h. (b) PC3 cells were transfected with lentiviral shRNA vectors to selectively knockdown integrin αV, and the trans fectants were established and validated by western blotting analysis. Cell viability assay with CCK-8 (c), cell proliferation assay with EdU staining (d, e), and western blotting of protein expression levels of PI3K/ AKT/mTOR signaling pathway-related proteins (f) and Cyclins/CDKs (g) within integrin αV stable knockdown PC3 cells post GO-M treatment at 10 μg/ mL for 24 h. Scale bar = 500 μm **: P < 0.001, relative to untreated control. NS, no significant difference.

Article Snippet: The antibodies (Ab) used in this study were: anti-PI3K(p110β) Ab (1:1000 dilution, CST, Danvers, MA, USA), anti-P-PI3K(Tyr458/Tyr199) Ab (1:1000 dilution, CST), anti-AKT(pan) Ab (1:1000 dilution, CST), anti-P-AKT(Ser473) Ab (1:2000 dilution, CST), anti-mTOR Ab (1:1000 dilution, CST), anti-P-mTOR(Ser2448) Ab (1:1000 dilution, CST), anti-Cyclin A2 Ab (1:2000 dilution, Proteintech, Chicago, IL, USA), anti-Cyclin B1 Ab (1:1000 dilution, Proteintech), anti-Cyclin D1 Ab (1:5000 dilution, Proteintech), anti-CDK1 Ab (1:500 dilution, Proteintech), anti-CDK2 Ab (1:1000 dilution, Proteintech), anti-CDK4 Ab (1:2000 dilution, Proteintech), anti-integrin αV Ab (1:2000 dilution, Abcam, Cambridge, UK), anti-integrin β1 Ab (1:1000 dilution, Proteintech), and anti-β-actin Ab (1:5000 dilution, Proteintech).

Techniques: Western Blot, Expressing, Transfection, shRNA, Knockdown, Viability Assay, CCK-8 Assay, Proliferation Assay, Staining, Control

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Low-dose graphene oxide promotes tumor cells proliferation by activating PI3K-AKT-mTOR signaling via cellular membrane protein integrin αV.

doi: 10.1016/j.envpol.2023.121817

Figure Lengend Snippet: Fig. 6. Effects of GO nanosheet on the proliferation of PC3 cells in vivo. (a) Subcutaneous tumor models were established by PC3 cells injection, which was pre-treated with GO nanosheet (GO-L, GO-M, and GO- S) at 10 μg/mL for 24 h in vitro. Optical images of mouse tumor models (b) and tumors removed from the body (c) on day 30 post tumor cells injection. The quantified data of tumor volumes (d) and tumor weights (e) of each group on day 30 post tumor cells injection, n = 4. Western blotting showed the expression levels of the PI3K/AKT/mTOR signal pathway-related proteins (f) and Cyclins/CDKs (g) within tumor tissues. (h) Histological examination of tumor tissues and representative IHC images of Ki67 and p21, n = 5. Scale bar = 200 μm *: P < 0.05, **: P < 0.001, relative to untreated control.

Article Snippet: The antibodies (Ab) used in this study were: anti-PI3K(p110β) Ab (1:1000 dilution, CST, Danvers, MA, USA), anti-P-PI3K(Tyr458/Tyr199) Ab (1:1000 dilution, CST), anti-AKT(pan) Ab (1:1000 dilution, CST), anti-P-AKT(Ser473) Ab (1:2000 dilution, CST), anti-mTOR Ab (1:1000 dilution, CST), anti-P-mTOR(Ser2448) Ab (1:1000 dilution, CST), anti-Cyclin A2 Ab (1:2000 dilution, Proteintech, Chicago, IL, USA), anti-Cyclin B1 Ab (1:1000 dilution, Proteintech), anti-Cyclin D1 Ab (1:5000 dilution, Proteintech), anti-CDK1 Ab (1:500 dilution, Proteintech), anti-CDK2 Ab (1:1000 dilution, Proteintech), anti-CDK4 Ab (1:2000 dilution, Proteintech), anti-integrin αV Ab (1:2000 dilution, Abcam, Cambridge, UK), anti-integrin β1 Ab (1:1000 dilution, Proteintech), and anti-β-actin Ab (1:5000 dilution, Proteintech).

Techniques: In Vivo, Injection, In Vitro, Western Blot, Expressing, Control