laser micro irradiation  (Addgene inc)

Journal: Heliyon

Article Title: Nuclear translocation of nucleotide enzyme Phosphoglucomutase 2 governs DNA damage response and anti-tumor immunity ☆

doi: 10.1016/j.heliyon.2024.e36415

Figure Lengend Snippet: Nuclear PGM2 is recruited to the DNA damage site. A. Immunofluorescence images displaying NLS- and NES-PGM2 in HeLa and U2OS sgPGM2 cells, with nuclei counterstained by DAPI. Scale bar: 10 μm. B. HeLa sgPGM2 cells were transfected with NLS- and NES-PGM2, followed by treatment with 0.001 % DMSO or 200 μM TMZ for 24 h. Then, the extent of DNA damage was evaluated by quantifying phospho-γH2A.X protein levels by WB. The levels of phospho-γH2A.X were measured using the DMSO-treated NLS group as the control. C. U2OS sgPGM2 cells were transfected with NLS- and NES-PGM2, followed by treatment with 0.001 % DMSO or 200 μM TMZ for 24 h. Subsequently, the extent of DNA damage was evaluated by quantifying phospho-γH2A.X protein levels by WB. The levels of phospho-γH2A.X were measured using the DMSO-treated NLS group as the control. D. Representative images showing phospho-γH2A.X and GFP-PGM2 in NLS- and NES-PGM2 HeLa sgPGM2 cells treated with 0.001 % DMSO or 200 μM TMZ for 24 h, with nuclei counterstained by DAPI. Scale bar: 5 μm. E. Representative images showing phospho-γH2A.X and GFP-PGM2 in NLS- and NES-PGM2 U2OS sgPGM2 cells treated with 0.001 % DMSO or 200 μM TMZ for 24 h, with nuclei counterstained by DAPI. Scale bar: 5 μm. F. Quantification of phospho-γH2A.X levels measured as mean intensity per nucleus in NLS- and NES-PGM2 sgPGM2 HeLa cells. At least 50 cells were measured in each group. G. Quantification of phospho-γH2A.X levels measured as mean intensity per nucleus in NLS- and NES-PGM2 sgPGM2 U2OS cells. At least 50 cells were measured in each group. H, I. Representative fluorescence micrographs of HeLa cells and U2OS cells expressing GFP or GFP-PGM2 individually coexpressing mCherry-PCNA as positive control, before and after laser micro-irradiation, following pretreatment with 10 μM BrdU for 24 h in HeLa cells and U2OS cells. Scale bar: 10 μm. J, K. Quantification of GFP intensity in the DNA laser micro-irradiation assay in Figure H, I. At least 10 cells were measured in each group. Statistical significance was denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Article Snippet: Laser micro irradiation was performed with Olympus FV3000 confocal microscope and a 405 nm laser with 60 % energy.

Techniques: Immunofluorescence, Transfection, Control, Fluorescence, Expressing, Positive Control, Irradiation

Journal: Heliyon

Article Title: Nuclear translocation of nucleotide enzyme Phosphoglucomutase 2 governs DNA damage response and anti-tumor immunity ☆

doi: 10.1016/j.heliyon.2024.e36415

Figure Lengend Snippet: Phosphorylation of PGM2 at S165 protects cells against DNA damage stress. A. The 165 serine phosphorylation site of PGM2 detected by MS is conservative. B. HeLa sgPGM2 cells were individually transfected with GFP-PGM2(S165A) and GFP-PGM2(S165D), followed by treatment with 0.001 % DMSO or 200 μM TMZ for 24 h. Subsequently, the extent of DNA damage was assessed by quantifying phospho-γH2A.X protein levels using Western blot analysis. The levels of phospho-γH2A.X were measured using the DMSO-treated S165A group as the control. C. U2OS sgPGM2 cells were individually transfected with GFP-PGM2(S165A) and GFP-PGM2(S165D), followed by treatment with 0.01 % DMSO or 200 μM TMZ for 24 h. Subsequently, the extent of DNA damage was assessed by quantifying phospho-γH2A.X protein levels using Western blot analysis. The levels of phospho-γH2A.X were measured using the DMSO-treated S165A group as the control. D. Representative images illustrating phospho-γH2A.X levels in PGM2 S165A and S165D HeLa sgPGM2 cells treated with 0.001 % DMSO or 200 μM TMZ for 24 h, with nuclei counterstained by DAPI. Scale bar: 5 μm. E. Representative images illustrating phospho-γH2A.X levels in PGM2 S165A and S165D U2OS sgPGM2 cells treated with DMSO or 200 μM TMZ for 24 h, with nuclei counterstained by DAPI. Scale bar: 5 μm. F. Quantification of phospho-γH2A.X levels measured as mean intensity per nucleus in Figure D. At least 50 cells were measured in each group. G. Quantification of phospho-γH2A.X levels measured as mean intensity per nucleus in Figure E. At least 50 cells were measured in each group. H. Representative fluorescence micrographs of HeLa cells individually expressing GFP-PGM2 S165A or GFP-PGM2 S165D, before and after laser micro-irradiation. Scale bar: 10 μm. I. Quantification of Figure H. At least 10 cells were measured in each group. J. Representative fluorescence micrographs of U2OS cells individually expressing GFP-PGM2 S165A or P-PGM2 S165D, before and after laser micro-irradiation. Scale bar: 10 μm. K. Quantification of Figure J. At least 10 cells were measured in each group. Statistical significance was indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Article Snippet: Laser micro irradiation was performed with Olympus FV3000 confocal microscope and a 405 nm laser with 60 % energy.

Techniques: Phospho-proteomics, Transfection, Western Blot, Control, Fluorescence, Expressing, Irradiation

Journal: Heliyon

Article Title: Nuclear translocation of nucleotide enzyme Phosphoglucomutase 2 governs DNA damage response and anti-tumor immunity ☆

doi: 10.1016/j.heliyon.2024.e36415

Figure Lengend Snippet: ROCK2 interacts with PGM2. A. Workflow regarding flag beads immunoprecipitation of Flag-PGM2. B. Identification of potential interacting proteins in Ctrl and etoposide-treatment group. C. 293T cells were separately transfected with GFP-Flag-PGM2 and mRFP-HA-ROCK2 or co-transfected with both. After 36 h of transfection, cell lysates were immunoprecipitated using anti-Flag beads. The input and immunoprecipitates were subjected to immunoblotting for the indicated proteins. D. 293T cells were separately transfected with GFP-Flag-PGM2 and mRFP-HA-ROCK2 or co-transfected with both. After 36 h of transfection, cell lysates were immunoprecipitated using anti-HA beads. The input and immunoprecipitates were subjected to immunoblotting for the indicated proteins. E. HeLa cells expressing mRFP-HA-ROCK2 were treated with 0.001 % DMSO or 5 μM etoposide for 24 h. Cell lysates were immunoprecipitated using anti-HA beads. The input and immunoprecipitates were subjected to immunoblotting for the indicated proteins. F. HeLa cells were treated with 0.001 % DMSO or 5 μM etoposide for 24 h. Cell lysates were immunoprecipitated using the anti-PGM2 antibody or IgG control antibody. The input and immunoprecipitates were subjected to immunoblotting for the indicated proteins. G. 293T cells were separately transfected with Flag-PGM2 wt, S165A mutant, and S165D mutant together with mRFP-ROCK2. After 36 h of transfection, cell lysates were immunoprecipitated using anti-HA beads. The input and immunoprecipitates were subjected to immunoblotting for the indicated proteins. H. HeLa cells expressing mRFP-HA-ROCK2 separately with GFP-Flag-PGM2 S165A or S165D were treated with 0.001 % DMSO or etoposide 5 μM for 24h. Cell lysates were immunoprecipitated using anti-HA beads. The input and immunoprecipitates were subjected to immunoblotting for the indicated proteins. I. Representative fluorescence micrographs of HeLa cells stably expressing GFP-PGM2 with/without ROCK2 knock down in before and after DNA laser micro-irradiation assay. Scale bar: 10 μm. J. Quantification of DNA laser micro-irradiation assay in Fig. I. At least 10 cells were measured in each group. Statistical significance was indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Article Snippet: Laser micro irradiation was performed with Olympus FV3000 confocal microscope and a 405 nm laser with 60 % energy.

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing, Control, Mutagenesis, Fluorescence, Stable Transfection, Knockdown, Irradiation