Journal: Nature communications

Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis.

doi: 10.1038/ncomms13781

Figure Lengend Snippet: Figure 2 | RAF1 ablation increases the number of cancer progenitor cells. (a) Quantification of Ki67 þ liver cells 8 (top panel) or 12 weeks (bottom panel) after DEN treatment. (b) Foci of altered hepatocytes (FAH) in F/F and Dhep or Dp/np livers isolated 12 weeks after DEN injection. Sections were stained with H&E or with the indicated antibodies. FAH are delimited by dotted circles (n ¼ 3 per genotype). Scale bars, 50 mm. (c) Percentage of cancer progenitor cells (CD44 þ /CD31 Ter119 CD45 ) present in non-aggregate and aggregate fractions of F/F and Dp/np livers, as determined by FACS analysis. Data are represented as mean±s.e.m., *Pr0.05, **Po0.01, ***Po0.005 according to Student’s t test. See also Supplementary Fig. 3.

Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), bcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357(62751) from Abcam; ROKa (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

Techniques: Isolation, Injection, Staining

Journal: Nature communications

Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis.

doi: 10.1038/ncomms13781

Figure Lengend Snippet: Figure 3 | Molecular characterization of RAF1-deficient lesions. (a) Immunoblotting of F/F and Dp/np livers collected 30 weeks after DEN treatment. The plots represents a densitometric quantification of the immunoblot performed using ImageJ. The data are expressed as relative band intensity adjusted to TUBA or ACTB, which serve as loading controls (upper plot). Phosphorylation is expressed as the ratio between the phosphospecific antibody signal and the signal obtained with the protein-specific antibody. In both cases, the data are normalized to the F/F non-tumour samples, which were arbitrarily set as 1. (b) Immunoblot analysis of signaling pathways in xenograft samples (n ¼ 3, analysed 40 days after transplant). The plots show a quantification of the immunoblots performed as described in (a). (c) YAP1 expression in the same patient cohort examined in Fig. 1a. Scale bar, 50 mm. Left panel, representative IHC image. Middle panel, comparison of YAP1 expression in matched tumour and non-tumour tissue. Right panel, YAP1 expression in tumours correlates positively with tumour grade and the ratio of RAF1/YAP1 expression in the same tumour negatively correlates with histological grade. (d) STAT3 expression in the same cohort. Left panel, representative IHC image. Middle panel, comparison of STAT3 expression in matched tumour and non-tumour tissue. Right panel, RAF1/YAP1 expression in the same tumour negatively correlated with the presence of medium-large clusters of STAT3 nuclear staining. Scale bar 50 mm. In (a,b), the data are represented as mean±s.e.m., *Pr0.05, **Po0.01, ***Po0.005 according to Student’s t-test. (c,d) Middle panels, In the box and whiskers plots (Tukey method), the box represents interquartile range, the middle bar the median, and the whiskers extend to 1.5 times the interquartile range. Statistical analysis was done using Wilcoxon signed rank test; the analysis in the right panels represents the Spearman correlation. rs and P values are indicated. See also Supplementary Fig. 4.

Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), bcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357(62751) from Abcam; ROKa (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

Techniques: Western Blot, Phospho-proteomics, Protein-Protein interactions, Expressing, Comparison, Staining

Journal: Nature communications

Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis.

doi: 10.1038/ncomms13781

Figure Lengend Snippet: Figure 4 | Molecular characterization of RAF1-deficient cells. (a) siRNA-mediated RAF1 silencing promotes the proliferation of Hep3B (n ¼ 6), HuH-7 (n ¼ 5) and HepG2 (n ¼ 4) cells and increases the expression of YAP1 and GP130 as well as STAT3 phosphorylation. siRAF1#1 targets the region around nucleotide 721, while siRAF1#2 is a mixture of siRNAs targeting the region from nucleotide 692 to 1,093 in the RAF1 mRNA. (b) PCR and immunoblotting analysis of F/F and RAF1D/D DIH. (c) Morphology (x200 magnification) and molecular characterization (d) of primary hepatocytes (P-HEPS) compared to DIH (AFP, a-fetoprotein; ALB, albumin; TUBA, loading control). The immunoblot is representative of two independent experiments. (e) Proliferation of DIH in decreasing amounts of FBS. (f) Molecular defects of RAF1-deficient P-HEPS and DIH treated with the indicated concentrations of IL6 for 30 min. TUBA serves as loading control. Data are presented as mean±s.e.m. *Pr0.05, **Po0.01, ***Po0.005 according to Student’s t test. See also Supplementary Fig. 5.

Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), bcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357(62751) from Abcam; ROKa (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: Nature communications

Article Title: A cell-autonomous tumour suppressor role of RAF1 in hepatocarcinogenesis.

doi: 10.1038/ncomms13781

Figure Lengend Snippet: Figure 6 | RAF1 ablation correlates with decreased YAP1 and GP130 protein turnover in Hep3B cells, primary hepatocytes (P-HEPS), and DIH. (a-c) qPCR analysis showing the expression of the YAP1 and Gp130 genes in Hep3B (a), P-HEPS (b) and DIH (c). qPCR data represent the mean (±s.e.m.) of three independent experiments; according to Student’s t test. (d-f) Cells were treated with cycloheximide for the indicated amount of time prior to lysis. YAP1 and GP130 expression levels were determined by immunoblotting. A quantification is shown in the right panel; the amount of protein present in each of the untreated samples (normalized to TUBA or ACTB as loading controls) is set as 1.

Article Snippet: The following antibodies were used for immunoblotting (1:1,000 unless otherwise stated): YAP1 (4912, 1:500), pYAP1-S127 (4911), pLATS1-T1079 (8654), LATS1 (3477), pMST1(T183)/MST2(T180) (3681), MST2 (3952), pERK1/2 (9101), ERK1/2 (9102), pSTAT3Y705 (9145), STAT3 (9139), PCNA (2586) all Cell Signaling Technology; GP130 (sc-656), ACTB (sc-1616), pCFL1S3 (sc-12912), ALB (sc-50536), ARAF (sc-408) BRAF (sc-9002), RAF1 (sc-133; all Santa Cruz Biotechnology; RAF1 (610152, 1:500), CD44 (550538), bcatenin (610153) from BD Biosciences; AFP (46799) and pYAP1Y357(62751) from Abcam; ROKa (04-841, Millipore)and TUBA (T9206, Sigma, 1:10,000).

Techniques: Expressing, Lysis, Western Blot

Journal: Molecular cell

Article Title: The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

doi: 10.1016/j.molcel.2018.12.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RAF1 Human Recombinant Protein , Origene , Cat# TP301983.

Techniques: Western Blot, Transduction, In Situ, Recombinant, Lysis, Protease Inhibitor, Staining, Magnetic Beads, Clone Assay, Bicinchoninic Acid Protein Assay, Isolation, Labeling, Viability Assay, RNA Sequencing Assay, Sequencing, Expressing, CRISPR, Mass Spectrometry, Mutagenesis, shRNA, Plasmid Preparation, Luciferase, Software, Blocking Assay