Journal: Oncotarget

Article Title: MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

doi: 10.18632/oncotarget.10709

Figure Lengend Snippet: Genes showing two-fold differential expression 30 hours after depletion of MYCN or TFAP4 by siRNA knockdown in BE(2)-C neuroblastoma cells

Article Snippet: TaqMan ® Assays for qPCR were: TFAP4 (Hs00231478_m1), MYCN (Hs00232074_m1); SDC1 (Hs00896423_m1); PRPS2 (Hs00267624_m1); SLC7A6 (Hs00938056_m1); CYLD (Hs00211000_m1); MFSD6 (Hs00214462_m1); FMR1 (Hs00924547_m1); C SGALNACT2 (Hs00603821_m1); DENND5B (Hs00958915_m1); SSU72 (Hs00982637_m1); LCORL (Hs00766084_m1); FAM73A (Hs01594834_m1), C19orf12 (Hs01107514_m1); OIP5-AS1 (Hs01587688_g1); COPS8 (Hs00991301_g1); HPRT (Hs02800695_m1); GUSB (Hs00939627_m1); PPIA (Hs99999904_m1). qPCR assays for TFAP4 expression using SYBR ® Green were performed using published primers [ ]. qPCR assays for EMT-associated genes using SYBR ® Green were performed using primers listed in .

Techniques: Quantitative Proteomics, Knockdown, Migration, Inhibition, De-Phosphorylation Assay, Mutagenesis, Ubiquitin Proteomics

Journal: Oncotarget

Article Title: MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

doi: 10.18632/oncotarget.10709

Figure Lengend Snippet: ( A – D ) Western blots of PRPS2 and SDC1 expression in BE(2)-C and SH-EP/S1 48 h after suppression of MYCN or TFAP4 or 72 h after suppression of MYCN expression in SH-EP/TET21/N cells ( n ≥ 2). ( E ) Quantitative ChIP assays on BE(2)-C cells. Fold enrichment is relative to the preimmune serum. Mean ± S.E. ( n = 5) in which each region was amplified by qPCR in triplicate. Bent arrow: transcriptional start site (TSS); Dist. (Distal region); grey arrow: TFAP4 binding site; black arrow: E-box (MYCN binding site); black boxes: amplicons indicated with a capital letter. The chromosome and coordinates (bp) are provided. ( F ) Luciferase activity was determined following transfection of reporter constructs into BE(2)-C transfected with TFAP4 and/or MYCN siRNA, or control siRNA. *** P < 0.001.

Article Snippet: TaqMan ® Assays for qPCR were: TFAP4 (Hs00231478_m1), MYCN (Hs00232074_m1); SDC1 (Hs00896423_m1); PRPS2 (Hs00267624_m1); SLC7A6 (Hs00938056_m1); CYLD (Hs00211000_m1); MFSD6 (Hs00214462_m1); FMR1 (Hs00924547_m1); C SGALNACT2 (Hs00603821_m1); DENND5B (Hs00958915_m1); SSU72 (Hs00982637_m1); LCORL (Hs00766084_m1); FAM73A (Hs01594834_m1), C19orf12 (Hs01107514_m1); OIP5-AS1 (Hs01587688_g1); COPS8 (Hs00991301_g1); HPRT (Hs02800695_m1); GUSB (Hs00939627_m1); PPIA (Hs99999904_m1). qPCR assays for TFAP4 expression using SYBR ® Green were performed using published primers [ ]. qPCR assays for EMT-associated genes using SYBR ® Green were performed using primers listed in .

Techniques: Western Blot, Expressing, Amplification, Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Control

Journal: Oncotarget

Article Title: MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

doi: 10.18632/oncotarget.10709

Figure Lengend Snippet: ( A – B ) Suppression of PRPS2 or SDC1 resulted in reduction of colony forming ability in BE(2)-C cells. ( C – D ) SDC1 suppression significantly decreased migration, while PRPS2 suppression showed no significant effect. Mean ± SD ( n = 3), ** P < 0.01.

Article Snippet: TaqMan ® Assays for qPCR were: TFAP4 (Hs00231478_m1), MYCN (Hs00232074_m1); SDC1 (Hs00896423_m1); PRPS2 (Hs00267624_m1); SLC7A6 (Hs00938056_m1); CYLD (Hs00211000_m1); MFSD6 (Hs00214462_m1); FMR1 (Hs00924547_m1); C SGALNACT2 (Hs00603821_m1); DENND5B (Hs00958915_m1); SSU72 (Hs00982637_m1); LCORL (Hs00766084_m1); FAM73A (Hs01594834_m1), C19orf12 (Hs01107514_m1); OIP5-AS1 (Hs01587688_g1); COPS8 (Hs00991301_g1); HPRT (Hs02800695_m1); GUSB (Hs00939627_m1); PPIA (Hs99999904_m1). qPCR assays for TFAP4 expression using SYBR ® Green were performed using published primers [ ]. qPCR assays for EMT-associated genes using SYBR ® Green were performed using primers listed in .

Techniques: Migration

Journal: Nature Chemical Biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O -GlcNAcylation

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: a , The scheme of PRPS-catalyzed reaction showing the metabolic pathway of de novo synthesis of nucleotides and NAD production from glucose-derived pentose phosphate pathway. b , c , H1299 cells were transfected with the indicated siRNAs and cultured with 1 μCi of 14 C-glucose. The de novo synthesized 14 C-RNA ( b ) or 14 C-DNA ( c ) was measured by liquid scintillation counting. Immunoblot with the indicated antibodies was performed to confirm the knockdown efficiency of siRNAs targeting OGT ( n = 3). d – g , The indicated siRNAs were introduced into H1299 cells. Cells were incubated in medium containing 13 C 6 -glucose. High-resolution LC–MS/MS was used to analyze 13 C 6 -labeled nucleotide monophosphates ( d ), R5P ( e ), PRPP ( f ) and NAD ( g ) ( n = 3). h , i , The mRNA levels ( h ) ( n = 4) and protein levels ( i ) ( n = 4) of PRPS1 and PRPS2 in H1299 and A549 cell lines were measured by qPCR and immunoblot analyses, respectively. j , The WT, PRPS1 knockout, PRPS2 knockout and double-knockout H1299 cells were incubated in medium containing 13 C 6 -glucose. LC–MS/MS was conducted to measure 13 C-PRPP levels ( n = 3). The immunoblotting was conducted with indicated antibodies. k , FLAG-PRPS1 was purified from indicated shRNA-transduced H1299 cells and subjected to PRPS1 enzymatic activity assay. The intensity of FLAG-PRPS1 immunoblot was calculated to quantify PRPS1 activity ( n = 3). Each error bar represents mean ± s.e.m. A two-tailed Student’s t -test was employed for statistical evaluation.

Article Snippet: The sgRNA oligonucleotide targeting human PRPS2 (Addgene, 78005) was obtained from Addgene.

Techniques: Derivative Assay, Transfection, Cell Culture, Synthesized, Western Blot, Knockdown, Incubation, Liquid Chromatography with Mass Spectroscopy, Labeling, Knock-Out, Double Knockout, Purification, shRNA, Enzyme Activity Assay, Activity Assay, Two Tailed Test

Journal: Nature Chemical Biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O -GlcNAcylation

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: a , b , The indicated siRNAs were introduced into A549 cells. Cells were incubated in medium containing 13 C 6 -glucose. LC/MS-MS analysis was performed to measure the levels of 13 C 6 labeled nucleotide monophosphates ( a ) and PRPP ( b ). (n = 3). c , The wild-type, PRPS1 knockout, PRPS2 knockout, and double knockout A549 cells were incubated in the medium containing 13 C 6 -glucose. LC/MS-MS analysis was performed to measure the incorporation of 13 C 6 -glucose into intracellular PRPP . The proteins purified from the cells were subject to immunoblot analyses with the indicated antibodies. (n = 3). d , The indicated plasmids were introduced into H1299 cells. FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assay. Immunoblotting was conducted with indicated antibodies. The intensity of FLAG-PRPS1 immunoblots was used to quantify PRPS1 activity. (n = 3). e , f , The indicated shRNAs were introduced into HEK293T cells (n = 3) ( e ). Myc-vector or Myc- OGT plasmids were introduced into HEK293T cells (n = 3) ( f ). The cells from all groups were transfected with FLAG-PRPS1 plasmids. FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assay. The intensity of FLAG-PRPS1 immunoblots was used to quantify PRPS1 activity. g , The indicated siRNAs were introduced into H1299 cells. Immunoblotting was conducted with indicated antibodies. The relative protein expression were quantified. (n = 3). h , i , The FLAG-PRPS1 plasmids were introduced into HEK293T cells. The cells were cultured with or without glucose for 24 h (n = 3) ( h ) or treated with or without 50 µM OSMI-1 for 24 h (n = 4) ( i ). FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assay. Immunoblotting was conducted with indicated antibodies. Intensities of FLAG-PRPS1 immunoblots were used to quantify the PRPS1 activity. Each error bar represents mean±SEM. Statistical analysis was done using a two-tailed Student’s t-test.

Article Snippet: The sgRNA oligonucleotide targeting human PRPS2 (Addgene, 78005) was obtained from Addgene.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Labeling, Knock-Out, Double Knockout, Purification, Western Blot, Enzyme Activity Assay, Activity Assay, Plasmid Preparation, Transfection, Expressing, Cell Culture, Two Tailed Test

Journal: Nature Chemical Biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O -GlcNAcylation

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: a , Reciprocal endogenous immunoprecipitation (IP) assay between OGT and PRPS1 in HEK293T cells ( n = 3). b , GST, GST-OGT and GST-PRPS1 proteins were expressed and purified from E. coli and subjected to in vitro pulldown of endogenous PRPS1 or OGT from HEK293T cells ( n = 3). c – f , The indicated siRNAs were introduced into H1299 cells ( c ); H1299 cells were then cultured with or without glucose for 24 h ( d ), transfected with or without HA-OGT plasmids ( e ) and transfected with or without HA-OGA plasmids ( f ). Cells from all groups were transfected with vector or FLAG-PRPS1 plasmids. IP and immunoblot analyses were conducted with anti-FLAG agarose beads and the indicated antibodies ( n = 3). g , h , PRPS1 O -GlcNAcylation analysis using a chemoenzymatic labeling method in HEK293T cells. g , Immunoblot of O -GlcNAcylated PRPS1 in elution and total PRPS1 in input. h , The indicated siRNAs were introduced into HEK293T cells. Immunoblotting was conducted with indicated antibodies ( n = 3). i , j , The recombinant proteins of GST-PRPS1 and GST-PRPS2 and enzymatic His-OGT domain (aa 323–1,041) were incubated in the in vitro O -GlcNAcylation assay reaction buffer. PRPS1 ( i ) and PRPS2 ( j ) O -GlcNAcylation was analyzed by the chemoenzymatic labeling method. Immunoblot analyses and Coomassie blue staining were performed ( n = 3). k , The indicated plasmids were introduced into HEK293T cells. FLAG-PRPS1 was immunoprecipitated and subjected to immunoblot analyses with indicated antibodies ( n = 4). l , HEK293T cells were genetically modified as indicated and treated with or without thiamet-G (2 μM) for 3 h. IP and immunoblotting were conducted with anti-FLAG agarose beads and indicated antibodies ( n = 3).

Article Snippet: The sgRNA oligonucleotide targeting human PRPS2 (Addgene, 78005) was obtained from Addgene.

Techniques: Immunoprecipitation, Purification, In Vitro, Cell Culture, Transfection, Plasmid Preparation, Western Blot, Labeling, Recombinant, Incubation, Staining, Genetically Modified

Journal: Nature Chemical Biology

Article Title: Direct stimulation of de novo nucleotide synthesis by O -GlcNAcylation

doi: 10.1038/s41589-023-01354-x

Figure Lengend Snippet: a - c , OGT mRNA levels in lung cancer and normal tissues, and the correlation with lung cancer malignancy and metastasis based on data analyses through ULCAN platform. Relative OGT mRNA levels in subgroups of lung adenocarcinoma and lung squamous cell carcinoma samples and in normal tissues ( a ); Relative OGT mRNA levels in the normal samples and patient samples of Stages 1-4 lung adenocarcinoma ( b ); Relative OGT mRNA levels according to nodal metastasis status in patients with lung adenocarcinoma ( c ). The box-whisker plots show interquartile ranges, namely minimum, first quartile, median, third quartile, and maximum values; Two-tailed Welch’s T-test. d , OGT , PRPS1 , and PRPS2 mRNA levels in normal adjacent tissue (N) and tumor tissue (T) from lung cancer patients. Each error bar represents mean±SEM. A two-tailed Student’s t-test was employed for statistical analysis. e , PRPS enzymatic activity in lung tumor tissue (T) compared with normal adjacent tissues (N) from lung cancer patients (n = 8). Two-tailed Wilcoxon test was performed for the paired samples. f , PRPS1 O -GlcNAcylation analysis in lung tumor tissue (T) and normal adjacent tissues (N) using chemoenzymatic labeling method. O -GlcNAcylated PRPS1 was blotted in elution and total PRPS1 in input (left panel); PRPS1 O -GlcNAcylation levels were calculated (right panel). (n = 3) Each error bar represents mean±SEM. Two-tailed Student’s t-tests were utilized for statistical analysis.

Article Snippet: The sgRNA oligonucleotide targeting human PRPS2 (Addgene, 78005) was obtained from Addgene.

Techniques: Whisker Assay, Two Tailed Test, Activity Assay, Labeling

Journal: RNA Biology

Article Title: HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer

doi: 10.4161/rna.25274

Figure Lengend Snippet: Figure 5. Identification of HuR metabolic target mRNAs through ribonucleotide immunoprecipitation assays. mRNA enriched by HuR immunoprecipitation were compared with an IgG control sample in MiaPaCa2 cells with an 84-gene glucose metabolism qPCR Array. (A) Heatmap/clustergram: red represents increased transcript levels with HuR RNP-IP compared with control, and green represents decreased levels. Genes enriched in the HuR sample at levels 4-fold or greater than the IgG sample are highlighted with a purple bar to the left of the heat map. (B) Eleven HuR targets are presented and grouped by pathway. RNA levels represent fold-change enrichment to HuR, relative to IgG. Data are normalized to GAPDH and the red dashed line (also present in panels C and D) indicate the predetermined cutoff used to define highly enriched HuR targets (greater than 4-fold enrichment in the HuR sample over IgG control). IDH1 was grouped with TCA cycle enzymes for the purposes of presentation, but technically is an isoenzyme of mitochondrial IDH and resides in the cytosol. GPI, IDH1 and PRPS2 were validated as HuR-bound mRNA targets by HuR RNP-IP and q-PCR in (C) BxPC3 and (D) PANC1 cell lines. HuR RNP-IP (gray bars) and IgG RNP-IP (black bars). PCR Arrays were normalized to GAPDH. qPCR assays were normalized to 18S.

Article Snippet: 30 Immunoblotting was also used to validate metabolic enzymes [e.g., GPI (TA501137), PRPS2 (TA308129), and IDH1(TA500610); OriGene Technologies], as well as GLUT1 (SLC2A1: Santa Cruz Biotechnology, sc-1605).

Techniques: Immunoprecipitation

Journal: Nature Communications

Article Title: Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor

doi: 10.1038/ncomms11457

Figure Lengend Snippet: ( a ) Immunoblot of HeLa lysates 48 h post doxycycline-induced expression of scrambled shRNA or ASNS shRNA in asparagine-free DMEM (−Asn) or DMEM supplemented with 0.1 mM asparagine (+Asn). Lysates were immunoblotted for mTOR activity marker pS6K, phospho-CAD (S1859), total CAD and tubulin. ( b ) Immunoblot of HeLa lysates with (shASNS+Dox) and without (shASNS−Dox) a 48-h induction of ASNS shRNA expression and HeLa lysates 48 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA (shASNS). Lysates were immunoblotted for ASNS, PRPS1, PRPS2 and tubulin. ( c ) Relative levels of asparagine and PRPP extracted from HeLa cells 48 and 72 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA expression as measured by LC-MS. ( d ) Percentages of the indicated metabolites labelled with 15 N extracted from HeLa cells labelled with exogenous 15 N-serine (50:50 15 N:14 N) for 24 h at 24 h post induction of scrambled shRNA (Scr) or ASNS shRNA expression. ( e ) Relative levels of the indicated intracellular metabolites extracted from HeLa cells 72 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA expression as measured by LC-MS. Error bars denote s.d. of the mean ( n =3). P values were calculated by the Student's t -test: * P <0.05; ** P <0.01; *** P <0.001. NS, not significant.

Article Snippet: Western blot analysis was performed using standard protocols, and the following commercial antibodies were used as probes: ASNS (Proteintech 14681-1-AP, 1:1,000), phospho-T389 S6 kinase (Cell Signaling 9234, 1:500), S6 kinase (Cell Signaling 2708, 1:1,000), phospho-S235/235 S6 ribosomal protein (Cell Signaling 4858, 1:3,000), S6 ribosomal protein (Cell Signaling 2217, 1:1,000), LC3A/B (Cell Signaling 4108, 1:1,000), PHGDH (Cell Signaling 13428, 1:1,000), PSAT1 (Abnova H00029968-A01, 1:500), PSPH (Sigma HPA020376, 1:500), SHMT1 (Abcam ab55736, 1:1,000), SHMT2 (Cell Signaling 12762, 1:1,000), PRPS2 (Abnova H00005634-A01, 1:1,000), 4E-BP1 (Cell Signaling 9452, 1:1,000) and α-tubulin (Sigma T6074, 1:10,000).

Techniques: Western Blot, Expressing, shRNA, Activity Assay, Marker, Liquid Chromatography with Mass Spectroscopy