Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: a–c Using the PBRM1 and UBE3A antibodies to performed the IP assay. Western blotting analysis the whole-cell lysates (WCL) of 293T ( a ), 786-O ( b ), and ACHN ( c ) cells. d Western blotting analysis of UBE3A proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-PBRM1 recombinant proteins. Asterisks indicated the corresponding protein band of GST-EV and GST-PBRM1. e A schematic diagram depicting a set of GST-UBE3A recombinant protein constructs. f Western blotting analysis of PBRM1 proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-UBE3A recombinant proteins. Asterisks indicated the corresponding protein band of GST-EV and GST-UBE3A recombinant proteins. g, h 786-O and ACHN cells were infected with indicates shRNAs for 72 h. Cells were harvested for western blotting analysis ( g ) and RT-qPCR assay ( h ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as mean ± SEM with three replicates ( n = 3). ns not significant. i – k IHC analysis of the tissue microarray with a cohort of patients with renal cell carcinoma by using the UBE3A and PBRM1 antibodies. The typical images of IHC were shown in ( i ). Heatmap showing the IHC score of PBRM1 and UBE3A in ( j ). Correlation analysis of the IHC score of PBRM1 and UBE3A proteins in ( k ).

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Western Blot, Recombinant, Construct, Infection, Quantitative RT-PCR, Microarray

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: a A schematic diagram depicted that UBE3A contained a consensus DAPK phosphorylation motif which was adjacent to the PKA phosphorylation site. b Western blotting analysis the whole-cell lysates (WCL) of 293T cells. c Western blotting analysis the WCL 786-O and ACHN cells. d Western blotting analysis of UBE3A proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-DAPK3 recombinant proteins. e Western blotting analysis of DAPK3 proteins in 786-O whole-cell lysates pulled down by GST-EV or GST-UBE3A recombinant proteins. f , g 786-O cells were transfected with indicated plasmids. Twenty-four hours post transfection, cells were harvested for Western blotting analysis ( f ) and RT-qPCR analysis ( g ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). ns not significant. h , i 786-O cells were transfected with indicated shRNAs. Seventy-two hours post infection, cells were harvested for western blotting analysis ( h ) and RT-qPCR analysis ( i ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). ns not significant. j 786-O cells were infected with indicated shRNAs. After 72 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. k 786-O cells were transfected with indicated plasmids. After 24 h, cells were treated with CHX, and cells were collected for western blot analysis at different timepoints. The GAPDH was recognized as the loading control. The protein level of PBRM1 was first normalized to loading control. The normalized values were further normalized to the values in 0 h group. Immunoblots (IB) are representative of results from two independent experiments ( n = 2). Statistical significance was determined by multiple student’s t -test at the time point of 5, 10, 15 h. Data presented as Mean ± SEM with two replicates. Ns not significant; *** P < 0.001. Data presented as Mean ± SEM with two replicates. Ns not significant; *** P < 0.001. l 786-O cells were infected with the indicated shRNAs. After 72 h, cells were collected for western blotting after treatment with MG132 for 8 h.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Phospho-proteomics, Western Blot, Recombinant, Transfection, Quantitative RT-PCR, Infection, Control

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Expressing

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: Identifying prognostic genes for developing a risk model. (A) Intersecting genes associated with colorectal cancer prognosis in TCGA and GSE39582 databases. (B) LASSO coefficient profiles of the 113 genes in TCGA data set. (C) Selection of the optimal parameter (λ) in the LASSO model. KM curves of the prognostic signature in (D) TCGA and (E) GSE39582 cohorts (analyzed using log-rank test). (F) Differential expression of the 15 genes in TCGA between different groups. *P<0.05, ***P<0.001, ****P<0.0001. (G) Distribution of risk score, survival duration and status of patients, and a heatmap of the 15 genes in the classifier. (H) A total of 15 genes were chosen for establishing a prognosis signature. The red circle indicates the target gene ZNF180. *P<0.05, **P<0.01, ***P<0.001. TCGA, The Cancer Genome Atlas; LASSO, least absolute shrinkage and selection operator; KM, Kaplan-Meier.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Selection, Quantitative Proteomics

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: ZNF180 expression is frequently downregulated in CRC tissues and cell lines. (A) mRNA expression levels of ZNF180 in CRC cell lines and HIEC-6 immortalized intestinal epithelial cells. (B) Protein expression levels of ZNF180 in CRC cell lines and HIEC-6 immortalized intestinal epithelial cells. (C) mRNA expression levels of ZNF180 in 12 paired CRC tissues and adjacent normal tissues from the SYSU6 cohort were confirmed by reverse transcription-quantitative PCR. **P<0.01. (D) Relative expression of ZNF180 in tumors was compared with the relative expression of ZNF180 in adjacent normal tissues. (E) Protein expression levels of ZNF180 in 12 paired CRC tissues and adjacent normal tissues from the SYSU6 cohort were confirmed by western blotting. (F) Levels of ZNF180 protein expression in CRC tissues were detected under high magnification microscopy. (G) Low ZNF180 expression rate was higher in CRC tissues; tissues from 40 patients in the SYSU6 cohort with both normal and tumor cells were assessed. (H) OS rate of patients in the SYSU6 cohort (n=220) was significantly higher in the high ZNF180 expression group. CRC, colorectal cancer; N, normal; OS, overall survival; SYSU6, The Sixth Affiliated Hospital of Sun Yat-sen University; T, tumor; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Microscopy

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: Association of ZNF180 expression and clinical characteristics in 220 patients with colorectal cancer form The Sixth Affiliated Hospital of Sun Yat-sen University cohort.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Expressing

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: ZNF180 is downregulated and its promoter hypermethylated in colorectal cancer. (A) Schematic representation of the CpG islands and bisulfite sequencing region in the ZNF180 promoter. HCT116 and RKO cells were subjected to 3 days of treatment with increasing doses of 5-AzaC; blue: CpG island of ZNF180 promoter; Input Sequence: Promoter region of ZNF180. (B) Protein expression levels were determined by western blotting. (C) mRNA expression levels were determined by RT-qPCR, respectively. The untreated group was used as the control group, and the relative expression levels of each group were divided by the relative expression levels of the control group. P-values were calculated using one-way ANOVA and Scheffe test, *P<0.05, ***P<0.001. (D) Correlation between ZNF180 mRNA expression level and ZNF180 methylation level in TCGA data set. (E) Overall survival rate was significantly higher in the low ZNF180 methylation group in TCGA dataset. (F) ZNF180 mRNA expression in patients with different DNA statuses in TCGA dataset. P-values were calculated using one-way ANOVA and Scheffe test, ***P<0.001. Relative (G) mRNA and (H) protein expression levels were determined by RT-qPCR and immunoblotting, respectively. P-values were calculated using unpaired Student's t-test, ***P<0.001. 5-AzaC, 5-azacytidine; RT-qPCR, reverse transcription-quantitative PCR; TCGA, The Cancer Genome Atlas; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Methylation Sequencing, Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Control, Methylation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: ZNF180 enhances cellular sensitivity and suppresses cell growth in vivo . (A) Cells were double-stained with PI and Annexin V, and analyzed by flow cytometry to evaluate apoptosis. (B) Quantification of the percentage of apoptotic cells. P-values were calculated using unpaired Student's t-test. *P<0.05, **P<0.01. (C) HCT116 vector cells and HCT116 ZNF180-overexpressing cells were treated with increasing doses of 5-FU for 48 h, and PARP and cleaved-PARP expression was analyzed by western blotting. GAPDH was used as the loading control. (D) HCT116 cells expressing the control vector or overexpressing ZNF180 were subcutaneously inoculated into nude mice. Images of isolated tumors are shown. (E) Growth curve indicates HCT116 growth suppression upon ZNF180 overexpression compared with vector in vivo . P-values were calculated using unpaired Student's t-test, *P<0.05 vs. ZNF180. (F) HCT116 (2×10 6 ) cells were subcutaneously injected into nude mice and were monitored every 2 days. (G) Weight of nude mice during tumorigenesis. (H) Representative immunohistochemistry and H&E staining images of ZNF180 and METTL14 expression in mice tumor tissues. 5-FU, 5-fluorouracil; H&E, hematoxylin and eosin; METTL14, methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: In Vivo, Staining, Flow Cytometry, Plasmid Preparation, Expressing, Western Blot, Control, Isolation, Over Expression, Injection, Immunohistochemistry

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: METTL14 is a candidate target gene of ZNF180. (A) Overlapping of the differentially expressed genes from TCGA, GSE39582 and GSE87211 cohorts; 478 genes were identified to be correlated with ZNF180. (B) Correlation between candidate target genes and ZNF180 in TCGA and GSE39582 cohorts. (C) mRNA expression levels of METTL14 in CRC cell lines and HIEC-6 immortalized intestinal epithelial cells. (D) Protein expression level of METTL14 in CRC cell lines and HIEC-6 immortalized intestinal epithelial cells. (E) Protein and (F) mRNA expression levels of METTL14 in 12 paired CRC tissues and noncancerous tissues from the SYSU6 cohort were confirmed by western blotting and reverse transcription-quantitative PCR, respectively. (G) mRNA expression levels of METTL14 in CRC tissues and noncancerous tissues in SYSU6 (12 pairs), TCGA (47 pairs) and GSE39582 (normal tissues=19, tumor tissue=566) cohorts. P-values were calculated using paired Student's t-test (a and b) or unpaired Student's t-test (c). *P<0.05, **P<0.01, ****P<0.0001. CRC, colorectal cancer; METTL14, methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit; N, normal; SYSU6, The Sixth Affiliated Hospital of Sun Yat-sen University; T, tumor; TCGA, The Cancer Genome Atlas; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: METTL14 is associated with ZNF180 in CRC cell lines and tissues. (A) mRNA expression levels of METTL14 in 12 paired CRC tissues and noncancerous tissues from the SYSU6 cohort, and the positive correlation between METTL14 and ZNF180 expression in the SYSU6 cohort. Pearson correlation coefficient was used for correlation analyses. **P<0.01. (B) ZNF180 expression was positively correlated with METTL14 expression in the GSE39582 dataset. (C) mRNA expression levels of ZNF180 and METTL14 in CRC tissues, the positive correlation between METTL14 and ZNF180 expression in TCGA dataset. Pearson correlation coefficient was used for correlation analyses. ****P<0.0001. (D) ZNF180 expression was positively correlated with METTL14 expression in the GSE87211 data set. (E) Positive correlation between METTL14 and ZNF180 expression in CRC cell lines. Pearson correlation coefficient was used for correlation analyses. ****P<0.0001. (F) Kaplan-Meier analysis indicated downregulation of METTL14 was significantly associated with poorer overall survival in patients with CRC in TCGA dataset. (G) Levels of METTL14 protein expression in CRC tissues under high magnifications microscopy. (H) Low METTL14 expression rate was higher in CRC tissues, and the high METTL14 expression rate was increased in the high ZNF180 expression group; tissues from 40 patients with CRC in the SYSU6 cohort with both tumor cells and normal cells were assessed. CRC, colorectal cancer; METTL14, methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit; SYSU6, The Sixth Affiliated Hospital, Sun Yat-sen University; TCGA, The Cancer Genome Atlas; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Microscopy

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: METTL14 is a candidate downstream molecule in ZNF180 signaling. (A) ZNF180 and METTL14 expression were measured by western blotting. (B) Localization of ZNF180 and METTL14 in HCT116 cells by confocal immunofluorescence analysis. (C) Overexpression of ZNF180 in HCT116 and RKO cells; ZNF180 and METTL14 mRNA expression levels were detected by RT-qPCR. P-values were calculated using unpaired Student's t-test, **P<0.01, ***P<0.001. (D) Agarose gel electrophoresis showed that DNA was digested into 100–750 bp. (E) Cells stably transfected with vector or flag-ZNF180 were analyzed by ChIP, and METTL14 DNA was qualitatively analyzed using DNA agarose gel electrophoresis. IgG refers to normal rabbit IgG (negative control) and H3 refers to histone H3 (positive control). (F) METTL14 DNA element was quantified by RT-qPCR. P-values were calculated using unpaired Student's t-test, **P<0.01, ***P<0.001. (G) METTL14-luciferase activity in 293T cells overexpressing ZNF180. P-values were calculated using unpaired Student's t-test, **P<0.01. (H) Protein expression levels of METTL14 were determined by immunoblotting. (I) Cell proliferation was determined using the MTS assay. P-values were calculated using unpaired Student's t-test, *P<0.05 vs. NC. (J) Cells were double-stained with PI and Annexin V, and analyzed by flow cytometry to evaluate apoptosis. (K) Quantification of the percentage of apoptotic cells. P-values were calculated using one-way ANOVA (parametric) test and Scheffe test, **P<0.01. METTL14, methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit; NC, negative control; OD, optical density; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Immunofluorescence, Over Expression, Quantitative RT-PCR, Agarose Gel Electrophoresis, Stable Transfection, Transfection, Plasmid Preparation, Negative Control, Positive Control, Luciferase, Activity Assay, MTS Assay, Staining, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Reports

Article Title: Zinc finger protein 180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer

doi: 10.3892/or.2024.8784

Figure Lengend Snippet: Mechanism of how ZNF180 induces an apoptotic phenotype by activating METTL14 transcriptional activity in colorectal cancer. METTL14, methyltransferase 14, N6-adenosine-methyltransferase non-catalytic subunit; ZNF180, zinc finger protein 180.

Article Snippet: The cells were then incubated with the following primary antibodies [1:200; diluted in QuickBlock buffer (cat. no. P0256; Beyotime Institute of Biotechnology)]: ZNF180 (cat. no. NBP1-92613; Novus Biologicals, Inc.) METTL14 (cat. no. 26158-1-AP; Proteintech Group, Inc.) and Ki67 (cat. no. CL594-27309; Proteintech Group, Inc.) at 4°C overnight, and with fluorescent secondary antibodies [(1:1,000; diluted in dilution buffer (cat. no. I917953, Macklin, Inc.)] at room temperature for 1 h. The following secondary antibodies were used: Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody with Alexa FluorTM 594 (cat. no. A-11012; Thermo Fisher Scientific, Inc.) and Alexa FluorTM 488 (cat. no. A-11008; Thermo Fisher Scientific, Inc.).

Techniques: Activity Assay

Journal: Biology Open

Article Title: Endoplasmic reticulum stress-induced cellular dysfunction and cell death in insulin-producing cells results in diabetes-like phenotypes in Drosophila

doi: 10.1242/bio.046524

Figure Lengend Snippet: Induction of UPR observed in wing imaginal discs and IPCs with ectopic Hsc70-3 DN expression. (A–B) Expression of Xbp1-GFP generated by ER stress-dependent splicing of xbp1*-GFP mRNA in wing imaginal discs. Phase contrast (A,B) and fluorescence (A′,B′) micrographs of wing imaginal discs. (A,A′) Control wing disc ( Bx>xbp1*-GFP ). (B,B′) Wing disc expressing a dominant-negative form of Hsc70-3 in the wing pouch region (arrow) ( Bx>hsc70-3 DN , xbp1*-GFP ). (C–E) Fluorescence micrograph of wing discs stained with DAPI (white). (C′–E′) Immunostaining of the wing discs with an anti-GRP78 antibody. (D″) Immunostaining of the wing disc with anti-HA antibody. (C,C′) Fluorescence micrograph of a control wing imaginal disc ( Bx-Gal4/+ ). (D–D″) Wing imaginal disc expressing control Hsc70-3 in the wing pouch region of the imaginal disc ( Bx>hsc70-3 ). (E,E′) Wing imaginal disc expressing a dominant-negative form of Hsc70-3 in the same region ( Bx>hsc70-3 DN ). Anti-GRP78 immunostaining is shown in white. Note that more intense immunofluorescence was observed exclusively in areas expressing Hsc70-3 DN , but not the control protein. (A–F) Relative intensity of anti-GRP78 immunostaining in wing imaginal discs. Immunofluorescence signal intensity in each wing imaginal disc with the control Hsc70-3 ( n =31) or Hsc70-3 DN ( n =25) expression was calculated and normalized to the control value, which was set as 1.0 ( Bx-Gal4/+ ) ( n =25; n.s., not significant, P >0.05; *** P <0.001, Student's t -tests). Error bars represent s.e.m. (G–I) Anti-GRP78 immunostaining of IPCs expressing GFPnls in brains from third-instar larvae. (G) Control IPCs ( ilp2>GFPnls ), (H) IPCs expressing the control Hsc70-3 ( ilp2>hsc70-3, GFPnls ), (I) IPCs expressing Hsc70-3 DN ( ilp2>hsc70-3 DN , GFPnls ). Anti-GRP78 immunostaining is colored in red (G–I; white in G′–I′). Nuclei of IPCs visualized by GFPnls expression are colored green (G–I; white in G″–I″). Arrows in H′ and H″ indicate positions of IPC cells. Note that remarkably higher immunostaining signal was observed in IPCs expressing Hsc70-3 DN , but not the control protein. (J) Relative intensities of anti-GRP78 immunostaining in larval IPCs. Immunofluorescence signal intensities in each IPC expressing Hsc70-3 ( n =25) or Hsc70-3 DN ( n =21) were calculated and normalized to the control value of 1.0 ( ilp2>GFPnls ) ( n =21, * P <0.05, *** P <0.001, Student's t -test). Error bars represent s.e.m. Scale bars: (A–E) 100 µm, (G–I) 50 µm.

Article Snippet: The following primary antibodies were used at the dilution described; rabbit anti-β-galactosidase (MP Biomedicals, #55976) at 1:1000, rabbit anti-GRP78 (Bip) (StressMarq Biosciences Inc., Cadboro Bay, Victoria, Canada) that could recognize Hsp70 family proteins including Hsc70-3 in Drosophila at 1:500, rabbit Cleaved Caspase-3 (Asp175) (#9661, Cell Signaling, Danvers, Massachusetts, USA) at 1:200 for larval brain immunostaining and at 1:150 for wing disc immunostaining, rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) (Cell Signaling, antibody #9578) at 1:500, and rabbit anti-phospho-SAPK/JNK (pThr183, pTyr185) (Calbiochem, La Jolla, CA, USA) at 1:200.

Techniques: Expressing, Generated, Fluorescence, Dominant Negative Mutation, Staining, Immunostaining, Immunofluorescence

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 7. Rac1 and -PIX siRNAs as well as the Rac1 inhibitor NSC23766 inhibit interaction of NOD2 with Erbin. Primary monocytes (A and B) or THP-1 cells (C) were either preincubated with the Rac1 inhibitor NSC23766 (NSC) or were transfected with Rac1 siRNA or -PIX siRNA, as indicated, and were stimulated with 10 g/ml MDP (MDP) for 40 min. Subsequently, immunoprecipitations with an Erbin Ab and subsequent im- munoblots with NOD2 and ERK2 Abs (A) or NOD2, Rac1, and Erbin Abs (B and C) were performed. One representative Western blot out of three is shown.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques: Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-PIX and Rac1 GTPase mediate trafficking and negative regulation of NOD2.

doi: 10.4049/jimmunol.181.4.2664

Figure Lengend Snippet: FIGURE 8. Schema of the molecular association among NOD2, -PIX, Rac1, and Erbin as discussed in the text.

Article Snippet: Membranes were exposed to Abs specific to Rac1 (Transduction Laboratories), NOD2 (ProSci), Nalp3 (Biozol), -Pix, c-Myc, Erbin, or ERK2 (Santa Cruz Biotechnology), respectively.

Techniques:

Journal: Cell Reports Medicine

Article Title: Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes

doi: 10.1016/j.xcrm.2021.100422

Figure Lengend Snippet:

Article Snippet: Human interphotoreceptor retinoid-binding protein (IRBP) peptide 161-180 , GenScript , Cat# RP20268.

Techniques: Recombinant, Synthesized, Diagnostic Assay, Software, Drug discovery, Flow Cytometry, Viability Assay, Histopathology