Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: G6PD, Cyclin E1 and MMP9 are overexpressed in human ccRCC tissues. ( A-E ) real-time RT-PCR ( A-C ), Western blot ( D ) and grayscale scanning ( E ) were employed for the detection of G6PD, Cyclin E1 and MMP9 expression levels in ccRCC tumor specimens and relevant adjacent normal tissues (n=20). β-actin was used as a loading control. Representative cropped gels and blots of the Western blot analysis were shown ( D ). The samples used for quantitative comparisons in the Western blot analysis were derived from the same experiment and that gels were processed in parallel ( E ). ( F-I ) IHC were conducted to analyze the expression of G6PD, Cyclin E1 and MMP9 in ccRCC and relevant adjacent normal tissues (n=20). Representative images were shown ( F ). Statistical analysis was conducted by paired Student's t -test for Western blot analysis ( E ) and by χ 2 test for IHC analysis ( G-I ), respectively. * p <0.05, ** p <0.01 vs. Normal tissues.

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Derivative Assay

Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: Cyclin E1 and MMP9 are positively correlated with G6PD and associated with poor outcomes in ccRCC patients. ( A-C ) mRNA expression levels of Cyclin D1, Cyclin E1 and MMP9 in normal kidney tissues (n=72) and ccRCC specimens (n=535) were analyzed by TCGA dataset mining (Mann-Whitney U test). ( D - F ) Spearman correlation analyses between G6PD and Cyclin D1, G6PD and Cyclin E1, G6PD and MMP9 at the mRNA expression levels were conducted in ccRCC and normal kidney tissues. ( G-O ) Kaplan-Meier analyses for overall survival of all ccRCC patients (n=528), patients with stage I/II ccRCC (n=320) and patients with stage III/IV ccRCC (n=205) in the TCGA cohort with high vs. low indicated gene mRNA expression levels were shown (log-rank test).

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: Expressing, MANN-WHITNEY

Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: Correlations between the expression of Cyclin D1, Cyclin E1, MMP9 and important clinicopathological variables in ccRCC.

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of the association of G6PD, Cyclin D1, Cyclin E1 and MMP9 expression and other clinicopathologic features with overall survival in ccRCC.

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: G6PD upregulates the expression of Cyclin E1 and MMP9 in vitro . ( A-C ) The expression of Cyclin E1 and MMP9 at the mRNA and protein level in stably transfected ACHN-G6PD OE , Caki-1- G6PD si and relevant control cells was analyzed by using real-time RT-PCR ( A ), Western blot and grayscale scanning assay ( B-C ), respectively. β-actin was used as a loading control. Representative cropped gels and blots of the Western blot analysis were shown ( B ). The samples used for quantitative comparisons in the Western blot analysis were derived from the same experiment and that gels were processed in parallel ( C ). ( D ) Relative MMP9 enzyme activities in ACHN-G6PD OE , Caki-1- G6PD si and relevant control cells were analyzed by using MMP9 activity kit in stable transfected ACHN or Caki-1 cells. All assays were done in at least triplicate. Bars represent the means ± SD. * p <0.05, ** p <0.01, *** p <0.001 vs. Control or Non-silencer (unpaired Student's t -test).

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: Expressing, In Vitro, Stable Transfection, Transfection, Quantitative RT-PCR, Western Blot, Derivative Assay, Activity Assay

Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: Cyclin E1 and MMP9 are involved in the G6PD-mediated ccRCC cells proliferation and migration. ( A-B ) The expression of Cyclin E1 at the mRNA and protein level in Caki-1, ACHN-G6PD OE and relevant control cells was analyzed by using real-time RT-PCR ( A ) and Western blot assay ( B ), respectively at 48 h after Cyclin E1 siRNA transfection. β-actin was used as a protein loading control. Representative cropped gels and blots of the Western blot analysis were shown ( B ). ( C-F ) Cyclin E1 siRNA transfected Caki-1, ACHN-G6PD OE and relevant control cells were subjected to cell cycle distribution analysis by PI staining and flow cytometry assay. ( G-H ) Cell proliferation abilities of Caki-1-Cyclin E1 si , ACHN-G6PD OE -Cyclin E1 si and relevant control cell lines were assessed by MTS assay at different time points. ( I-M ) Caki-1, ACHN-G6PD OE cells following treatment with the MMP9 inhibitor JNJ-0966 (10 μM, 24 h) and relevant control cells were subjected to Transwell assays. Representative images ( I , K ) and quantification analyses ( J , M ) are shown. All assays were done in at least triplicate. Bars represent the means ± SD. * p <0.05, ** p <0.01, *** p <0.001 vs. relevant control (Mixed ANOVA for G-H , unpaired Student's t -test for others).

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Staining, Flow Cytometry, MTS Assay

Journal: International Journal of Medical Sciences

Article Title: G6PD upregulates Cyclin E1 and MMP9 to promote clear cell renal cell carcinoma progression

doi: 10.7150/ijms.58902

Figure Lengend Snippet: G6PD upregulates Cyclin E1 and MMP9 to enhance ccRCC progression in vivo . ( A ) Stably transfected ACHN-G6PD OE , Caki-1- G6PD si and relevant control cells were subcutaneous injected in the nude mice, respectively. Representative xenografted mice images were shown. ( B-C ) The protein expression of G6PD, Cyclin E1 and MMP9 in the mice tumor tissue were analyzed by Western blot analysis ( B ) and grayscale scanning ( C ). β-actin served as a loading control. Representative cropped gels and blots of the Western blot analysis were shown ( B ). The samples used for quantitative comparisons in the Western blot analysis were derived from the same experiment and that gels were processed in parallel ( C ). ( D ) Relative MMP9 enzyme activities in the mice tumor tissue were analyzed by using MMP9 activity kit. The data represent three independent experiments. Each bar represented the mean ± SD. ** p <0.01, *** p <0.001 vs. Control or Non-silencer (unpaired Student's t -test).

Article Snippet: The following antibodies were used: G6PD antibody (ab133525, Abcam), Cyclin E1 antibody (bsm-52048R, Bioss, Beijing, China), MMP9 antibody (ab76003, Abcam).

Techniques: In Vivo, Stable Transfection, Transfection, Injection, Expressing, Western Blot, Derivative Assay, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: NVP-BEZ235 inhibits thyroid cancer growth by p53- dependent/independent p21 upregulation

doi: 10.7150/ijbs.37592

Figure Lengend Snippet: NVP-BEZ235 induces cell cycle arrest in thyroid cancer cells. (A) IHH4, K1, BCPAP and C643 cells were treated with 200 nM NVP-BEZ235 for 48 hours, respectively. DNA content was measured by flow cytometry to determine cell cycle fractions. The fraction of cells in each cell cycle phase was indicated in the figures. (B) The protein expressions of cyclin E1 and cyclin-dependent kinase 2 (Cdk2) were examined by western blot in IHH4, K1, BCPAP and C643 cells with 200 nM NVP-BEZ235 treatment (left). Relative protein levels were quantified by ImageJ software (right). * p < 0.05, ** p < 0.01, *** p < 0.001, respectively, NVP-BEZ235 versus control.

Article Snippet: Anti-cyclin E1 antibodies was purchased from Sino Biological (Beijing, China).

Techniques: Flow Cytometry, Western Blot, Software

Journal: International Journal of Biological Sciences

Article Title: NVP-BEZ235 inhibits thyroid cancer growth by p53- dependent/independent p21 upregulation

doi: 10.7150/ijbs.37592

Figure Lengend Snippet: p21 knockdown abolished the effect of NVP-BEZ235 in both p53 wild type and mutant thyroid cancer cells. ( A) MTT assays showed that the inhibitory effect of cell growth on IHH4, K1, BCPAP and C643 cells after NVP-BEZ235 treatment (200 nM) was abolished by p21 knockdown (si-p21). (B) Cell cycle arrest induced by NVP-BEZ235 (200 nM) was reversed by p21 knockdown (si-p21) in IHH4, K1, BCPAP and C643 cell lines. (C) p21 knockdown (si-p21) was performed in IHH4, K1, BCPAP and C643 cells and then cells were treated with NVP-BEZ235. The protein expressions of p21, cyclin E1 and Cdk2 were determined by Western blot (left). Relative protein levels were quantified by ImageJ software (right). GAPDH was used as loading control. 235, NVP-BEZ235; NC, Negative control. * p < 0.05, ** p < 0.01, *** p < 0.001, respectively, 235 + si-p21 versus 235 + NC.

Article Snippet: Anti-cyclin E1 antibodies was purchased from Sino Biological (Beijing, China).

Techniques: Mutagenesis, Western Blot, Software, Negative Control

Journal: Annals of Translational Medicine

Article Title: The immunoglobulin superfamily member 3 (IGSF3) promotes hepatocellular carcinoma progression through activation of the NF-κB pathway

doi: 10.21037/atm.2020.02.14

Figure Lengend Snippet: Primary antibodies with indicated concentration for WB, IHC, IF

Article Snippet: Primary antibodies with indicated concentration for WB, IHC, IF ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Dilution Company Cat No. WB IHC IF IGSF3 1:400 1:400 1:50 Bioss Bs-9196R PCNA 1:250 BOSTR BM0104 cyclin A 1:250 BOSTER A00700 cyclin E 1:400 BOSTER A00543-1 CDK1 1:400 BOSTER BM0027 CDK2 1:400 BOSTER PB0562 p-IKBα 1:400 Wanleibio WL03120 IKBα 1:400 Wanleibio WL02495 IKBα 1:200 Beyotime AF1282 p-NF-κB 1:400 Wanleibio WL02169 NF-κB 1:400 Wanleibio WL01273b NF-κB 1:200 Beyotime AF1234 GAPDH 1:800 BOSTER BM3876 Ki-67 1:200 BOSTER PB0065 Lamin B 1:1,000 Bioss bs-1840R β-action 1:800 Bioss bsm-33036M Open in a separate window caption a8 Primary antibodies with indicated concentration for WB, IHC, IF Cell proliferation, migration, and Matrigel invasion assay We used the Costar 24 well insert system (Life Technologies, corning, USA) to perform cell migration assay.

Techniques: Concentration Assay

Journal: Annals of Translational Medicine

Article Title: The immunoglobulin superfamily member 3 (IGSF3) promotes hepatocellular carcinoma progression through activation of the NF-κB pathway

doi: 10.21037/atm.2020.02.14

Figure Lengend Snippet: IGSF3 affects HCC cell proliferation in vitro. (A,B) Western blot analyses of cyclin A, cyclin E, CDK1, and CDK2 levels in HCLLM3 and hepG2 cells. Bar graph shows cyclin A, cyclin D, CDK1, and CDK2 levels in HCLLM3 and hepG2 cells transfected with either shCON or shIGSF3; (C,D) FACS analysis results showing proportion of cells in each phase of the cell cycle. Bar graph shows the results of the number of cells in each phase of the cell cycle. All experiments were conducted three times. Results were indicated as mean ± SD (*, P<0.05; **, P<0.01; ***, P<0.001).

Article Snippet: Primary antibodies with indicated concentration for WB, IHC, IF ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Dilution Company Cat No. WB IHC IF IGSF3 1:400 1:400 1:50 Bioss Bs-9196R PCNA 1:250 BOSTR BM0104 cyclin A 1:250 BOSTER A00700 cyclin E 1:400 BOSTER A00543-1 CDK1 1:400 BOSTER BM0027 CDK2 1:400 BOSTER PB0562 p-IKBα 1:400 Wanleibio WL03120 IKBα 1:400 Wanleibio WL02495 IKBα 1:200 Beyotime AF1282 p-NF-κB 1:400 Wanleibio WL02169 NF-κB 1:400 Wanleibio WL01273b NF-κB 1:200 Beyotime AF1234 GAPDH 1:800 BOSTER BM3876 Ki-67 1:200 BOSTER PB0065 Lamin B 1:1,000 Bioss bs-1840R β-action 1:800 Bioss bsm-33036M Open in a separate window caption a8 Primary antibodies with indicated concentration for WB, IHC, IF Cell proliferation, migration, and Matrigel invasion assay We used the Costar 24 well insert system (Life Technologies, corning, USA) to perform cell migration assay.

Techniques: In Vitro, Western Blot, Transfection

Journal: Annals of Translational Medicine

Article Title: The immunoglobulin superfamily member 3 (IGSF3) promotes hepatocellular carcinoma progression through activation of the NF-κB pathway

doi: 10.21037/atm.2020.02.14

Figure Lengend Snippet: IGSF3 promotes HCC cell cycle and NF-B signaling pathways in vivo. (A) Western blot analyses of cyclin A, cyclin E, CDK1, and CDK2 in liver tissue from tumor xenografts; (B) bar graph indicates data from cyclin A, cyclin D, CDK1, and CDK2 expression; (C) Western blot analyses NF-κB, p-NF-κB, IKBα and p-IKBα in liver tissue from tumor xenografts; (D) bar graph show the results of p-IKBα/IKBα and p-NF-κB/NF-κB expression. All experiments were performed three times. Results were represented as mean ± SD (*, P<0.05; **, P<0.01; ***, P<0.001).

Article Snippet: Primary antibodies with indicated concentration for WB, IHC, IF ( ). table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Dilution Company Cat No. WB IHC IF IGSF3 1:400 1:400 1:50 Bioss Bs-9196R PCNA 1:250 BOSTR BM0104 cyclin A 1:250 BOSTER A00700 cyclin E 1:400 BOSTER A00543-1 CDK1 1:400 BOSTER BM0027 CDK2 1:400 BOSTER PB0562 p-IKBα 1:400 Wanleibio WL03120 IKBα 1:400 Wanleibio WL02495 IKBα 1:200 Beyotime AF1282 p-NF-κB 1:400 Wanleibio WL02169 NF-κB 1:400 Wanleibio WL01273b NF-κB 1:200 Beyotime AF1234 GAPDH 1:800 BOSTER BM3876 Ki-67 1:200 BOSTER PB0065 Lamin B 1:1,000 Bioss bs-1840R β-action 1:800 Bioss bsm-33036M Open in a separate window caption a8 Primary antibodies with indicated concentration for WB, IHC, IF Cell proliferation, migration, and Matrigel invasion assay We used the Costar 24 well insert system (Life Technologies, corning, USA) to perform cell migration assay.

Techniques: In Vivo, Western Blot, Expressing

Journal: Cell reports

Article Title: eIF4A3 Phosphorylation by CDKs Affects NMD during the Cell Cycle.

doi: 10.1016/j.celrep.2019.01.101

Figure Lengend Snippet: Figure 1. CDK1 and CDK2 Phosphorylates the T163 Residue of eIF4A3 (A) Experimental schematic for identification of possible phosphorylation sites within human eIF4A3 in HEK293T cells. Immunopurified FLAG-eIF4A3 was analyzed by SDS-PAGE (center) and LC-MS/MS. Specific phosphorylation at T163 was demonstrated by western blotting with a lab-made antibody against eIF4A3 phosphorylated at T163 (bottom). To demonstrate that western blotting was semiquantitative, 3-fold serial dilutions of total cell extracts were loaded in the four leftmost lanes. The immunoglobulin heavy chain is indicated by an asterisk. (B) IP of FLAG-eIF4A3 using extracts of cells depleted of endogenous CDK1. Representative images from two biological replicates (n = 2) are shown. (C) IP of FLAG-eIF4A3 using extracts of cells overexpressing CDK1-HA-WT or -DN; n = 3. (D) An in vitro kinase assay involving a purified recombinant CDK1-cyclin B1 complex and either His-eIF4A3-WT or -T163A. The relative amount and integrity of each protein were demonstrated by silver staining. The extent of T163 phosphorylation was determined by western blotting (WB) with the anti-p-eIF4A3 antibody. n = 2. (E) An in vitro kinase assay by means of the purified recombinant CDK2-cyclin A2 complex and either His-eIF4A3-WT or -T163A; n = 2. See also Figure S1.

Article Snippet: Primary antibodies against the following proteins served for western blotting or IP: eIF4A3 [Santa Cruz Biotechnology or (Cho et al., 2012)], MLN51 (Abcam), MAGOH (Abcam), Y14 (Abnova), PYM (Abcam), CWC22 (Abnova and Sigma), CBP80 (Cho et al., 2012), eIF4E (Cell Signaling Technology), FLAG (Sigma), HA (Roche), MYC (Calbiochem), CDK1 (Bethyl Laboratories), CDK2 (Bethyl Laboratories), eIF4A1 (Abcam), b-actin (Sigma), snRNP70 (Santa Cruz Biotechnology), cyclin B1 (Cell Signaling Technology), cyclin E1 (Bethyl Laboratories), cyclin A2 (Cell Signaling Technology), UPF1 (a gift from Dr. Lynne E. Maquat), UPF2 (Cho et al., 2012), UPF3X (Kim et al., 2012), and GAPDH (AbFrontier).

Techniques: Residue, Phospho-proteomics, SDS Page, Liquid Chromatography with Mass Spectroscopy, Western Blot, In Vitro, Kinase Assay, Recombinant, Silver Staining

Journal: Frontiers in Pharmacology

Article Title: Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells

doi: 10.3389/fphar.2023.1271435

Figure Lengend Snippet: CTSV depletion attenuates growth of breast cancer cells by stalling progression through G2/M phase. (A) CTSV depletion in MCF-7 and ZR75-1 cells results in reduced cell growth when assessed by MTT assay. (B) PI staining with flow cytometry analysis identified that CTSV depletion results in fewer cells in G1, with a concomitant increase in G2/M phase. Rescue experiments were undertaken by restoring CTSV expression in sh1 cells, where cell cycle profiles were restored to that of the NTC cells. (C) Western blotting analysis of CTSV depleted cells shows that cyclin B1 expression is elevated in comparison to control cells, whereas cyclins D1/D2 and E1 remain unchanged. Tubulin expression was used as an internal loading control, with presented blots representative of at least three independent experiments. The average and standard deviation (SD) values are representative of three independent experiments, with statistical analysis was determined by two-way-ANOVA with a Tukey’s post hoc test for multiple comparisons performed using GraphPad Prism 8.

Article Snippet: The following antibodies were used in this study; goat polyclonal CTSV (BioTechne, AF1080), goat polyclonal cyclin D1/D2 (AF4196, BioTechne), mouse monoclonal cyclin E1 (MAB68101, BioTechne), rabbit monoclonal cyclin B1 (MAB60001, BioTechne), rabbit polyclonal HDAC1 (2062S, Cell Signaling), mouse monoclonal histone H1 (NBP2-45184, BioTechne), rabbit polyclonal histone H2a (NB100-56346, BioTechne), rabbit polyclonal histone H2b (NB100-56633, BioTechne), rabbit monoclonal histone H3 (4499S, Cell Signaling), rabbit monoclonal histone H4 (NBP2-80444, BioTechne), mouse monoclonal Hsc70 (MAB4148, BioTechne), mouse monoclonal Hsp90 (ab13492, Abcam), mouse monoclonal Asf1b (NBP2-61684, BioTechne), rabbit monoclonal NASP (ab181169, Abcam), mouse monoclonal GATA3 (BioTechne, MAB6330), goat polyclonal GAPDH (BioTechne, AF5718), mouse monoclonal CTSL (BioTechne, MAB9521) and rat monoclonal α-Tubulin (ab6160, Abcam).

Techniques: MTT Assay, Staining, Flow Cytometry, Expressing, Western Blot, Comparison, Standard Deviation

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 3 CLOCK interference promotes GCs proliferation. A The interference efficiency of CLOCK was measured using RT-qPCR. Data are expressed as mean ± SEM (n = 5), **P < 0.01. B Western blotting reveals the expression levels of CLOCK. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 5), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 5), *P < 0.05. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A). GAPDH as a housekeeping protein. H Quantifying the Western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

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

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 6 ASB9 interference promotes GCs proliferation. A RT-qPCR detected the interference efficiency of ASB9. Data are expressed as mean ± SEM (n = 6), **P < 0.01. B Western blotting reveals the expression levels of ASB9. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 4), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 16), ****P < 0.0001. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (ASB9, CCNB1, CCNE1, CDK4, and CDKN1A). H Quantifying the western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I RNA expression of ASB9 in GCs. ZT: zone time

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection, RNA Expression

Journal: Nature Communications

Article Title: Sox17 is required for endothelial regeneration following inflammation-induced vascular injury

doi: 10.1038/s41467-019-10134-y

Figure Lengend Snippet: Activation of Sox17 at onset of EC regeneration and Sox17-mediated Cyclin E1 expression. a qPCR analysis of gene expression in sorted CD31 + cells from mTmG- Scl mice before and after injury (12 mg/kg i.p.). Sox17 , Vegfr2 , and Ccne1 increased significantly at day 2 post-LPS compared to baseline. n = 3. Color scale: the fold change increases from red to white to green color. b Western blot analysis in fresh isolated ECs from wild-type mice and quantification c showed a 5-fold increase in Sox17 protein expression within 1 day following injury compared to baseline and followed by recovery within 3 days post-LPS. n = 3. d , e Western blot analysis of cultured HLMVECs in which Sox17 was overexpressed showed 2.5x fold increase in Cyclin E1 protein expression relative to control cells. n = 3. OE, overexpression. f Representation of the CCNE1 promoter region with Sox17 binding sites (circled numbers) and their sequences. g HLMVECs were retrovirally transduced with Sox17 or control plasmid for 3 days, and Ch-IP assay followed by qPCR was performed to amplify Sox17 binding sites in the CCNE1 promoter. n = 3. h 293T cells were transfected with a Sox17 overexpression plasmid containing CCNE1 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. ** P < 0.01 and *** P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for ( c ) and two-way ANOVA with Bonferroni post-tests for ( e , g , h )

Article Snippet: Immunoblotting was performed with Sox17 (1:250, Origene #AM32707PU-N for mouse Sox17; 1:200, R&D #AF1924 for human Sox17), HIF-1α (1:200, Cayman Chemical #10006421), HIF-2α (1:500, Novus Biologicals #NB100–122), Flag M2 (1:1000, Sigma #F1804), Cyclin E1 (1:500, R&D #AF6810) primary antibody using Bio-Rad Protein Electrophoresis and Blotting system.

Techniques: Activation Assay, Expressing, Western Blot, Isolation, Cell Culture, Over Expression, Binding Assay, Transduction, Plasmid Preparation, Transfection, Luciferase, Construct

Journal: Nature Communications

Article Title: Sox17 is required for endothelial regeneration following inflammation-induced vascular injury

doi: 10.1038/s41467-019-10134-y

Figure Lengend Snippet: Overexpression of Sox17 in ECs induces EC proliferation and regeneration. Mixture of 50 μg plasmid with 100 μl liposomes was injected i.v. 3 h after LPS challenge (12 mg/dose i.p.) in wild-type mice. This plasmid has a Flag-tag added to the N-terminus of Sox17 protein coding region and expression is under the regulation of a mouse Cdh5 promoter. a Confocal microscopy of flag staining with CD31 and DAPI co-staining for nuclei in lung cryo-sections from mice receiving a control vector or a Sox17-construct to over-express Sox17. Scale bar = 50 μm (original panel) and 20 μm (enlarged panel). n = 6. OE, overexpression. b Co-localization coefficient for the fraction of Flag in CD31 + cells assesses the transgene expression in the endothelium. The Pearson correlation coefficient is significantly increased in Sox17-overexpressing mice compared to control mice. n = 6. c Western blot analysis and its quantification d showed a significant increase in the flag and Cyclin E1 expression in the pulmonary endothelial cells of mice with 3 days of Sox17 overexpression compared to vector mice. n = 3. e Quantification of BrdU + nuclei in each field of 425 μm 2 area in lung cryo-sections from vector-overexpressing and Sox17-overexpressing mice. n = 5 per group and 6 technical replicates per sample. Slides are co-stained with CD31-AF594, BrdU-AF488, and DAPI. Both groups show increased BrdU + ECs at day 3 post-LPS as compared to baseline and the response was significantly greater in mice in which ECs overexpressed Sox 17. f Lung transvascular albumin permeability pre-LPS and post-LPS challenge in mice overexpressing endothelial Sox17 and control mice. n = 5. Mice overexpressing Sox17 in ECs showed significantly reduced vascular leakiness post-LPS when compared to control mice. g Survival curve of LPS challenge in control mice and mice over-expressing Sox17 in the endothelium. n = 11 per group. At this lethal dose of LPS (20 mg/kg), the death rate for control mice is 60% while for Sox17-overexpressed mice is 10%. h Model. LPS induces tissue hypoxia due to local oxygen depletion by infiltrating activated neutrophils, thereby stabilizing HIF-1α resulting in upregulation Sox17 expression and Sox17 mediated expression of Cyclin E1. This activates cell cycle re-entry and EC proliferation, and restoration of endothelial integrity. ** P < 0.01 and *** P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for ( d – f ) and Log-rank (Mantel-Cox) test for ( g )

Article Snippet: Immunoblotting was performed with Sox17 (1:250, Origene #AM32707PU-N for mouse Sox17; 1:200, R&D #AF1924 for human Sox17), HIF-1α (1:200, Cayman Chemical #10006421), HIF-2α (1:500, Novus Biologicals #NB100–122), Flag M2 (1:1000, Sigma #F1804), Cyclin E1 (1:500, R&D #AF6810) primary antibody using Bio-Rad Protein Electrophoresis and Blotting system.

Techniques: Over Expression, Plasmid Preparation, Injection, FLAG-tag, Expressing, Confocal Microscopy, Staining, Construct, Western Blot, Permeability