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