Journal: International Journal of Molecular Sciences

Article Title: Vitamin B12-Induced Autophagy Alleviates High Glucose-Mediated Apoptosis of Islet β Cells

doi: 10.3390/ijms242015217

Figure Lengend Snippet: Vitamin B12 induced autophagy in RIN-m5F cells. ( A ) RIN-m5F cells were subjected to varying concentrations of vitamin B12, and cytotoxicity levels were assessed utilizing a CCK-8 kit assay. ( B ) The cells were treated with vitamin B12 concentrations of 2, 4, and 8 μM for 24 h and stained utilizing an anti-LC3 antibody (scale = 5 μm). ( C ) The cells were cultured as described in section ( B ), and the mathematical statistical methods were conducted to calculate the number of autophagosomes in each cell. At least 30 cells were counted for statistical analysis utilizing the t -test. ( D , F , H ) The impact of vitamin B12 at 2, 4, and 8 μM concentrations on the expression of autophagy-related proteins (anti-LC3, anti-p62, anti-GAPDH, anti-S6K, and an-ti-p-S6K) in cells were carried out using Western blotting. ( E , G , I ) The LC3-II/LC3-I, p62/GAPDH, and p-S6K/S6K ratios in ( D , F , H ), respectively, were analyzed by integrated optical density (IOD) using Image-Pro Plus 6.0 software. All experiments were conducted a minimum of three times. The error bars represent the standard deviation, ** p < 0.01 reveals significant differences in comparison to the control group.

Article Snippet: The following antibodies were purchased from various sources: anti-LC3 polyclonal antibody (PM036), anti-LC3 monoclonal antibody (M186-3), and anti-p62 antibody (PM045) from Medical Biological Laboratory; anti-p70S6K antibody (2708) and anti-phosphorylated p70S6K antibody (9206) from Cell Signaling Technology (Beverly, MA, USA); and anti-3-phosphoglyceraldehyde dehydrogenase (GAPDH) anti-body (ZB002) from YTHX Biotechnology Co., Ltd. (Beijing, China).

Techniques: CCK-8 Assay, Staining, Cell Culture, Expressing, Western Blot, Software, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Vitamin B12-Induced Autophagy Alleviates High Glucose-Mediated Apoptosis of Islet β Cells

doi: 10.3390/ijms242015217

Figure Lengend Snippet: The influence of high glucose on autophagy in RIN-m5F cells. ( A ) RIN-m5F cells were exposed to the indicated concentrations of glucose, and cytotoxicity was assessed utilizing a CCK-8 kit assay. ( B ) The cells were cultured under conditions of 35, 45, and 55 mM glucose for 36 h and then stained with anti-LC3 antibody (scale = 5 μm). ( C ) The cells were treated according to section ( A ), mathematical statistical methods were conducted to calculate the number of autophagosomes per cell, and at least 30 cells were counted by statistical analysis based on a t -test. ( D ) The cells were treated according section ( A ), and Western blotting analysis was conducted to investigate the expression levels of autophagy-related proteins with specific antibodies including anti-LC3, anti-p62, anti-GAPDH, anti-S6K, and anti-p-S6K. ( E , F ) Subsequent to the procedure described in section ( D ), the ratios of p62/GAPDH and p-S6K/S6K were assessed through IOD measurements utilizing Image-Pro Plus 6.0 software. All experiments were conducted a minimum of three times. The error bars represent the standard deviation, ** p < 0.01 reveals significant differences in comparison to the control group.

Article Snippet: The following antibodies were purchased from various sources: anti-LC3 polyclonal antibody (PM036), anti-LC3 monoclonal antibody (M186-3), and anti-p62 antibody (PM045) from Medical Biological Laboratory; anti-p70S6K antibody (2708) and anti-phosphorylated p70S6K antibody (9206) from Cell Signaling Technology (Beverly, MA, USA); and anti-3-phosphoglyceraldehyde dehydrogenase (GAPDH) anti-body (ZB002) from YTHX Biotechnology Co., Ltd. (Beijing, China).

Techniques: CCK-8 Assay, Cell Culture, Staining, Western Blot, Expressing, Software, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Vitamin B12-Induced Autophagy Alleviates High Glucose-Mediated Apoptosis of Islet β Cells

doi: 10.3390/ijms242015217

Figure Lengend Snippet: Vitamin B12 induced autophagy under conditions of high glucose stress. ( A ) RIN-m5F cells were treated with varying concentrations of vitamin B12 (2, 4, and 8 μM) in conjunction with 45 mM glucose for 36 h, and the number of autophagosomes was determined through immunofluorescence analysis (scale = 5 μm). ( B ) The cells were treated according to section ( A ), and mathematical statistical methods were conducted to calculate the number of autophagosomes in each cell. At least 30 cells were counted by t test analysis. ( C , E , G ) The cells were treated according to section ( A ), and Western blotting was conducted to investigate the expression levels of proteins with specific anti-LC3, anti-p62, anti-GAPDH, anti-S6K, and anti-p-S6K antibodies. ( D , F , H ) The ratio of LC3-II/LC3-I, p62/GAPDH and p-S6K/S6K ratios in ( C , E , G ), respectively, were assessed through IOD measurements utilizing Image-Pro Plus 6.0 software. All experiments were conducted a minimum of three times. The error bars represent the standard deviation, ** p < 0.01, * p < 0.05 reveals significant differences in comparison to the control group.

Article Snippet: The following antibodies were purchased from various sources: anti-LC3 polyclonal antibody (PM036), anti-LC3 monoclonal antibody (M186-3), and anti-p62 antibody (PM045) from Medical Biological Laboratory; anti-p70S6K antibody (2708) and anti-phosphorylated p70S6K antibody (9206) from Cell Signaling Technology (Beverly, MA, USA); and anti-3-phosphoglyceraldehyde dehydrogenase (GAPDH) anti-body (ZB002) from YTHX Biotechnology Co., Ltd. (Beijing, China).

Techniques: Immunofluorescence, Western Blot, Expressing, Software, Standard Deviation, Comparison, Control

Journal: International Journal of Molecular Sciences

Article Title: Vitamin B12-Induced Autophagy Alleviates High Glucose-Mediated Apoptosis of Islet β Cells

doi: 10.3390/ijms242015217

Figure Lengend Snippet: 3-MA suppressed vitamin B12-induced autophagy under the condition of high glucose stress. ( A ) RIN-m5F cells were cultivated under various conditions: either in the presence or absence of 45 mM glucose, supplemented with 10 mM 3-MA, a combination of 45 mM glucose and 10 mM 3-MA, 2 μM vitamin B12, 45 mM glucose plus 2 μM vitamin B12, or a concoction of 20 mM glucose, 2 μM vitamin B12, and 10 mM 3-MA, cultured for 36 h and stained with anti-LC3 anti-body (scale bar = 5 μm). ( B ) The cells were treated according to section ( A ), and mathematical statistical methods were conducted to calculate the number of autophagosomes in each cell. At least 30 cells were analyzed. ( C ) The cells were treated according to section ( A ), and Western blotting was performed to investigate the expression levels of proteins with specific antibodies against LC3 and GAPDH. ( D ) The LC3-II/LC3-I ratio in ( C ) was analyzed by IOD analysis utilizing Image-Pro Plus 6.0 software. All experiments were conducted a minimum of three times. The error bars represent the standard deviation, ** p < 0.01, * p < 0.05 reveals significant differences in comparison to the control group. ## p < 0.01 reveals significant differences in comparison to the group treated with vitamin B12 combined with high glucose through one-way ANOVA.

Article Snippet: The following antibodies were purchased from various sources: anti-LC3 polyclonal antibody (PM036), anti-LC3 monoclonal antibody (M186-3), and anti-p62 antibody (PM045) from Medical Biological Laboratory; anti-p70S6K antibody (2708) and anti-phosphorylated p70S6K antibody (9206) from Cell Signaling Technology (Beverly, MA, USA); and anti-3-phosphoglyceraldehyde dehydrogenase (GAPDH) anti-body (ZB002) from YTHX Biotechnology Co., Ltd. (Beijing, China).

Techniques: Cell Culture, Staining, Western Blot, Expressing, Software, Standard Deviation, Comparison, Control