mgmt  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Loss of MGMT increases autophagy in BMMs. WT and MGMT-KO BMM were pretreated with bafilomycin A1 (500 nM) for 1 h. The cells were stimulated with poly (I:C) (20 µg/ml) and incubated for 24 h. ( A , B ) Cells were fixed and stained for LC3B and nuclei. At least 50 cells per condition per independent experiment were quantified for LC3B puncta. The data are presented as the means ± SEMs from three independent experiments. Scale bar = 5 µM. *** P < 0.001, ** P < 0.01 ( C ) Cells were treated as indicated, and the cell lysates were processed for Western blotting for LC3Bii.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Staining, Western Blot

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Loss of MGMT enhances mitophagy in macrophages. ( A , B ) WT and MGMT KO BMMs were treated with bafilomycin A1 (500 nM) and incubated for 24 h. The cells were fixed and stained for HSP60 and LC3B and processed for confocal microscopy analysis. The colocalization of HSP60 and LC3B was quantified in at least 50 cells per condition per independent experiment. The data are presented as the means ± SEMs from three independent experiments. Scale bar = 5 µM. *** P < 0.001, ** P < 0.01 and * P < 0.05. ( C , D ) Cells were treated as described above and incubated with LysoTracker Deep Red (100 nM) for 1 h. The cells were then fixed and stained for HSP60 and processed for confocal microscopy analysis. At least 50 cells per condition per independent experiment were quantified for colocalization between lysosomes and HSP60. The data are presented as the means ± SEMs from three independent experiments. *** P < 0.001, ** P < 0.01 and * P < 0.05.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Loss of MGMT increases autophagy in BMMs. WT and MGMT-KO BMM were pretreated with bafilomycin A1 (500 nM) for 1 h. The cells were stimulated with poly (I:C) (20 µg/ml) and incubated for 24 h. ( A , B ) Cells were fixed and stained for LC3B and nuclei. At least 50 cells per condition per independent experiment were quantified for LC3B puncta. The data are presented as the means ± SEMs from three independent experiments. Scale bar = 5 µM. *** P < 0.001, ** P < 0.01 ( C ) Cells were treated as indicated, and the cell lysates were processed for Western blotting for LC3Bii.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Staining, Western Blot

Journal: Scientific Reports

Article Title: Loss of O 6 -methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

doi: 10.1038/s41598-024-78885-3

Figure Lengend Snippet: Loss of MGMT enhances mitophagy in macrophages. ( A , B ) WT and MGMT KO BMMs were treated with bafilomycin A1 (500 nM) and incubated for 24 h. The cells were fixed and stained for HSP60 and LC3B and processed for confocal microscopy analysis. The colocalization of HSP60 and LC3B was quantified in at least 50 cells per condition per independent experiment. The data are presented as the means ± SEMs from three independent experiments. Scale bar = 5 µM. *** P < 0.001, ** P < 0.01 and * P < 0.05. ( C , D ) Cells were treated as described above and incubated with LysoTracker Deep Red (100 nM) for 1 h. The cells were then fixed and stained for HSP60 and processed for confocal microscopy analysis. At least 50 cells per condition per independent experiment were quantified for colocalization between lysosomes and HSP60. The data are presented as the means ± SEMs from three independent experiments. *** P < 0.001, ** P < 0.01 and * P < 0.05.

Article Snippet: The cells were incubated with a rabbit anti-γ-H2AX antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-LC3B antibody (Cell Signaling Technology, USA) at a dilution of 1:200, a rabbit anti-MGMT antibody (Cell Signaling Technology, USA) at a dilution of 1:60, or a mouse anti-HSP60 antibody (DSHB, USA) at a concentration of 1 μg/mL in blocking solution at 4 °C overnight.

Techniques: Incubation, Staining, Confocal Microscopy