Journal: International Journal of Molecular Sciences

Article Title: Proteomic Identification of Target Proteins of Thiodigalactoside in White Adipose Tissue from Diet-Induced Obese Rats

doi: 10.3390/ijms160714441

Figure Lengend Snippet: List of proteins showing differential expression between control and thiodigalactoside (TDG)-treated rats.

Article Snippet: The following antibodies were used in this study: anti-goat UCP-1, MDH2, PGM1, PPARγ, and CA3; anti-mouse CK and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and anti-rabbit ANXA2 (Cell Signaling Technology, Beverly, MA, USA) and PGC-1α (Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Control, Sequencing, Transduction, Membrane

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: PGM2 translocates into the nucleus by chemo-drug treatment. A. PGM2 functions in ribose-phosphate conversion. B. Immunofluorescence staining of endogenous PGM2 in U251 cells using PGM2 antibody and co-localization profiling of PGM2 with DAPI of line-indicated section. Scale bar: 10 μm. C. Immunofluorescence staining of endogenous PGM2 in U87 cells using PGM2 antibody and co-localization profiling of PGM2 with DAPI of line-indicated section. Scale bar: 10 μm. D. Immunofluorescence staining of endogenous PGM2 in HeLa cells using PGM2 antibody and co-localization profiling of PGM2 with DAPI of line-indicated section. Scale bar: 10 μm. E. Immunofluorescence staining of HA-tagged PGM2 in HeLa cells using HA antibody and co-localization profiling of PGM2 with DAPI of line-indicated section. Scale bar: 10 μm. F. Immunofluorescence images of PGM2 staining in ctrl, TMZ-treated, and Doxo-treated U251 cells, with nuclei counterstained by DAPI. U251 cells were treated with 0.001%DMSO, 400 μM TMZ, or 2 μM Doxo for 24 h. G. Quantification of PGM2 intensity (Nuclear intensity/Cytoplasmic intensity) in figure F. At least 30 cells were measured in each group. H. Representative images of live-cell imaging in U2OS cells. Scale bar: 20 μm. U2OS cells were transfected with GFP or GFP-PGM2, followed by live-cell imaging with or without 5 μM etoposide for 24 h. I. Quantification of relative GFP intensity (Nuclear intensity/Cytoplastic intensity) of live-cell imaging in U2OS cells, with 10 cells measured in each group. J. Immunofluorescence images displaying GFP or GFP-PGM2 and αTubulin in 0.001%DMSO-treated, TMZ-treated, and etoposide-treated HeLa cells, with nuclei counterstained by DAPI. HeLa cells were transfected separately with GFP or GFP-PGM2, and then treated with 0.001%DMSO, 400 μM TMZ, or 5 μM etoposide for 24 h. K. Quantification of relative GFP intensity (Nuclear intensity/Cytoplasmic intensity) of GFP or GFP-PGM2 in figure J. At least 20 cells were measured in each group. Statistical significance was denoted as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

Article Snippet: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

Techniques: Immunofluorescence, Staining, Live Cell Imaging, Transfection

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: PGM2 depletion enhances the sensitivity of GBM cells to DNA damage. A. The relative mRNA expression of PGM2 in TCGA and GTEx datasets. B. U251 cells were subjected to knockout of PGM2 using two independent sgRNAs, and the effectiveness of the knockout was confirmed through Western blot analysis. C. sgScramble and sgPGM2 U251 cells were treated with TMZ in a dose-dependent manner (0.001 % DMSO, TMZ 200 μM, 400 μM). DNA damage was assessed by quantifying phospho-γH2A.X protein levels. D. sgScramble and sgPGM2 U251 cells were treated with 0.001%DMSO or 400 μM TMZ in a time-dependent manner (24h, 48h). DNA damage was assessed by quantifying phospho-γH2A.X protein levels. E. U251 cells transfected with sgScramble or sgPGM2 were subjected to the neutral comet assay. Microphotographs representing the results of the neutral comet assay. Scale bar: 0.2 cm. F. Quantification of the tail moment of figure E. Data were compiled from approximately 100 cells in each group. G. Representative images of phospho-γH2A.X staining in U251 cells. Scale bar: 30 μm. U251 cells transfected with sgScramble or sgPGM2 were treated with 0.001 % DMSO or TMZ 400 μM for 48h, and after treatment, the level of phospho-γH2A.X was assessed through immunofluorescence staining. H. Quantification of phospho-γH2A.X intensity per nucleus of figure G. At least 50 cells were measured in each group. I. Representative images of TP53BP1 staining in U251 cells. Scale bar: 20 μm. U251 cells transfected with sgScramble or sgPGM2 were treated with 0.001 % DMSO or TMZ 400 μM for 48h, and after treatment, the level of TP53BP1 was assessed through immunofluorescence staining. J. Quantification of TP53BP1 foci per nucleus in figure I. At least 50 cells were counted 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: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

Techniques: Expressing, Knock-Out, Western Blot, Transfection, Neutral Comet Assay, Staining, Immunofluorescence

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: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

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: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

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: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing, Control, Mutagenesis, Fluorescence, Stable Transfection, Knockdown, 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: PGM2 knockdown sensitizes glioma to TMZ in vivo by activating the immune microenvironment. A. Implementation of PGM2 knockdown in GL261 cells using two distinct shRNAs; effectiveness of the knockdown ascertained via qRT-PCR analysis. B. Display of representative phospho-γH2A.X stained GL261 cells expressing either scramble shRNA (shScramble), shPGM2-1, or shPGM2-2 with/without 400 μM TMZ treatment for 24h. Scale bar: 5 μm. C. Quantification of phospho-γH2A.X levels measured as mean intensity per nucleus in figure B. At least 50 cells were measured in each group. D. Representative bioluminescence imaging of C57/B6J mice bearing GL261-luc gliomas expressing either shScramble or shPGM2-2 with/without TMZ treatment. E. Quantitative assessment of the bioluminescent signal intensity in C57/B6J mice bearing GL261 gliomas expressing either shScramble or shPGM2-2 with/without TMZ treatment. F. Quantitative analysis of ISGs expression in CT2A cells expressing either shScramble or shPGM2-2 treated by 100 μM and 200 μM TMZ, performed via qRT-PCR. G. Representative brain slices image of GL261 glioma-bearing mice stained with DAPI for nuclear (Left panel). Kaplan-Meier survival analysis of C57/B6J mice intracranially implanted with GL261 cells expressing either shScramble or shPGM2-2 with/without TMZ treatment (Right panel). H. Fluorescence imaging of the distribution of CD3 + from mice bearing GL261 gliomas expressing either shScramble or shPGM2-2 with/without TMZ treatment. Scale bar: 20 μm. I. Fluorescence imaging of CD8 + GranzB + cells within frozen sections from mice bearing GL261 gliomas expressing either shScramble or shPGM2-2 with/without TMZ treatment. Scale bar: 20 μm. J. Fluorescence imaging of CD8 + IFNγ + cells within frozen sections from mice bearing GL261 gliomas expressing either shScramble or shPGM2-2 with/without TMZ treatment. Scale bar: 20 μm. K, L, M. Quantification of CD3 + cells, CD8 + GranzB + cells, and CD8 + IFNγ + cells within fields of view of frozen sections from mice bearing GL261 gliomas expressing either shScramble or shPGM2-2 with/without TMZ treatment in Figure H, I, J. Three mice were counted 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: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

Techniques: Knockdown, In Vivo, Quantitative RT-PCR, Staining, Expressing, shRNA, Imaging, Fluorescence

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 inhibition sensitizes glioma to TMZ and PD-L1 in vivo by activating the immune environment. A. Representative brain slice image of GL261 glioma-bearing mice treated with TMZ, Belumosudil, or the combination of TMZ and Belumosudil stained with DAPI for nuclear. B. Kaplan-Meier survival analysis of C57/B6J mice intracranially implanted with GL261 cells treated with TMZ, Belumosudil, or the combination of TMZ and Belumosudil. C. Fluorescence imaging of the distribution of CD3 + from mice bearing GL261 gliomas treated with TMZ, Belumosudil, or the combination of TMZ and Belumosudil. Scale bar: 20 μm. D. Fluorescence imaging of CD8 + GranzB + cells within frozen sections from mice bearing GL261 gliomas treated with TMZ, Belumosudil, or the combination of TMZ and Belumosudil. Scale bar: 20 μm. E. Fluorescence imaging of CD8 + IFNγ + cells within frozen sections from mice bearing GL261 gliomas treated with TMZ, Belumosudil, or the combination of TMZ and Belumosudil. Scale bar: 20 μm. F, G, H. Quantification of CD3 + cells, CD8 + GranzB + cells, and CD8 + IFNγ + cells within fields of view of frozen sections from mice bearing GL261 glioma treated with TMZ, Belumosudil, or the combination of TMZ and Belumosudil in Figure C, D, E. Three mice were counted in each group. I. Representative brain slices image of GL261 glioma-bearing mice treated with PD-L1, Belumosudil, or the combination of PD-L1 and Belumosudil stained with DAPI for nuclear. J. Kaplan-Meier survival analysis of C57/B6J mice intracranially implanted with GL261 cells treated with PD-L1, Belumosudil, or the combination of PD-L1 and Belumosudil. K. Fluorescence imaging of the distribution of CD3 + from mice bearing GL261 gliomas treated with PD-L1, Belumosudil, or the combination of PD-L1 and Belumosudil. Scale bar: 20 μm. L. Fluorescence imaging of CD8 + GranzB + cells within frozen sections from mice bearing GL261 glioma treated with PD-L1, Belumosudil, or the combination of PD-L1 and Belumosudil. Scale bar: 20 μm. M. Fluorescence imaging of CD8 + IFNγ + cells within frozen sections from mice bearing GL261 glioma treated with PD-L1, Belumosudil, or the combination of PD-L1 and Belumosudil. Scale bar: 20 μm. N, O, P. Quantification of CD3 + cells, CD8 + GranzB + cells, and CD8 + IFNγ + cells within fields of view of frozen sections from mice bearing GL261 glioma treated with PD-L1, Belumosudil, or the combination of PD-L1 and Belumosudil. Three mice were counted in each group. Q. Diagrammatic representation of the non-canonical roles of PGM2 and the impact of PGM2 on glioma progression. Statistical significance was indicated as * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001.

Article Snippet: Antibodies used in this study: PGM2 Polyclonal antibody, Proteintech, Cat#11022-1-AP; Phospho-Histone H2A.X (Ser 139) (20E3) Rabbit mAb, CST, Cat#9718; ROCK2 Monoclonal antibody, Proteintech, Cat#66633-1-Ig; HRP-conjugated HA Tag Recombinant antibody, Proteintech, Cat#HRP-81290; Monoclonal ANTI-FLAG® M2-Peroxidase (HRP) antibody produced in mouse, Sigma Aldrich, Cat#A8592; GFP-Tag (7G9) Mouse mAb, Abmart, Cat#M20004; Alpha Tubulin Monoclonal antibody, Proteintech, Cat#66031-1-Ig; Beta Actin Recombinant antibody, Proteintech, Cat#81115-1-RR; HRP-conjugated GAPDH Monoclonal antibody, Proteintech, Cat#HRP-60004; 53BP1 Antibody, CST, Cat#4937; HA-Tag (C29F4) Rabbit mAb, CST, Cat#3724; DYKDDDDK Tag (9A3) Mouse mAb, CST, Cat#8146; Lamin B1 Monoclonal antibody, Proteintech, Cat# 66095-1-Ig; HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L), Proteintech, Cat#SA00001-1; HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L), Proteintech, Cat#SA00001-2.

Techniques: Inhibition, In Vivo, Slice Preparation, Staining, Fluorescence, Imaging