Journal: Cancer & Metabolism

Article Title: Tissue of origin dictates GOT1 dependence and confers synthetic lethality to radiotherapy

doi: 10.1186/s40170-019-0202-2

Figure Lengend Snippet: GOT1 dependence exhibits tissue specificity. a Schematic of the GOT1 pathway in PDA. b Colony number after dox treatment in PDA (red) and CRC (blue) cell lines expressing dox-inducible (iDox) shRNAs against GOT1 (two independent hairpins; shGOT1 #1, shGOT1 #3) relative to a non-targeting hairpin (shNT). Error bars represent s.d. from biological replicates ( n = 3). Mutations in KRAS , BRAF , and TP53 are presented in the table below the bar graph. WT, wild type; SM, silent mutation. c Western blots (left) and quantification (right) for GOT1 and vinculin (VCL) loading control from iDox-shGOT1 #1 PDA and CRC tumors. d , e Tumor growth curves and f , g final tumor weights from subcutaneous PDA xenografts ( n = 8, BxPC-3 +/−dox tumors; n = 6, PA-TU-8902 +/−dox tumors). Error bars represent s.d. h , i Tumor growth curves and j , k final tumor weights from subcutaneous CRC xenografts ( n = 5, DLD-1 +/−dox, HCT 116 +dox tumors; n = 4, HCT 116 −dox tumors). Error bars represent s.d. Tumor growth curves for the corresponding iDox-shNT lines are presented in Additional file : Figure S2b. l Western blot (left) and quantification (right) for GOT1 pathway components from a in wild-type PDA and CRC cell lines. AcCoA, acetyl-CoA; αKG, alpha-ketoglutarate; Asp, aspartate; Cit, citrate; Fum, fumarate; Glu, glutamate; GOT1, glutamate oxaloacetate transaminase 1; GOT2, glutamate oxaloacetate transaminase 2; Iso, isocitrate; Mal, malate; MDH1, malate dehydrogenase 1; ME1, malic enzyme 1; NADP+, oxidized nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; OAA, oxaloacetate; Pyr, pyruvate; Suc, succinate. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; Student’s t test (unpaired, two-tailed)

Article Snippet: The following antibodies were used: anti-aspartate aminotransferase (anti-GOT1) at a 1:1000 dilution (Abcam, ab171939), anti-GOT2 at a 1:1000 dilution (Atlas Antibodies Sigma-Aldrich, HPA018139), anti-ME1 at a 1:1000 dilution (Santa Cruz, sc-100569), anti-MDH1 at a 1:10,000 dilution (Abcam, ab180152), and loading control vinculin at a 1:10,000 dilution (Cell Signaling Technology, 13901) or GAPDH at a 1:1000 dilution (Cell Signaling Technology, 2118).

Techniques: Expressing, Mutagenesis, Western Blot, Two Tailed Test

Journal: Scientific reports

Article Title: Adipose cell size changes are associated with a drastic actin remodeling.

doi: 10.1038/s41598-019-49418-0

Figure Lengend Snippet: Figure 2. Representative TIRF images of isolated adipocytes from chow or HFD-fed mice (chow, HFD) that were co-stained with phalloidin to detect filamentous actin and different markers of stress fibers, actin organization and focal adhesions (non-muscle myosin (NMM) IIA isoforms MYH9 and MYH11, α-actinin, Filamin A, FAK, Vinculin). Each marker (black) shown in right panel for each condition, and phalloidin (black) shown in left panel for each condition. Scale bar = 20 µm.

Article Snippet: Arp2, Arp3, profilin-1, non-muscle myosin (NMM) IIA (MYH9 and MYH11), Vinculin and Vimentin antibodies were from Abcam (Cambridge, UK), α-actinin, focal adhesion kinase (FAK), IRS-1 total, pS362/365 and pY612, AS160 pT642, MYPT1 pT696, YAP pS127, Akt total and pS473 and pT308, were from Cell Signaling Technologies (Danvers, USA), agarose-conjugated IQGAP1 antibody and, cofilin-1 total and pS3 antibodies were from Santa Cruz (Dallas, USA).

Techniques: Isolation, Staining, Marker

Journal: Nature Communications

Article Title: p38-mediated phosphorylation at T367 induces EZH2 cytoplasmic localization to promote breast cancer metastasis

doi: 10.1038/s41467-018-05078-8

Figure Lengend Snippet: EZH2 and vinculin interact in a phosphorylation-dependent manner. a Proximity ligation images depicting co-localization with the indicated proteins by red fluorescent dots in MDA-MB-231 cells. Scale bars=10 μm. b Quantitative analysis of the direct interaction between recombinant EZH2 and vinculin proteins using BLI. EZH2 was immobilized on a sensor chip, and a concentration series of vinculin protein was added. Sensorgrams and corresponding fitting curves for kinetics constants and affinity determination (left) and corresponding plot of steady state response against concentration (right) for determination of binding affinity. c Co-immunoprecipitation experiment from whole cell extracts demonstrating interaction between endogenous EZH2 and vinculin after treatment with doxycycline to induce p38 activation. d Western blot of MDA-MB-231 cells transduced with dox-inducible MKK6EE to activate p38α activity. e Western blot analysis comparing phospho-vinculin(Y100) in MDA-MB-231 knockdown-rescue WT-EZH2 and T367A-EZH2 cells. f Our working model of pEZH2(T367) function in breast tumorigenesis

Article Snippet: For immunofluorescence imaging and quantitation of phospho-vinculin Y100 focal adhesion, 8-well chambered slides slides (Thermo Fisher Lab-Tek Cat #154534) were first coated with fibronectin (Sigma Fibronectin F0895) per the manufactuer’s coating protocol at a dilution of 2 μg ml −1 , and immunofluorescence was carried out as outlined above.

Techniques: Phospho-proteomics, Ligation, Recombinant, Concentration Assay, Binding Assay, Immunoprecipitation, Activation Assay, Western Blot, Transduction, Activity Assay, Knockdown