Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 2. Expression of renalase in rats was increased after 6-OHDA treatment. (A) Detection of serum renalase concentration from the 2nd to 5th week after 6-OHDA application by ELISA kit (n = 6 rats per group). (B) qRT-PCR detection of relative renalase expression in NG, NTS, and heart tissues of Parkinson’s rats and sham group (4th week after 6-OHDA application, n = 5 rats per group). (C,D) Western blot assay showing relative protein levels of renalase in NG, NTS, and heart tissues of Parkinson’s rats and sham group (n = 4 rats per group). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. sham.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 3. PC12 cells transfected with RNLS overexpression plasmid (or empty vector control) continued to overexpress and metabolize DA. (A,B) Transient transfection of PC12 cells with RNLS overexpression plasmid (or empty vector control) expressing the protein. Western blot assay showing relative protein levels of RNLS in PC12 cells at different time points (n = 6). (C) qRT-PCR detection of relative RNLS at different time points after PC12 cells transfected with (n = 6). (D) Detection of relative dopamine (DA) concentration in PC12 cells at different time points by ELISA kit following RNLS overexpression or NC, showing altered DA levels (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 4. Increased levels of DA metabolites after RNLS overexpression of in PC12 cells. (A,B) Detection of DOPAL content at different time points in PC12 cells after RNLS overexpres- sion by high performance liquid chromatography (n = 3). (C) qRT-PCR detection of relative ALDH expression at different time points after PC12 cells were transfected with (n = 6). (D,E) Western blot analysis showing relative protein levels of ALDH at different time points after PC12 cells were transfected with (n = 6). Data are presented as mean ± SD, * p< 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Over Expression, High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing, Transfection, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 6. α-Syn aggregated in PC12 cells transfected with RNLS for 96 h. (A,B) Representative graph of Tri-αS and higher molecular weight α-Syn aggregates in PC12 cells transfected with RNLS for 96 h (n = 6). (C(a,b)) Immunofluorescent photomicrographs of transfected cells (overexpressing RNLS protein for 96 h), cytoplasmic aggregates in the cell neurotic processes are highlighted by white arrows in the figure. (C(c,d)) Untreated control PC12 cells. Scale bar: 20 µm. Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Molecular Weight, Control

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 5. Changes in α-Syn content at different time points in RNLS-overexpressing PC12 cells. (A) qRT-PCR detection of relative α-Syn expression at different time points after PC12 cells trans- fected with RNLS (n = 6). (B) Representative image of immunofluorescence with enhanced α-Syn fluorescence after transfection of PC12 cells. Scale bar: 20 µm. (C) Immunohistochemical quantitative statistics (n = 60). (D–G) Western blot analysis showing relative protein levels of monomer of α-Syn (Mon-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). (E–H) Western blot analysis showing relative protein levels of dimer of α-Syn (Dim-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). (F–I) Western blot analysis showing relative protein levels of trimer of α-Syn (Tri-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Fluorescence, Transfection, Immunohistochemical staining, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 7. The content of tyrosine hydrolase (TH) decreased after PC12 cells were transfected with RNLS. (A) qRT-PCR detection of relative TH expression at different time points after PC12 cells transfected with RNLS (n = 6). (B,C) Western blot analysis showing relative protein levels of TH at different time points after PC12 cells were transfected withR NLS (n = 6). (D) Representative graph of immunofluorescent (IF) results of TH in PC12 cells at different time points administered with RNLS overexpression. Scale bar: 20 µm. (E) Immunohistochemical quantitative statistics (n = 60). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Immunohistochemical staining

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 8. Cell viability decreased after PC12 cells were transfected with RNLS. (A) Live/dead cell viability assay of cultured PC12 cells. The cells were transfected with plasmids and cultured for 24, 48, 72, or 96 h and then stained with the Calcein-AM/PI Double Staining Kit. The live and dead cells exhibited green and red fluorescence. Scale bar: 10 µm. (B) Immunohistochemical quantitative statistics (n = 3). (C) CCK-8 was used to determine PC12 cells viability following overexpression or NC for 24, 48, 72 or 96 h (n = 12). Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Viability Assay, Cell Culture, Staining, Double Staining, Fluorescence, Immunohistochemical staining, CCK-8 Assay, Over Expression

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 9. Decreased expression of axonal transporters after RNLS overexpression in PC12 cells. (A) qRT-PCR detection of relative dynein (DYN) expression at different time points after PC12 cells transfected with RNLS (n = 6). (B,C) Western blot analysis showing relative protein levels of dynein at different time points after PC12 cells were transfected with RNLS (n = 6). (D) mRNA expression of relative kinesin in PC12 cells at different time points (n = 6). (E,F) Western blot analysis showing relative protein levels of KHC at different time points after PC12 cells were transfected with RNLS (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Transfection, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 10. Ultrastructural damage after RNLS overexpression in PC12 cells. (A,B) The relative ∆Ψ m in PC12 cells for each treatment at different time points, as determined by JC-1 staining (n = 15). Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl; scale bar: 20 µm; ∆Ψm: mitochondrial membrane potential. (C) Representative images of transmission microscopy at different time points after PC12 cells were transfected with RNLS. Scale bar: 1 µm and 2 µm, direct magnification: ×1200, ×2000 and ×4000, the red arrow indicates mitochondria.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Over Expression, Staining, Membrane, Transmission Assay, Microscopy, Transfection

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 11. Numerous clues to PD peripheral pathology. (A) Vagal application of DOPAL (3,4- dihydroxyphenylacetaldehyde) to simulate PD-like autonomic dysfunction strongly suggests that DOPAL will likely induce the expression and aggregation of α-Syn/oligomers at the injection site, which will then be transported to the heart and the NG. Axonal transportation is likely to be impaired by DOPAL and DOPAL-mediated oligomer α-Syn [22]. (B) Typical PD model causes the RNLS up-regulation. (C) The pathological α-Syn protein fibers were injected into the mouse intestine, and it was found that the α-Syn protein eventually diffused into the substantia nigra pars compacta, where it degraded dopaminergic neurons. (D) Orthostatic hypotension (OH) has been linked to a higher risk of Parkinson’s disease in studies [48,49]. OH is a predictor of motor decline in individuals with early PD [50]. The experiment confirmed that the change in BP also occurred before the movement disorder in rats treated with 6-OHDA. (E) Current mechanisms and central nervous system origins of PD: MAO-mediated DA metabolism in the central nervous system. (F) The role of RNLS in peripheral etiology of PD.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Injection

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

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: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: High CDK7 protein expression is associated with a poor prognosis of GIST. A Based on data from the GSE136755 dataset, the mRNA levels of CDK4, CDK7 and CDK9 were relatively higher than those of other CDKs for CDK1-10. The mRNA level was calculated via GEO2R. B CDK7 expression was significantly elevated in high-risk GISTs based on the data from GSE136755. C Representative scanned images of GIST samples with low or high CDK7 protein expression, as determined by IHC. D Kaplan–Meier survival curves with a risk table showing that high CDK7 protein expression was significantly positively related to poor recurrence-free survival in GIST patients ( P = 0.044)

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: Knockdown of CDK7 decreases cell viability and proliferation and induces cell cycle arrest. A Immunoblotting analysis of CDK7 expression after targeting siRNA-mediated CDK7 knockdown in GIST-T1 and GIST-882 cells. A nontargeting siRNA and two independent siRNAs (siRNA1 and siRNA2) are represented by siNC, siCDK7-1, and siCDK7-2. B CCK-8 cell viability assay after CDK7 knockdown in GIST-T1 and GIST-882 cells. C Colony formation assays of GIST-T1 and GIST-882 cells after CDK7 knockdown. D Flow cytometry analysis was used to detect and analyse the cell cycle distribution after CDK7 knockdown. E Immunoblotting analysis of cyclin-D1, CDK4 and γH2AX expression after CDK7 knockdown

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Knockdown, Western Blot, Expressing, CCK-8 Assay, Viability Assay, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: CDK7 inhibition leads to the suppression of RNA transcription in GIST cells. A Heatmap showing the change in global active transcripts in GIST-T1 cells following treatment with 50 nmol THZ1 and DMSO for 6 h. B Heatmap showing the transcriptional changes in cell cycle-related genes from the gene set (GOBP_CELL_CYCLE.v7.5.1) in GIST-T1 cells after THZ1 treatment. C The enriched GO functional terms of the transcripts that were reduced over twofold in GIST-T1 cells following treatment with 50 nmol/L THZ1 for 6 h. D Immunoblot analyses of RNAPII, RNAPII CTD phosphorylation (S2, S5, and S7), and CDK7 in GIST-T1 and GIST-882 cells treated either with THZ1 or DMSO at the indicated concentrations for 24 h

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Inhibition, Functional Assay, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: CDK7 inhibition leads to the suppression of c-KIT transcriptional activity and protein expression in GIST cells. A, B qRT–PCR and immunoblotting analysis of c-KIT expression after siRNA-mediated CDK7 knockdown in GIST-T1 and GIST-882 cells. C , D qRT–PCR and immunofluorescence assays showed that c-KIT expression changed after THZ1 treatment in GIST-T1 and GIST-882 cells. E Immunoblotting analysis showed that the expression of c-KIT and downstream ERK and AKT signalling pathway components was inhibited following THZ1 treatment at the indicated concentration in GIST-T1 and GIST-882 cells for 24 h. F Immunoblotting analysis indicated that combination treatment with imatinib and THZ1 enhanced c-KIT expression inhibition in GIST-T1 and GIST-882 cells for 24 h

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Inhibition, Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Immunofluorescence, Concentration Assay

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: CDK7 inhibits c-KIT expression through OSR1 in GIST. A Volcano plot of RNA-seq data displaying the distribution of differential gene expression between THZ1 treatment and DMSO treatment. The upregulated and downregulated genes are highlighted in red and blue, respectively. The results illustrated that OSR1 was the top downregulated gene with the lowest P value. B , C qRT–PCR and immunoblotting analyses showed that OSR1 expression was inhibited by CDK7 knockdown or THZ1 treatment in a dose-dependent manner. OSR1 expression was inhibited by CDK7 knockdown in GIST-T1 and GIST-882 cells. D Gene expression profile in the Oncomine database. The results showed that OSR1 was significantly upregulated in GIST compared with normal gastric tissue. E Gene expression profile in the MediSapiens IST Online transcriptome database. The results showed that OSR1 was uniquely overexpressed in GIST and prostate cancer compared with other cancer types. F Immunoblot analyses of OSR1 expression in clinical sample tissues of GIST, gastrointestinal leiomyoma and schwannoma. The expression of OSR1 was significantly higher in GIST than in leiomyoma and schwannoma. G Data from the MediSapiens database showed that c-KIT and OSR1 expression levels were positively correlated (n = 77, r = 0.407, P < 0.001). H , I qRT–PCR and immunoblotting analyses showed that OSR1 knockdown significantly inhibited c-KIT mRNA and protein expression in GIST-T1 and GIST-882 cells. J Immunoblot analyses showed that OSR1 overexpression promoted c-KIT expression in GIST-T1 and GIST-882 cells. K The inhibitory effect of c-KIT expression was reversed when CDK7 siRNA and OSR1 plasmid were cotransfected into GIST-T1 and GIST-882 cells

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Expressing, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Western Blot, Knockdown, Over Expression, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours

doi: 10.1186/s12964-022-00928-x

Figure Lengend Snippet: Relationship between CDK7 expression and clinicopathological characteristics of GIST patients

Article Snippet: IHC staining was performed with anti-CDK7 antibody (#2916, CST).

Techniques: Expressing, Modification

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