pgm3  (Proteintech)

Journal: Science Advances

Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth

doi: 10.1126/sciadv.adq0334

Figure Lengend Snippet: ( A ) Schematic illustration of the HBP. ( B and C ) Proliferation (B) and colony formation (C) of patient-derived primary GBM cells after treatment with AZA at indicated concentrations and times. Scale bar, 50 μm. ( D ) Colony formation of patient-derived primary GBM cells after knockdown of GFAT1 with shRNA. The GFAT1 knockdown was confirmed by Western blotting. ( E ) Western blotting analysis of the expression of different HBP enzymes in patient-derived primary GBM cells. ( F ) Western blotting analysis of HBP enzyme expression in GBM30 cells after siRNA transfection for 48 hours. ( G ) Colony formation of GBM cells after knockdown of HBP enzymes with siRNA. ( H ) Western blotting analysis of GFAT2 expression in patient-derived primary GBM cells. ( I ) Western blotting analysis of GFAT2 expression in GBM30 cells after siRNA transfection for 48 hours. ( J ) Neurosphere growth of GBM30 cells after knockdown of GFAT2 with siRNA. Scale bar, 50 μm. ( K ) Colony formation of GBM30 cells after knockdown of GFAT1, GFAT2, and NAGK alone or in combination with siRNA. ( L and M ) LC-MS/MS measurement of 13 C-UDP-GlcNAc isotopomers in GBM30 cells incubated with 13 C 6 -glucose (L) and 13 C 2 -UDP-GlcNAc in GBM30 cells incubated with 13 C 2 -GlcNAc labeled on the acetyl group (M) after knockdown of GFAT1, NAGK, GFAT1/NAGK, or PGM3 by shRNA. For data analysis of (B to D), (G), and (J to M), the results are shown as means ± SD [ n = 4 for (J), (M), and (L); n = 3 for other panels]. For (B), (J), and (L), the significance was determined by two-way analysis of variance (ANOVA) with Tukey’s test as compared with control group. For (C), (D), (G), (K), and (M), the significance was determined by one-way ANOVA with Tukey’s test as compared with control group. ns, not significant.

Article Snippet: Transient gene silencing experiments were performed with siRNAs for GFAT1 (Santa Cruz Biotechnology, #sc-60618), GFAT2 (Santa Cruz Biotechnology, #sc-91875), GNPNAT1 (Santa Cruz Biotechnology, #sc-60709), PGM3 (Santa Cruz Biotechnology, #sc-95517), UAP1 (Dharmacon, #L-017160-01-0005), and NAGK (Santa Cruz Biotechnology, #sc-75136) using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, #13778150).

Techniques: Derivative Assay, Knockdown, shRNA, Western Blot, Expressing, Transfection, Liquid Chromatography with Mass Spectroscopy, Incubation, Labeling, Control

Journal: Science Advances

Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth

doi: 10.1126/sciadv.adq0334

Figure Lengend Snippet: ( A ) Representative IHC images of human GBM, grade II and III glioma, and normal brain tissues stained with GFAT1, NAGK, and PGM3. Scale bar, 50 μm. ( B ) Comparisons of gene expression levels between GBM tumor and normal brain tissues using the online tool GEPIA2 based on the TCGA and GTEx datasets. * P < 0.05. ( C to F ) IHC staining of the GFAT1, NAGK, and PGM3 in a human glioma TMA. Scale bars, 2 mm (C). Representative images of different levels of anti-GFAT1, anti-NAGK or anti-PGM3 staining and scoring. Scale bar, 200 μm (D). (E) Expression levels were quantified by ImageJ and presented by the heatmap. (F) The expression distribution of these three enzymes in same patients was summarized at the table.

Article Snippet: Transient gene silencing experiments were performed with siRNAs for GFAT1 (Santa Cruz Biotechnology, #sc-60618), GFAT2 (Santa Cruz Biotechnology, #sc-91875), GNPNAT1 (Santa Cruz Biotechnology, #sc-60709), PGM3 (Santa Cruz Biotechnology, #sc-95517), UAP1 (Dharmacon, #L-017160-01-0005), and NAGK (Santa Cruz Biotechnology, #sc-75136) using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, #13778150).

Techniques: Staining, Gene Expression, Immunohistochemistry, Expressing

Journal: Science Advances

Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth

doi: 10.1126/sciadv.adq0334

Figure Lengend Snippet: ( A ) Overall glycosylated proteins in whole lysates of GBM30 cells were probed by the lectin WGA using Western blotting after PGM3 knockdown with shRNA for 72 hours. The signal intensity was quantified by ImageJ. ( B and C ) Representative confocal fluorescence microscopy imaging (B) or flow cytometry analysis (C) of GBM30 cells stained with Rhodamine-labeled PHA-L lectin after PGM3 knockdown with shRNA for 72 hours. Fluorescence intensity was quantified by ImageJ (B) or the mean fluorescence intensity (MFI) was analyzed with the FlowJo V.10.10 software (C). Scale bar, 10 μm. ( D and E ) Western blotting (D) or real-time qPCR analysis (E) of the expression of HBP enzymes in different GBM cells after PGM3 knockdown with shRNA for 72 hours. ( F ) Western blotting analysis of SCAP N-glycosylation levels in GBM30 cells after PGM3 knockdown with shRNA for 72 hours (for details, please see Materials and Method). ( G ) Western blot analysis of membrane extracts for SCAP and PDIA1; nuclear extracts for SREBP-1 N-terminal form (N) and Lamin A; and cytoplasmic proteins FASN, SCD1, and β-actin from different GBM cells after PGM3 knockdown with shRNA for 72 hours. ( H ) Real-time qPCR analysis of gene expression in GBM cells after knockdown of PGM3 with shRNA for 72 hours. For data analysis in (A), (B), (C), (E), and (H), all the results are shown as means ± SD [ n = 30 in (B); n = 3 in (A), (C), (E) and (H)]. The significance was determined by comparing each treatment with the control group (shCon). For (A) to (C), the data were analyzed with one-way ANOVA with Tukey’s test. For (E) and (H), the data were analyzed with two-way ANOVA with Tukey’s test.

Article Snippet: Transient gene silencing experiments were performed with siRNAs for GFAT1 (Santa Cruz Biotechnology, #sc-60618), GFAT2 (Santa Cruz Biotechnology, #sc-91875), GNPNAT1 (Santa Cruz Biotechnology, #sc-60709), PGM3 (Santa Cruz Biotechnology, #sc-95517), UAP1 (Dharmacon, #L-017160-01-0005), and NAGK (Santa Cruz Biotechnology, #sc-75136) using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, #13778150).

Techniques: Western Blot, Knockdown, shRNA, Fluorescence, Microscopy, Imaging, Flow Cytometry, Staining, Labeling, Software, Expressing, Glycoproteomics, Membrane, Gene Expression, Control

Journal: Science Advances

Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth

doi: 10.1126/sciadv.adq0334

Figure Lengend Snippet: ( A ) Representative IHC images of human GBM and normal brain tissues stained with different antibodies against indicated proteins. Scale bar, 50 μm. ( B and C ) Western blotting (B) or real-time qPCR (C) analysis of the protein or mRNA levels in GBM cells after treatment with Fatostatin for 24 hours. ( D and E ) Western blot (D) or real-time qPCR (E) analysis of the protein or mRNA levels in GBM cells after knockdown of SREBP-1 with shRNA for 72 hours. ( F to I ) The schemes showed the putative SREBP-1-binding sites (SREs) on GFAT1 (F), GNPNAT1 (G), PGM3 (H), and UAP1 (I) gene promoters. The DNA fragment located at −9547/−9468-bp upstream of the first exon of PGM3, which was predicted with no SRE, was used as the negative binding sites (NS) for all the four promoters. ChIP-PCR analysis of SREBP-1 binding to SREs located in respective gene promoters in GBM30 or GBM83 cells was shown in columns. ( J ) Promoter reporter luciferase activity for respective gene promoters containing SREs shown in the schemes cloned in the pGL3-basic vector that were transfected into HEK 293FT cells together with Renilla and infected with Ads expressing N-terminal SREBP-1a (Ad-1a N), SREBP-1c (Ad-1c N), or control (Ad-null) virus for 48 hours. The promoter activities were normalized with Ad-null group. For statistical analysis, all the results are shown as means ± SD ( n = 3). Gene expression in (C) was compared by two-tailed Student’s t test, and all the other data were analyzed by two-way ANOVA with Tukey’s test. For (E), the significance was determined by comparing each treatment with the group of shCon. For (J), the significance was determined by comparing each treatment with the group of Ad-null.

Article Snippet: Transient gene silencing experiments were performed with siRNAs for GFAT1 (Santa Cruz Biotechnology, #sc-60618), GFAT2 (Santa Cruz Biotechnology, #sc-91875), GNPNAT1 (Santa Cruz Biotechnology, #sc-60709), PGM3 (Santa Cruz Biotechnology, #sc-95517), UAP1 (Dharmacon, #L-017160-01-0005), and NAGK (Santa Cruz Biotechnology, #sc-75136) using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, #13778150).

Techniques: Staining, Western Blot, Knockdown, shRNA, Binding Assay, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Transfection, Infection, Expressing, Control, Virus, Gene Expression, Two Tailed Test

Journal: Science Advances

Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth

doi: 10.1126/sciadv.adq0334

Figure Lengend Snippet: ( A ) Western blotting analysis of the protein levels in GBM30-luciferase cells after genetic knockdown of GFAT1, NAGK, or PGM3 by shRNA, respectively, for 48 hours. ( B ) Luminescence imaging of intracranial tumor implanted with GBM30 cells described in (A) (5 × 10 4 cells per mouse) at day 18 post-implantation. The luminescent intensities are shown as means ± SD ( n = 6). The data were analyzed by one-way ANOVA with Tukey’s test, and the significance was determined by comparing each treatment with the shCon group. ( C ) H&E staining ( n = 3) of whole brain sections from GBM30 intracranial xenograft mice with different genetic knockdown at day 22 post-implantation. Scale bar, 2 mm. ( D ) GBM30-bearing mouse overall survival was assessed by Kaplan-Meier analysis. The significance was analyzed with log-rank test by comparing the gene knockdown groups with the shCon group. ( E to G ) Western blotting analysis of FLAG–nSREBP-1a expression in GBM30-luciferase cells infected with Ad expressing with/without FLAG-tagged N-terminal active SREBP-1a form after knockdown of PGM3 by lentivirus-mediated shRNA (48 hours). The cells (5 × 10 4 cells per mouse) were implanted into nude mouse brains and examined by luminescence imaging at day 18 post-implantation (F). The luminescent intensities are shown as means ± SD ( n = 6). The significance was determined by Student’s t test. Mouse overall survival was assessed by Kaplan-Meier analysis (G). The significance was analyzed with log-rank test between different groups.

Article Snippet: Transient gene silencing experiments were performed with siRNAs for GFAT1 (Santa Cruz Biotechnology, #sc-60618), GFAT2 (Santa Cruz Biotechnology, #sc-91875), GNPNAT1 (Santa Cruz Biotechnology, #sc-60709), PGM3 (Santa Cruz Biotechnology, #sc-95517), UAP1 (Dharmacon, #L-017160-01-0005), and NAGK (Santa Cruz Biotechnology, #sc-75136) using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, #13778150).

Techniques: Western Blot, Luciferase, Knockdown, shRNA, Imaging, Staining, Expressing, Infection