Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Schematic depicting generation of the ENZ-driven resistance model. (b) Tumour volume and serum PSA of PSA+ (T49) and PSA− (T42) ENZ-resistant tumours at time following ENZ treatment. (c) Immunoblot of AR and PSA in cell lines derived from CRPC (16DCRPC) and ENZ-resistant AR-driven (49FENZR) and lineage plastic (42DENZR, 42FENZR) tumours. (d) Frequency of activating AR F876L mutation in CRPC and ENZ-resistant cell lines. (e) PCA of the global transcriptome in the indicated cell lines. (f) Significantly enriched (p < 0.05) gene ontology pathways in CRPC and ENZ-resistant cell lines ranked by normalized enrichment score. The following keywords were used to define functional categories: Plasticity (morphogenesis, plasticity, differentiation, mesenchymal); Neuronal (cerebral, axon, synap, neuro); Migration (chemotaxis, migration); Hormone (androgen, hormone); Translation. (g) Expression of ‘Core 9’ embryonic stem cell genes and neuronal lineage markers in the indicated cell lines, reported relative to LNCaP. (h) ASC scores in the indicated prostate cancer cell lines (n = 3) and AR+/NE+ and AR−/NE+ patient tumours from Aggarwal et al. Statistical analysis was performed using a two-tailed unpaired t-test. Error bars represent mean ± SD. (i) Transcript expression of RB1 and TP53 in cell lines and SU2C patient samples with wild-type RB1/TP53 (SU2CWT) or biallelic RB1 and TP53 deletion (SU2CRB1/TP53). An RB1/TP53 signature score was applied to cell lines and tumours (higher score indicative of functional RB1/TP53 loss). (j) Immunoblot of pRB1-S780 in the indicated cell lines. (k) Partial least squares discriminant analysis (PLS-DA) of global transcriptome separates AR+/NE+ and AR−/NE+ patient tumours from the Labrecque et al cohort (AR+/NE+, n = 11; AR−/NE+, n = 11; GEO: GSE126078). RNA-seq data from cell lines were projected on the PLS-DA plot. Probability ellipse=95% confidence. (l) Spheroid formation quantified at 8 days following seeding of single cells from the indicated cell lines (mean ± SD; two-tailed unpaired t-test, n = 3). Phase contrast images are shown. Scale bar, 100 μm. (m) Flow cytometry plots of CD44 and NCAM1 cell surface expression (top) and ALDH activity (bottom) in the indicated cell lines (mean ± SD). Diethylaminobenzaldehyde (DEAB) is as a control for background fluorescence.

Article Snippet: The following antibodies were used for immunoblotting: AR (clone D6F11; 1:1,000; Cell Signaling 5153, lot no. 9), CDK1 (clone POH1; 1:1,000; Cell Signaling 9116, lot no. 7), pCDK1-T161 (1:500; Cell Signaling 9114, lot no. 4), pCDK1-Y15 (1:1,000, Cell Signaling 9111, lot no. 9), EED (1:1,000; Millipore 17–10034, lot no. 3577475), EZH2 (1:2,000; Active Motif 39933, lot no. 2962006), pEZH2-T350 (1:1,000; generated in this study), pEZH2-S21 (1:500; Bethyl, lot no. IHC-00388–6), H3K27Me3 (1:2,000; Millipore 07–449, lot no. 3317006–3170806), Myc tag (1:1,000; Abcam ab9106, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR130480","term_id":"238373784","term_text":"GR130480"}} GR130480 –22), NANOG (1:500; Abcam ab80892, lot no. CR3280357–1), NSE/ENO2 (clone BBS/NC/VI-H14; 1:1,000; Agilent M0873), OCT4 (clone D7O5Z; Cell Signalling 75463 S, lot no. 1), PSA (clone D6B1; 1:5,000; Cell Signaling 5365, lot no. 4), RB1 (Clone IF9, 1:500; Santa Cruz sc-73598, lot no. C0619), pRB1-S780 (1:1,000; Cell Signaling 9307), SOX2 (clone D6D9; 1:1,000; Cell Signaling 3579 S, lot no. QL230817), SUZ12 (clone D39F6; 1:1,000; Cell Signaling 3737 S, lot no. 8). β-actin (clone AC-74; 1:25,000; Sigma A2228, lot no. 00959896) and vinculin (Clone hvin-1; 1:1,000; Cell Signaling 4650, lot no. 118M4777V) were used for loading controls.

Techniques: Western Blot, Derivative Assay, Mutagenesis, Functional Assay, Migration, Chemotaxis Assay, Expressing, Two Tailed Test, RNA Sequencing Assay, Flow Cytometry, Activity Assay, Fluorescence

Journal: Journal of Neuroscience Research

Article Title: The antiviral cytokine interferon‐gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation

doi: 10.1002/jnr.23987

Figure Lengend Snippet: IFNγ modulates the expression of cell cycle checkpoint proteins and the phosphorylation of pRb in NSPCs. A: Expression of cyclins D1, D2, D3, E, and cdk2 were measured using western blot and fluorescence signals were normalized to GAPDH as a loading control. For cyclin D1, the top band (open arrowhead) corresponds to the phosphorylated form of cyclin D1 and the bottom band (closed arrowhead) corresponds to the unphosphorylated form of cyclin D1. B: Expression of total retinoblastoma protein (pRb) and associated pRb phosphorylation at different serine residues (S780, S795, and S807/811) was measured. The fluorescence signal for each band was normalized to GAPDH as a loading control. For pRb S795, normalization was also performed against total pRb. Quantitation of samples is shown as the average with SEM. Statistical analysis was applied using repeated measures one‐way ANOVA with Bonferroni multiple comparisons post‐hoc analysis (****p < 0.0001, *** p < 0.001, **p < 0.01 *p < 0.5; n = 3‐5).

Article Snippet: Primary antibodies used were as follows: anti‐phospho STAT1 (Y701, #612133), anti‐STAT1 (N‐terminus, #610120), anti‐STAT3 (610190) from BD Biosciences; anti‐phospho STAT1 (S727, #9177) anti‐Cyclin D1 (#2978), anti‐Cyclin D3 (#2936); pRb S780 (#8180), pRb S807/811 (#8516), pRb S780 (#9307), Rb‐total (#9313) anti‐phospho STAT3 (#9131) form Cell Signaling Technology; pRb S795 (1:500, ab47474) from Abcam; and anti‐cdk4 (MAB8879), anti‐cdk2 (#07‐631) anti‐cyclin E (#07‐687) from Millipore.

Techniques: Expressing, Phospho-proteomics, Western Blot, Fluorescence, Control, Quantitation Assay

Journal: Journal of Neuroscience Research

Article Title: The antiviral cytokine interferon‐gamma restricts neural stem/progenitor cell proliferation through activation of STAT1 and modulation of retinoblastoma protein phosphorylation

doi: 10.1002/jnr.23987

Figure Lengend Snippet: Characterization of Antibodies Used in Flow Cytometry and Western Blot Analyses.

Article Snippet: Primary antibodies used were as follows: anti‐phospho STAT1 (Y701, #612133), anti‐STAT1 (N‐terminus, #610120), anti‐STAT3 (610190) from BD Biosciences; anti‐phospho STAT1 (S727, #9177) anti‐Cyclin D1 (#2978), anti‐Cyclin D3 (#2936); pRb S780 (#8180), pRb S807/811 (#8516), pRb S780 (#9307), Rb‐total (#9313) anti‐phospho STAT3 (#9131) form Cell Signaling Technology; pRb S795 (1:500, ab47474) from Abcam; and anti‐cdk4 (MAB8879), anti‐cdk2 (#07‐631) anti‐cyclin E (#07‐687) from Millipore.

Techniques: Flow Cytometry, Western Blot, Molecular Weight, Concentration Assay, Recombinant, Infection, Expressing, Immunofluorescence, Virus, Protein-Protein interactions, Derivative Assay, Phospho-proteomics, Activation Assay, Activity Assay, Purification

Journal: Nature medicine

Article Title: Aurora kinase A drives the evolution of resistance to third generation EGFR inhibitors in lung cancer

doi: 10.1038/s41591-018-0264-7

Figure Lengend Snippet: a Immunoblot analysis of total and phosphorylated AURKA in PC9 and H1975 parental and acquired resistant cell lines treated with 1uM of the indicated inhibitors for 24 h. b Mean of cell proliferation of PC9 or H1975 cells transfected with plasmids expressing the indicated genes and treated 1uM EGFR-TKI for 72 h compared to DMSO treated cells performed in n=3 biologically independent samples. Significance based on comparison to LacZ control. c Immunoblot analysis 4 parental and 8 acquired resistant cell lines. Quantified intensities for pAURKA and TPX2 relative to the parental cell line is shown. d Proliferation compared to DMSO of PC9 and H1975 parental or acquired resistant cells treated with 1uM of osimertinib or rociletinib, 30nM of MLN8237 or the combination for 72 h. Mean over n=3 biologically independent samples. e Apoptosis measured by YO-PRO-1 positivity in the same models and drug treatments for 72 h. Shown is mean from n=3 biologically independent samples. f Mean tumor volume (mm 3 ) of PC9-RR xenografts during treatment with rociletinib (100mg/kg), MLN8237 (10mg/kg) or the combination. n=10 tumors in the vehicle and rociletinib arm, and n=7 in the MLN8237 and combination arm. g Percent change in tumor volume compared to baseline for individual PC9-OR cell xenografts teated for 11 days with osimertinib (5mg/kg), MLN8237 (10mg/kg) or the combination. P-value comparing combo to single agent osimertinib treatment. h Immunoblot of lysates from parental PC9, PC9-OR and PC9-RR cells treated with the indicated inhibitors or DMSO for 24 h. i Proposed mechanism for the efficacy of the combination in acquired resistant cells. P-values based two-tailed Student’s t-test. Error bars represent s.e.m. Blots are representative of at least two independent experiments. Full blots shown in .

Article Snippet: Antibodies for pEGFR (Y1068; 3777), pERK1/2 (T202/Y204; 4370), ERK1/2 (9102), pAKT (S473; 4060), AKT (9272), PARP (5625), pAURKA (T288; 3079), AURKA (4718) pRb (S780; 9307), BIM (2933), pBIM (S69; 4585), BAX (5023), Vimentin (5741), p-p65 (S536; 3033), p65 (8242), Pan phospho-AURKA/B/C (T288/T232/T198; 2914), CD44 (3570), Histone H3 (9715) were purchased from Cell Signaling Technology; TPX2 (HPA005487) and pan total AURKA/B/C (HPA002636) were purchased from Sigma; EGFR (SC-03), NEDD9 (sc-33657), AJUBA (sc-398008), PAK1 (sc-16617), CD24 (sc-19585), CD133 (sc-30219), B-tubulin (sc-9104), p53 (sc-126) were purchased from Santa Cruz biotechnology and FZR1/CDH1 (ab3242) from Abcam and V5 tag (46–0705) from Thermofisher.

Techniques: Western Blot, Transfection, Expressing, Comparison, Control, Two Tailed Test

Journal: Clinical Proteomics

Article Title: The impact of ultraviolet- and infrared-based laser microdissection technology on phosphoprotein detection in the laser microdissection-reverse phase protein array workflow

doi: 10.1186/s12014-020-09272-z

Figure Lengend Snippet: Confidence levels for the ten phosphoprotein abundances assessed by reverse phase protein array

Article Snippet: Samples were probed with a total of ten antibodies targeting the phosphorylated forms of Akt S473 (Cell Signaling catalog #9271; 1:100), c-Abl T735 (Cell Signaling catalog #2864; 1:50), EGFR Y1068 (Cell Signaling catalog #2234; 1:50), HER2 Y1248 (Imgenex catalog #90189-1; 1:500), HER3 Y1289 (Cell Signaling catalog #4791; 1:200), ERK1/2 T202/Y204 (Cell Signaling catalog #9101; 1:1000), p70S6K T389 (Cell Signaling catalog #9205; 1:100), PDGFR Y751 (Cell Signaling catalog #3161; 1:50), Rb S780 (Cell Signaling catalog #3590; 1:2000), and RET Y905 (Cell Signaling catalog #3221; 1:100).

Techniques: