Journal: The Indian Journal of Medical Research

Article Title: Downregulated inhibitor of growth 3 (ING3) expression during colorectal carcinogenesis

doi:

Figure Lengend Snippet: Immunostaining examination of ING3 protein in colorectal lesions. ING3 immunoreactivity was detectable in colorectal mucosa (A, B) , adenoma (C, D) , carcinoma (E, F) and infiltrating inflammatory cells (A, F) . (X 200 magnification).

Article Snippet: For immunobloting, the membrane was incubated for 1 h with rabbit anti- ING3 antibody (Protein Tech Group Inc, USA; 1: 500) or mouse anti-β-actin (Santa Cruz, USA).

Techniques: Immunostaining

Journal: The Indian Journal of Medical Research

Article Title: Downregulated inhibitor of growth 3 (ING3) expression during colorectal carcinogenesis

doi:

Figure Lengend Snippet: Correlation between ING3 status and prognosis of the colorectal carcinoma patients. Kaplan-Meier curves for cumulative survival rate of patients with colorectal carcinomas according to ING3 expression status.

Article Snippet: For immunobloting, the membrane was incubated for 1 h with rabbit anti- ING3 antibody (Protein Tech Group Inc, USA; 1: 500) or mouse anti-β-actin (Santa Cruz, USA).

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 positively regulates mitochondrial respiration in tubular epithelial cells. HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. (A,B) The transfection efficiencies were evaluated by qPCR and Western blotting, respectively. (C–F) Following transfection, mitochondrial OXPHOS of HK2 cells were detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A&R, antimycin and rotenone. (G,H) mtDNA-encoded components for complex I, III, IV, and V and 16S rRNA were determined by qPCR. (I) mtDNA-encoded components for complex I, III, IV, and V were determined by Western blotting. (J) The mitochondria were stained with MitoTracker (red) and ING2 (green) followed by DAPI (blue) re-dyeing. (K) The mitochondria DNA (mtDNA) copy number was determined by qPCR with G6PC serving as internal reference. Data were from three individual experiments and presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: MRPL12 mediated the effects of ING2 on mitochondrial OXPHOS in tubular epithelial cells. (A,B) HK2 cells were transfected with ING2-shRNA or ING2-overexpression plasmid. The mRNA and protein levels of TFAM and MRPL12 were evaluated by qPCR and Western blotting. (C,D) HK2 cells were divided into control group, ING2 overexpression group, MRPL12 knockout group, and MRPL12 knockout plus ING2 overexpression group. Mitochondrial OXPHOS was detected using Seahorse. OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A&R, antimycin and rotenone. (E) Western blotting to validate the altered expression of mtDNA-encoded components of mitochondria complex. Data were from three individual experiments and presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, Western Blot, Knock-Out, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 inhibited the ubiquitination of MRPL12. (A) HK2 cells were transfected with control or ING2-shRNA, followed by MG132 treatment for 6 h. Protein levels of ING2 and MRPL12 were evaluated by Western blotting. (B) A ubiquitin modified site was found at 58 lysine residue (red, upper panel), predicted using predictor of protein ubiquitination sites, UbPred. (C,D) The ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) and immunoprecipitation, respectively, after transfected ING2-overexpression plasmid. (E) By using ASEB, a web tool for predicting protein acetylation site, a acetylation site was detected at the 185 lysine residue (green, lower panel). (F) The acetylation of MRPL12 was determined by immunoprecipitation after transfected ING2-overexpression plasmid. IP, immunoprecipitation; WB, Western blotting. Data were from three individual experiments.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Transfection, shRNA, Western Blot, Modification, Proximity Ligation Assay, Immunoprecipitation, Over Expression, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 ameliorated the ischemia induced mitochondrial OXPHOS defects and tubular cell apoptosis. (A) Immunostaining of kidney tissue section from healthy individual and AKI patients with antibodies against ING2. At least two patients for each. (B) Immunostaining of kidney tissue section from healthy and AKI mice with antibodies against ING2 and MRPL12. HK2 cells were subjected to serum deprivation for 48 h, and protein contents of both ING2, MRPL12, and mtDNA-encoded components of mitochondria complex (C) were figured out by Western blotting. The apoptosis of HK2 cells was evaluated by flow cytometry (D) . HK2 cells were divided into control group, ING2 overexpression group, serum deprivation group, and serum deprivation plus ING2 overexpression group. MRPL12 and mtDNA-encoded components of mitochondria complex were detected through Western blotting (E) . Mitochondrial OXPHOS was detected using Seahorse (F,G) . The apoptosis of HK2 cells was evaluated by flow cytometry (H) , and the ubiquitination of MRPL12 was determined by proximity ligation assay (PLA) (I) . OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; A&R, antimycin and rotenone. Data were from three individual experiments and presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Immunostaining, Western Blot, Flow Cytometry, Over Expression, Proximity Ligation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

doi: 10.3389/fcell.2021.700195

Figure Lengend Snippet: ING2 overexpression effectively ameliorated ischemic kidney injury. The mice were injected with rAAV-con or rAAV-ING2 followed by the induction of IRI with sham operation group as control. (A) Immunofluorescent staining of kidneys after the intra-renal injection of rAAV vectors with ING2 (red) and DAPI (blue). (B) Immunostaining of renal tissue sections with antibodies against ING2, MRPL12, ND2, and COX II. (C) Western blot analysis of renal tissue ING2, MRPL12, ND2, and COX II. (D) Levels of creatinine in the serum or urine of IRI and sham operation mice. Data were from two individual experiments and presented as mean ± SEM. rAAV-con + IRI, n = 6; rAAV-ING2 + IRI, n = 6; rAAV-con + sham, n = 4; rAAV-ING2 + sham, n = 4. ** p < 0.01. ns, not significant.

Article Snippet: Sections were stained with rabbit antibodies against ING2 (1:200, Proteintech), MRPL12 (1:200, Proteintech), ND2 (1:200, Proteintech), or COX II (1:200, Proteintech) overnight at 4°C and incubated with the secondary antibody (HRP labeled goat anti-rabbit IgG, 1:1,000) for 1 h at RT.

Techniques: Over Expression, Injection, Staining, Immunostaining, Western Blot