Journal: Journal of Clinical and Translational Hepatology

Article Title: Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis

doi: 10.14218/jcth.2020.00185

Figure Lengend Snippet: Fig. 1. Effect of arsenic on H3K4 methylation in L02 hepatocytes. Cells were exposed to arsenic at 100 µmol/L for 24 h. Proteins, prepared from whole cell extracts, were analyzed by western blotting. The activities of H3K4me1/me2 were determined with respective specific antibodies. (A) SET7/9 protein knock-down efficiency. H3K4me1 expression was measured in control, model and SET7/9 knock-down L02 cells. (B) Data shown are mean±standard deviation of three independ- ent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group. (C) LSD1 protein knock-down and overexpression efficiency. H3K4me1/me2 expression was measured in control, model and LSD1 knock-down and OG-L002 group of L02 cells. (D) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group.

Article Snippet: Fetal bovine serum, Dulbecco’s modified Eagle’s medium (GIBCO, New York, NY, USA), antibodies against GRP78, C/EBP-homologous protein (CHOP), LSD1, H3 and H3K4me1 (Abcam, Cambridge, UK), antibodies against H3K4me2 (Active Motif, Carlsbad, CA, USA), GAPDH (Bioprimacy Biotechnology, Wuhan, China), secondary antibodies (Boster Biological Engineering, Wuhan, China), SET7/9-specific small interfering RNAs (siRNAs) were purchased from Shanghai Jima Gene (China), negative small hairpin (sh)RNA and LSD1 overexpressed plasmids were obtained from Shanghai Jima Gene.

Techniques: Methylation, Western Blot, Knockdown, Expressing, Control, Standard Deviation, Transfection, Over Expression

Journal: Journal of Clinical and Translational Hepatology

Article Title: Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis

doi: 10.14218/jcth.2020.00185

Figure Lengend Snippet: Fig. 2. Effect of arsenic on ERS-related proteins in L02 hepatocytes. Cells were exposed to arsenic at 100 µmol/L for 24 h. Proteins, prepared from whole cell extracts, were analyzed by western blotting. The activities of GRP78, CHOP, and LSD1 were determined with respective specific antibodies. (A) SET7/9 protein knock-down efficiency. CHOP expression was measured in control, model and SET7/9 knock-down in L02 cells. (B) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group. (C) LSD1 protein knock-down and overexpression efficiency. GRP78, CHOP, and LSD1 expression was measured in control, model and LSD1 knock-down and OG-L002 group of L02 cells. (D) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group.

Article Snippet: Fetal bovine serum, Dulbecco’s modified Eagle’s medium (GIBCO, New York, NY, USA), antibodies against GRP78, C/EBP-homologous protein (CHOP), LSD1, H3 and H3K4me1 (Abcam, Cambridge, UK), antibodies against H3K4me2 (Active Motif, Carlsbad, CA, USA), GAPDH (Bioprimacy Biotechnology, Wuhan, China), secondary antibodies (Boster Biological Engineering, Wuhan, China), SET7/9-specific small interfering RNAs (siRNAs) were purchased from Shanghai Jima Gene (China), negative small hairpin (sh)RNA and LSD1 overexpressed plasmids were obtained from Shanghai Jima Gene.

Techniques: Western Blot, Knockdown, Expressing, Control, Standard Deviation, Transfection, Over Expression

Journal: Journal of Clinical and Translational Hepatology

Article Title: Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis

doi: 10.14218/jcth.2020.00185

Figure Lengend Snippet: Fig. 3. Effect of arsenic on apoptosis and cycles in L02 hepatocytes. (A) The changes of apoptosis in the LO2 cells of different groups were analyzed by flow cy- tometry. LSD1 protein knock-down and overexpression efficiency. Apoptosis was measured in control, model and LSD1 knock-down and OG-L002 group of L02 cells. (a: control; b: model; c: negative transfection; d: LSD1 overexpression group; e: OG L002; f: the apoptosis rate of different groups) Data shown are mean±standard devia- tion of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group. (B) The cell cycle distribution of each group. SET7/9 protein knock-down efficiency. Cell cycles were assessed in control, model and SET7/9 knock-down of L02 cells. (a: cycles diagram for each group; f: the cycles of different groups) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group. (C) The cell cycle distribution of each group. Cycles were assessed in control, model and LSD1 knock-down and OG-L002 group of L02 cells. (a: cycles diagram for each group; f: the cycles of different groups) Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group.

Article Snippet: Fetal bovine serum, Dulbecco’s modified Eagle’s medium (GIBCO, New York, NY, USA), antibodies against GRP78, C/EBP-homologous protein (CHOP), LSD1, H3 and H3K4me1 (Abcam, Cambridge, UK), antibodies against H3K4me2 (Active Motif, Carlsbad, CA, USA), GAPDH (Bioprimacy Biotechnology, Wuhan, China), secondary antibodies (Boster Biological Engineering, Wuhan, China), SET7/9-specific small interfering RNAs (siRNAs) were purchased from Shanghai Jima Gene (China), negative small hairpin (sh)RNA and LSD1 overexpressed plasmids were obtained from Shanghai Jima Gene.

Techniques: Knockdown, Over Expression, Control, Transfection, Standard Deviation

Journal: Journal of Clinical and Translational Hepatology

Article Title: Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis

doi: 10.14218/jcth.2020.00185

Figure Lengend Snippet: Fig. 4. Effect of arsenic on growth in L02 hepatocytes. (A) Real-time cellular analysis was employed to detect the SET7/9 protein knock-down efficiency. Pro- liferation was measured in control, model and SET7/9 knock-down of L02 cells. (B) The proliferation rate in control, model and SET7/9 knock-down. Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfection group. (C) Real-time cellular analysis was employed to detect the LSD1 protein knock-down and overexpression efficiency. D: The proliferation rate was measured in control, model and LSD1 knock-down and overexpression LSD1 group. Data shown are mean±standard deviation of three independent experiments, *p<0.05 vs. control group; #p<0.05 vs. negative transfec- tion group.

Article Snippet: Fetal bovine serum, Dulbecco’s modified Eagle’s medium (GIBCO, New York, NY, USA), antibodies against GRP78, C/EBP-homologous protein (CHOP), LSD1, H3 and H3K4me1 (Abcam, Cambridge, UK), antibodies against H3K4me2 (Active Motif, Carlsbad, CA, USA), GAPDH (Bioprimacy Biotechnology, Wuhan, China), secondary antibodies (Boster Biological Engineering, Wuhan, China), SET7/9-specific small interfering RNAs (siRNAs) were purchased from Shanghai Jima Gene (China), negative small hairpin (sh)RNA and LSD1 overexpressed plasmids were obtained from Shanghai Jima Gene.

Techniques: Knockdown, Control, Standard Deviation, Transfection, Over Expression

Journal: Frontiers in Oncology

Article Title: OTUB2 Facilitates Tumorigenesis of Gastric Cancer Through Promoting KDM1A-Mediated Stem Cell-Like Properties

doi: 10.3389/fonc.2021.711735

Figure Lengend Snippet: Otubain 2 (OTUB2) increases the stability of KDM1A. (A) Relative protein levels of KDM1A in GC cells transfected with OTUB2 overexpression plasmids (MKN-45 and SGC-7901 cells) or shRNA targeting OTUB2 (HGC-27 and AGS cells). Actin served as internal control. (B) co-immunoprecipitation was conducted with anti-Flag (Flag-OTUB2) or anti-HA (HA-KDM1A) antibody and was analyzed by western blotting in 293T cells. (C) after transfected with shOTUB2, cells were treated with or without MG132. The ubiquitination level of KDM1A in MKN-45 and HGC-27 cells was measured by Western blot. Actin served as internal control. (D) After transfected with OTUB2 overexpression plasmids (MKN-45 cells) or shOTUB2, cells were treated with or without cycloheximide (CHX) for 0, 1, 2, 4, and 8 h, respectively. The protein expression levels of KDM1A were assessed by Western blot. Actin was used as internal control. Data was represented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For deubiquitination assay, polyUb was purified by IP using anti-KDM1A antibody (1 μg, Boster, Catalog No. M00532; Beijing, China) from cells transfected with OTUB2 shRNA after treated with MG-132.

Techniques: Transfection, Over Expression, shRNA, Control, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing

Journal: Frontiers in Oncology

Article Title: OTUB2 Facilitates Tumorigenesis of Gastric Cancer Through Promoting KDM1A-Mediated Stem Cell-Like Properties

doi: 10.3389/fonc.2021.711735

Figure Lengend Snippet: The expression of otubain 2 (OTUB2) and KDM1A in GC cells are positively correlated. (A) The expression of OTUB2, KDM1A, and CD44 was measured by immunohistochemistry in GC tissues collected from patients with GC. (B) The correlation between the expression of OTUB2, KDM1A, and CD44 in human tumor tissues was analyzed. (C) After OTUB2 was overexpressed (SGC-7901 cells) or downregulated (HGC-27 cells), the expression of KDM1A and CD44 was detected by immunohistochemistry in tumor tissues obtained from nude mice.

Article Snippet: For deubiquitination assay, polyUb was purified by IP using anti-KDM1A antibody (1 μg, Boster, Catalog No. M00532; Beijing, China) from cells transfected with OTUB2 shRNA after treated with MG-132.

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: OTUB2 Facilitates Tumorigenesis of Gastric Cancer Through Promoting KDM1A-Mediated Stem Cell-Like Properties

doi: 10.3389/fonc.2021.711735

Figure Lengend Snippet: OTUB2 promotes GC stem cell properties through regulating KDM1A expression. (B) After MKN-45 cells were co-transfected with OTUB2 overexpression plasmids/NC and shKDM1A/Scr. The HGC-27 cells were co-transfected with shOTUB2/shNC and OV-KDM1A/vector, the cell viability was detected by MTT assay (A) ; The spheroid-forming ability of GC cells were detected by sphere formation assay (B) . Wound healing assays (C) and Transwell assays (D) were used to determine the changes in migratory and invasive abilities of GC cells; Relative protein levels of KDM1A, Nanog, ALDH, and Oct4 IN GC cells were measured by Western blot (E) . Data was represented as mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001. # P < 0.05, ## P < 0.01, and ### P < 0.001.

Article Snippet: For deubiquitination assay, polyUb was purified by IP using anti-KDM1A antibody (1 μg, Boster, Catalog No. M00532; Beijing, China) from cells transfected with OTUB2 shRNA after treated with MG-132.

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, MTT Assay, Tube Formation Assay, Western Blot