Journal: International Journal of Molecular Sciences

Article Title: High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity

doi: 10.3390/ijms12096116

Figure Lengend Snippet: List of marker genes, their references and Taqman probes used in this study.

Article Snippet: 22 , Pgam2 , phosphoglycerate mutase 2 , NM_018870.3 , Mm00450782_g1 , heart , [ ] .

Techniques: Marker, Binding Assay, Ubiquitin Proteomics, Inhibition, Control

Journal: Oncotarget

Article Title: Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

doi: 10.18632/oncotarget.4044

Figure Lengend Snippet: Figure 6: Correlation of ActD-stimulated changes of PGAM localization in NSCLC cells with blockade of the cell cycle cell in G2 phase. Upper panel: the PGAM2 localization in the cells incubated with 0.04 µg/ml (low) or 2.5 µg/ml (high) actinomycin D (ActD). Lower panel: the cells in G1 (red) and G1/S (yellow) phase in control conditions and blockade of the ActD-treated cells in G2 phase (green fluorescence) as determined by FUCCI Cell Cycle Sensor. Bar=10 µm.

Article Snippet: NHS-SS-Diazirine cross-linker (spacer arm length 1.35 nm) was from Thermo Scientific Pierce; protein G-agarose from Roche; siRNA-A (sc-37007) and mouse PGAM2 siRNA (sc-152183) were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Control, Fluorescence

Journal: Oncotarget

Article Title: Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

doi: 10.18632/oncotarget.4044

Figure Lengend Snippet: Figure 7: The effects of KLN-205 cells transfection with PGAM2. The cells were transfected with PGAM2-FITC in normal conditions and in the presence of RNase. As a control, cells were transfected with BSA-FITC. Bar=10 µm.

Article Snippet: NHS-SS-Diazirine cross-linker (spacer arm length 1.35 nm) was from Thermo Scientific Pierce; protein G-agarose from Roche; siRNA-A (sc-37007) and mouse PGAM2 siRNA (sc-152183) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Control

Journal: Oncotarget

Article Title: Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

doi: 10.18632/oncotarget.4044

Figure Lengend Snippet: Figure 8: The effects of PGAM2 silencing on nucleolar morphology and new RNA production in KLN-205 cells. A. in PGAM2-silenced cells, unlike control cells, stained with propidium iodide (red), only one centrally located nucleolus can be found and nucleolar PGAM staining (green) is undetectable; B. the level of new RNA (red) synthesis in PGAM2-silenced cells is reduced as compared to control cells. To visualize DNA the cells were counterstained with Hoechst 33342 (blue). Bar=10 µm.

Article Snippet: NHS-SS-Diazirine cross-linker (spacer arm length 1.35 nm) was from Thermo Scientific Pierce; protein G-agarose from Roche; siRNA-A (sc-37007) and mouse PGAM2 siRNA (sc-152183) were obtained from Santa Cruz Biotechnology.

Techniques: Control, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: PGAM2 is a SUMO-1 substrate with SUMO acceptor sites at K49 and K146. (A) Results of Ni-NTA pulldown experiments performed in HeLa cells transfected with PGAM2-V5-his and one of the following expression vectors: HA-SUMO-1-wt, mut (i.e., deficient for conjugation), HA-SUMO-2-wt, or HA-SUMO-2-mut, as indicated. The top panel was probed with V5-HRP, and the bottom panel was probed with HA-HRP. Notably, the strong, slowly migrating band (red arrow) was observed only in the presence of both PGAM2-V5-his and HA-SUMO-1-wt, and the weak, slowly migrating band (red asterisk) was observed in the presence of both PGAM2-V5-his and HA-SUMO-1-wt. (B) Pias family proteins do not promote SUMO moiety conjugation to PGAM2. Top two panels: Ni-NTA pulldown experiments were performed in HeLa cells transfected with PGAM2-V5-his and flag-SUMO-1-wt in the presence of Pias family proteins, as indicated. Lower two panels: Western blot analysis was performed to evaluate the expression levels of HA-tagged Pias family proteins after the transfection of HeLa cells. β-actin was used as an internal control. (C) SENP2-wt deconjugates SUMO1-conjugated PGAM2. Ni-NTA pulldown experiments were performed as described above with the indicated expression vectors. Note that SUMO1-PGAM2 disappeared only in the lane with the presence of flag-SENP2-wt but in not others (top panel). The second panel shows that flag-SENP2-wt also abolishes global SUMO-1 conjugation. Western blot analysis was performed to reveal the comparable expression of flag-SENP2-wt, flag-SENP2-mut, flag-SENP5-wt, and flag-SENP5-mut (third panel). β-actin was used as a loading control (the bottom panel). (D) SUMO plot shows the different scores for the predicted sumoylation sites on PGAM2 (K146, K49, and K176). (E) Ni-NTA pulldown experiments were performed in HeLa cells transfected with vectors encoding PGAM2-wt or one of its point mutants, K49R, K146R, or K176R, together with flag-SUMO-1 expression vector. SUMO-conjugated PGAM2 is indicated with red arrows. HMW, high molecular weight conjugates.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Transfection, Expressing, Conjugation Assay, Western Blot, Control, Plasmid Preparation, High Molecular Weight

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: PGAM2 expression is increased during myogenic differentiation. (A) Results of RNA sequencing analysis showing that the expression of PGAM2 but not PGAM1 was significantly increased at differentiation day (d)4 when compared with d0. The increased expression of MHC4 at d4 compared with d0 was used as a positive control. n = 3 per group. **** p < 0.001 vs. d0. (B) Results of real-time PCR analysis of differentiating C2C12 cells at different days confirming increased PGAM2 expression during myogenic differentiation. n = 4 per group. * p < 0.05 vs. d0. (C) Protein levels of PGAM2 were significantly increased in differentiating WT C2C12 cells at day d4 compared with d0. Western blot analysis performed on whole-cell lysates purified from d0 and d4 WT C2C12 cells. Cav3 was used as a differentiation marker. β-actin was used as a loading control. n = 6 per group.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Expressing, RNA Sequencing, Positive Control, Real-time Polymerase Chain Reaction, Western Blot, Purification, Marker, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: The PGAM2-K176R mutation impairs myogenic differentiation. (A,B) Western blot analysis of whole-cell lysates from WT and C2C12 K176R/K176R cells (#207) at differentiation day (d)0 (i.e., undifferentiated), d2, d4 and d6. Cells were plated and cultured in growth medium (GM) overnight in 6-cm plates and then switched to differentiation medium (DM). DM was replenished every 2 days. β-Actin was used as a control. * p < 0.05, ** p < 0.01 vs . WT. (C) Immunofluorescence staining for MHC was performed in WT and C2C12 K176R/K176R cells at differentiation days as indicated. Red, MHC; blue, DAPI. * p < 0.05, vs. WT. Magnification, ×20.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Mutagenesis, Western Blot, Cell Culture, Control, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: PGAM2-K176R mutation decreases the glycolytic rate. For measuring the glycolytic rate, C2C12 cells were seeded into 96-well Seahorse cell culture plates and cultured overnight. The Seahorse XFe96 Extracellular Analyzer was used to record three basal rate measurements, followed by injections with Rot/AA according to the manufacturer’s protocol. Data were automatically generated by the Seahorse XF Glycolytic Rate Assay Report Generator and Wave program. (A) One representative mitochondrial respiration curve from seven independent experiments is shown. (B) PGAM2 K176R/K176R mutant cells exhibited a reduced basal glycolytic rate, basal PER, and compensatory glycolysis. No significant difference was observed in % PER from glycolysis (basal) between WT and PGAM2 K176R/K176R mutant cells. Data are expressed as the mean ± SD. n = 6 wells per group. ** p < 0.01 vs WT.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Mutagenesis, Cell Culture, Generated

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: PGAM2 K176R/K176R mutation impairs mitochondrial function. For measuring the oxygen consumption rate (OCR), C2C12 cells were seeded into 96-well Seahorse cell culture plates and cultured overnight. The Seahorse XFe96 Extracellular Analyzer was used to record OCR at baseline and three times at 6-min intervals after injections with 5 µM oligomycin, 3 µM FCCP, and 4 µM rotenone/antimycin A (Rot/AA), respectively, according to the manufacturer’s protocol. Data were automatically generated by Seahorse XF Cell Mito Stress Test Report Generator. (A) One representative mitochondrial respiration curve is shown. (B) PGAM2 K176R/K176R mutant cells exhibited reduced basal OCR, spare respiratory capacity, proton leak, and ATP production compared with WT cells. Data are expressed as mean ± SD. n = 6 wells per group. * p < 0.05, *** p < 0.001 vs WT.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Mutagenesis, Cell Culture, Generated

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: Dysregulated gene expression in PGAM2-K176R mutant C2C12 cells at differentiation day 0. Results of RNA sequencing analysis of WT and PGAM2-K176R mutant C2C12 cells at differentiation day (d)0. (A) Volcano plot with a filter of padj <0.05 and a |log2 fold change| = 1 shows the changes in differentially expressed genes (DEGs) in WT and PGAM2-K176R mutant C2C12 cells. (B) Heatmap providing a global view of DEGs in WT and PGAM2-K176R mutant cells. (C) Top 10 most enriched GO terms in the biological pathways (BPs) associated with downregulated (top panel) and upregulated (bottom panel) genes in PGAM2-K176R mutant cells compared with WT cells. (D) Top 20 most downregulated (left panel) and upregulated (right panel) genes in PGAM2-K176R mutant cells compared with WT cells.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Gene Expression, Mutagenesis, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: Dysregulated gene expression in PGAM2-K176R mutant C2C12 cells at differentiation day 4. Results of RNA sequencing analysis for WT and PGAM2-K176R mutant C2C12 cells at differentiation day (d)0. (A) Volcano plot with a filter of padj <0.05 and a |log2 fold change| = 1 shows the changes in differentially expressed genes (DEGs) in WT and PGAM2-K176R mutant C2C12 cells. (B) Heatmap providing a global view of DEGs in WT and PGAM2-K176R mutant cells. (C) Top 10 most enriched GO terms in BPs associated with downregulated (top panel) and upregulated (bottom panel) genes in PGAM2-K176R mutant cells compared with WT cells. (D) Top 20 most downregulated (left panel) and upregulated (right panel) genes in PGAM2-K176R mutant cells compared with WT cells.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Gene Expression, Mutagenesis, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Sumoylation-deficient phosphoglycerate mutase 2 impairs myogenic differentiation

doi: 10.3389/fcell.2022.1052363

Figure Lengend Snippet: PGAM2 with missense mutations linked with human disease exhibit reduced sumoylation. A Ni-NTA pulldown experiments were performed on C2C12 cells transfected with PGAM2-V5-wt, PGAM2-V5-E89A, or PGAM2-V5-R90W in the presence of flag-SUMO-1-wt or flag-SUMO-1-ΔGG mutant, respectively. Top panel, anti-V5 blot; bottom panel, anti-flag blot. HMW, high molecular weight conjugates. (B) Statistical analysis of data shown in (A) . Data for sumoylated PGAM2 were normalized to those for free PGAM2, and PGAM2-V5-wt sumoylation was given a value of 100%. n = 4 per group. *** p < 0.001 vs WT.

Article Snippet: Rabbit anti-PGAM2 antibody (Cat# A7917, 1:1000) was from ABclonal (Manhattan Beach, CA, United States).

Techniques: Transfection, Mutagenesis, High Molecular Weight