Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: The list of antibodies used for Western blotting.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: The sequences of primers used for real-time PCR.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: (A) Decreasing mRNA expression levels of RASAL1 upon the time of high Pi treatment after 24, 48 and 72 hours. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, *P<0.05, **P<0.01, ***P<0.001). (B) Western blot confirming the decreased protein expression of RASAL1 in Pi treated cells. (C) MeDIP result showing the methylated promoter of RASAL1 along the treatment time, correlated with the reduced expression of RASAL1 in both mRNA and protein level. (D) Ras activity was measured by ELISA assay, untreated cells served as controls. Pi- treated cells showed the hyper-activation of total Ras.

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Expressing, Western Blot, Methylated DNA Immunoprecipitation, Methylation, Activity Assay, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: Cells were treated with Pi 3mM for 72 hours. (A) MeDIP result showing the demethylated promoter of RASAL1 by using PFA, correlated with restored mRNA expression of RASAL1. (B) qPCR analysis showed the restored RASAL1 mRNA expression levels by using phosphate transporter inhibitor (PFA). Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, **P<0.01). (C) ELISA assay indicating the normalization of total Ras with combination of PFA or RAS inhibitor farnesylthiosalicylic acid (FTS). (D) bright-field images showing the morphology change of HCAEC cells cultured under normal control condition, high Pi conditions, and Pi combined with FTS or PFA. Scale bars 25 μm. (E) Western blot analysis showing the expression of endothelial cell marker CD31 and fibroblast cell marker S100A4 in HCAEC cells exposed to normal control condition, high Pi conditions, and Pi combined with FTS or PFA (F) qRT-PCR data showing the mRNA expression levels of EndMT transcriptional factors (SNAIL, SLUG, and TWIST) and FSP1 in HCAEC cells under four conditions indicated above. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, ***P<0.001).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Methylated DNA Immunoprecipitation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Western Blot, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: (A) Simplified schematic showing the human RASAL1 promoter along with exons (black boxes), translational start site (black arrow), locations of RASAL1 chip primer for amplicon1 and amplicon2, and amplicon2 primers serve as negative control chip primer. (B-C) The binding properties of HDAC2 and DNMT1 to the RASAL1 promoter region were analyzed by chromatin immunoprecipitation (ChIP) assay and detected by qRT-PCR in Pi treated (B) or in control cells (C). IgG purified from the same species serve as negative control for ChIP (expression are presented as means ± s.d., n = 3 independent experiments, **P<0.01, ***P<0.001, n.s. no significance).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Negative Control, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, Purification, Expressing

Journal: PLoS ONE

Article Title: DNMT1 and HDAC2 Cooperate to Facilitate Aberrant Promoter Methylation in Inorganic Phosphate-Induced Endothelial-Mesenchymal Transition

doi: 10.1371/journal.pone.0147816

Figure Lengend Snippet: In the physiological conditions, the CpG islands located in RASAL1 promoter are unmethylated (top panel) as indicated by open circles, and RASAL1 is transcriptional active. Under pathological conditions, initially when endothelial cells are exposed to stimulus, such as TGFβ1 or high concentration of Pi resulting in RASAL1 silencing through condensed chromatin structure at RASAL1 promoter mediated by HDAC2. While the RASAL1 promoter remains unmethylated (middle panel) and RASAL1 is transiently silenced. When the cells are continuously exposed to the stimulus, the CpG islands located in RASAL1 promoter are methylated (indicated by filled circles) by DNMT1 recruited through the interaction with HDAC2. Therefore, RASAL1 is permanently silenced due to promoter hypermethylation (lower panel).

Article Snippet: RASAL1 , 46–269 , 1:1000 , ProSci.

Techniques: Concentration Assay, Methylation

Journal: Stem cell research

Article Title: Branched-chain amino acid aminotransferase-1 regulates self-renewal and pluripotency of mouse embryonic stem cells through Ras signaling.

doi: 10.1016/j.scr.2020.102097

Figure Lengend Snippet: Fig. 4. Bcat1 regulates Rasal1 to control the activation of Ras signaling. [A] Heat-map showing genes differentially expressed in two Bcat1−/−and WT E14 cells; [B] Volcano diagram showing gene expression changes in Bcat1−/−cells relative to WT ESCs. Purple plot denotes Rasal1; [C] GO analysis of genes differentially up- regulated in Bcat1−/−relative to WT E14 cells; [D] GSEA analysis of RNA sequencing data that involve the stem cell differentiation and cell development; [E] GO pathway analyses showing differentially up-regulated in Bcat1−/−cells; [F, G] The mRNA and protein levels of Rasal1 in Bcat1−/−[F] and Bcat1-OE cells [G]; [H] Western blotting showing changes in p-Mek and p-Erk in Bcat1−/−and WT E14 cells; **p < 0.01, t-test. See also Supp. Fig. 5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies included those specific for Nanog, Oct4, Sox2, α-tubulin, Rasal1, p-Erk, and p-Mek from Santa Cruz Biotechnology, β-actin from Abmart and Bcat1 from Abcam.

Techniques: Control, Activation Assay, Gene Expression, RNA Sequencing, Cell Differentiation, Western Blot

Journal: Stem cell research

Article Title: Branched-chain amino acid aminotransferase-1 regulates self-renewal and pluripotency of mouse embryonic stem cells through Ras signaling.

doi: 10.1016/j.scr.2020.102097

Figure Lengend Snippet: Fig. 5. Bcat1 deletion alters global DNA methylation. [A] The relative intracellular α-KG levels in WT and BKO cells. [B] Average methylation levels in WT and Bcat1−/−cells. ChrM, mitochondrial DNA; [C] DNA methylation patterns across the entire transcriptional units at whole genome level; [D] Heat-map representation of methylation levels in different genomic regions. Black lines denote the median methylation levels of CpGs at the given local density. Red color gradient indicates abundance of CpGs that fall into bins of given methylation levels and CpG densities. Blue bar charts above each heat-map show distribution of CpG densities. Green bar charts to the right of heat-maps show the distribution of methylation levels; [E] Methylation levels in Rasal1 loci. The green shaped region represents promoter of Rasal1; [F] Bisulfite analysis to detect methylation at the Rasal1 promoter in two BKO cell lines and WT E14 cells; [G] Pathway analyses showing DMRs between WT and BKO 1# cells in coding gene bodies; [H] Pathway analyses showing DMRs between WT and BKO 1# cells in coding gene promoters;**p < 0.01, t-test. See also Supp. Fig. 6. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies included those specific for Nanog, Oct4, Sox2, α-tubulin, Rasal1, p-Erk, and p-Mek from Santa Cruz Biotechnology, β-actin from Abmart and Bcat1 from Abcam.

Techniques: DNA Methylation Assay, Methylation

Journal: Stem cell research

Article Title: Branched-chain amino acid aminotransferase-1 regulates self-renewal and pluripotency of mouse embryonic stem cells through Ras signaling.

doi: 10.1016/j.scr.2020.102097

Figure Lengend Snippet: Fig. 6. Bcat1 function in mouse ESCs is mediated by Rasal1. [A] RNA levels of pluripotency markers in indicated Rasal1 knockdown cells are shown; [B] Repre sentative images of EdU analysis of Rasal1 knockdown in BKO cells (left), and quantification of EdU incorporations based on flow cytometry (right). Scale bar, 50 μm; [C] Representative images of indicated BKO, RKD, and WT E14 cells cultured 2 days in differentiation medium. (Top row) cells cultured in medium lacking 2i only; (bottom row) cells cultured in medium lacking 2i and LIF. Quantification is presented in Supp. Fig 7d. Scale bar represents 50 μm; [D, E] mRNA levels of differ entiation markers in indicated cells in the absence of either 2i [D] or 2i and LIF [E]; [F] Representative images of EdU analysis of Rasal1-OE cells (left), and quantification of EdU incorporations based on flow cytometry (right). Scale bar, 50 μm; [G] RNA levels of pluripotency markers in indicated Rasal1-OE cells or Bcat1- OE E14 cells; [H] Representative images of indicated Bcat1-OE, Rasal1-OE and negative control cells cultured 2 days in differential medium. (Top row) cells cultured in medium lacking 2i only; (bottom row) cells cultured in medium lacking 2i and LIF. Quantifications are presented in Supp. Fig 8b, c. Scale bar, 50 μm; [I, J] mRNA level of differentiation markers in indicated Rasal1- OE, Bcat1-OE and control cells cultured in the absence of either 2i [I] or 2i and LIF [J]. *p < 0.05, **p < 0.01, NS, not significant, t-test. See also Supp. Figs. 7 and 8.

Article Snippet: Antibodies included those specific for Nanog, Oct4, Sox2, α-tubulin, Rasal1, p-Erk, and p-Mek from Santa Cruz Biotechnology, β-actin from Abmart and Bcat1 from Abcam.

Techniques: Knockdown, Flow Cytometry, Cell Culture, Negative Control, Control

Journal: Theranostics

Article Title: Twist1 contributes to developing and sustaining corticosteroid resistance in ulcerative colitis

doi: 10.7150/thno.62256

Figure Lengend Snippet: Higher TW1 and Ras expression in colon tissue neutrophils of UC patients with CR . Surgically removed colon tissues were obtained from UC patients of CS (n=12) and CR (n=34), and NC subjects (n=8). Neutrophils, eosinophils, B cells, T cells and epithelial cells were isolated from the samples by FACS. A, RNAseq results show 15 DEGs in neutrophils. B, heatmaps show gene expression levels between TW1 and other DEGs. C, RNAs were extracted from isolated immune cells and epithelial cells; TW1 mRNA levels were assessed by conventional RT-qPCR. Violin plots show TW1 mRNA levels. ***, p<0.001 (Mann Whitney test), compared with NC group. The data of violin plots are presented as median, IQR and data range, and represent 6 independent experiments.

Article Snippet: TW1 CRISPR/Cas9 KO plasmids, Abs of RASAL1 (B-2), STAT3 (F-2), pSTAT3 (23G5), KRas (F234), CD11b (44; AF488), HRP-His tag (H-3), Ly6G Ab (RB6-8C5; AF594), CD45 (35-Z6, AF790), CD11c (3.9, AF680), CD3 (PS1, AF700), CD19 (B-1, AF594), EpCAM (C-10, AF647) and Farnesyl thiosalicylic acid (FTS) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Expressing, Isolation, Gene Expression, Quantitative RT-PCR, MANN-WHITNEY

Journal: Theranostics

Article Title: Twist1 contributes to developing and sustaining corticosteroid resistance in ulcerative colitis

doi: 10.7150/thno.62256

Figure Lengend Snippet: TW1 interferes with steroid effects on regulating neutrophil activities . A-C, protein extracts were prepared with colon tissue-isolated neutrophils collected from CR UC patients (n = 19), CS UC patients (n = 12), and NC subjects (n = 8), and analyzed by co-IP with antibodies of GRα or TW1 as the precipitation Ab. The immunoblots show a complex of TW1 and GRα (the data are from one experiment that represent 3 independent experiments with pooled protein samples obtained from all cases per group). D-E, proteins were extracted from neutrophils isolated from surgical colon samples were analyzed by Western blotting. Immunoblots show the GRα (D) and GRβ (E) levels in neutrophils. F-G, isolated colon neutrophils were treated with the agents listed below the bar graph. CS: Corticosteroid sensitive UC patients. CR: Corticosteroid resistant UC patients. PMA: PMA in the culture at 50 nM. LPS: LPS in the culture at 250 ng/ml. Dex: Dexamethasone in the culture at 1 µM. TW1d: TW1-deficient neutrophils (by CRISPR). Control: Neutrophils were treated with control CRISPR reagents. The bars show neutrophil elastase (NE) levels in the culture. H, the immunoblots show TW1 protein in neutrophils (the data are from one experiment that represent 3 independent experiments). I, competitive efficiency of binding to GR between cortisol and TW1. ***, p<0.001 (ANOVA followed by the Dunnett's test), compared with the CS alone group. ###, p<0.001 (the Student t -test), compared with the group of neutrophils treated with PMA and Dex. H, The bars show eosinophil cation protein (ECP) levels in the culture supernatant. I, gated FACS plots show apoptotic neutrophils after exposure to PMA in the culture.

Article Snippet: TW1 CRISPR/Cas9 KO plasmids, Abs of RASAL1 (B-2), STAT3 (F-2), pSTAT3 (23G5), KRas (F234), CD11b (44; AF488), HRP-His tag (H-3), Ly6G Ab (RB6-8C5; AF594), CD45 (35-Z6, AF790), CD11c (3.9, AF680), CD3 (PS1, AF700), CD19 (B-1, AF594), EpCAM (C-10, AF647) and Farnesyl thiosalicylic acid (FTS) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Isolation, Co-Immunoprecipitation Assay, Western Blot, CRISPR, Control, Binding Assay

Journal: Theranostics

Article Title: Twist1 contributes to developing and sustaining corticosteroid resistance in ulcerative colitis

doi: 10.7150/thno.62256

Figure Lengend Snippet: TW1 is involved in CR development in neutrophils in the mouse colon mucosa . Mice were treated with hypoxia or normoxia for one week. Neutrophils were isolated from the colon tissues by FACS. A-B, the violin plots show TW1 mRNA levels in neutrophils. The immunoblots show TW1 proteins in neutrophils. C-E, protein extracts of neutrophils were analyzed by Western blotting (C, D) and co-IP (E). Immunoblots show the protein levels of GRα (C), GRβ (D) and complexes of TW1/GRα, but not TW1/GRβ (E). F-G, isolated neutrophils were cultured overnight in the conditions denoted below the violin plots. PMA: PMA in the culture (50 nM). LPS: LPS in the culture (250 ng/ml). Dex: Dex in the culture (1 µM). TW1/KO: Mice with TW1 condition knockout neutrophils. TW1/KO/1: The TW1-knockout was initiated before hypoxia treatment. TW1/KO/2: The TW1-knockout was activated after the hypoxia treatment. The violin plots indicate the NE levels in the culture supernatant. The data of violin plots are presented as median (IQR). Each bubble in violin plots presents data obtained from one experiment. ***, p<0.001, compared with the normoxia group [the Student t -test (A) and ANOVA followed by the Dunnett's test (F, G)]. ###, p<0.001, compared with the group treated with hypoxia/PMA/Dex (F), or hypoxia/LPS/Dex (G). The data of B-E are from one experiment, respectively; each of them represents 6 independent experiments.

Article Snippet: TW1 CRISPR/Cas9 KO plasmids, Abs of RASAL1 (B-2), STAT3 (F-2), pSTAT3 (23G5), KRas (F234), CD11b (44; AF488), HRP-His tag (H-3), Ly6G Ab (RB6-8C5; AF594), CD45 (35-Z6, AF790), CD11c (3.9, AF680), CD3 (PS1, AF700), CD19 (B-1, AF594), EpCAM (C-10, AF647) and Farnesyl thiosalicylic acid (FTS) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Isolation, Western Blot, Co-Immunoprecipitation Assay, Cell Culture, Knock-Out

Journal: Theranostics

Article Title: Twist1 contributes to developing and sustaining corticosteroid resistance in ulcerative colitis

doi: 10.7150/thno.62256

Figure Lengend Snippet: Ras activation sustains TW1 expression in neutrophils of the colon tissues . A-E, neutrophils were isolated from blood samples obtained from healthy subjects and cultured with the treatment denoted below the bars of panel A. IL-6: 50 ng/ml. TGF-β: 10 ng/ml. FTS: 75 µM. A, TW1 mRNA levels in neutrophils. B, TW1 protein levels in neutrophils. C-E, bars show levels of Ras.GTPase, Ras.GDPase and Ras activation in neutrophils. F-H, neutrophils were isolated from surgically removed colon tissues of patients with UC and colon cancer, including subjects with corticoid resistance (CR group; n=19), or corticoid sensitiveness (CS group; n=12). The cells were analyzed by Ras-specific ELISA. The bars show levels of Ras.GTPase, Ras.GDPase and Ras activation in neutrophils. I, the immunoblots show STAT3 phosphorylation in naive neutrophils after the treatment denoted below the bars. J, the immunoblots show a protein complex of STAT3/RASAL1 in neutrophils. K, the bars show the binding efficacy of recombinant (r) RASAL1 to rRas after exposing to rSTAT3 at gradient concentrations. L, the bars show NE levels in culture supernatant. M, a flow chart show effect-flow between Ras, TW1 and RASAL1. Harmine: Harmine (a TW1 inhibitor; 10 µM) in the culture. * p<0.05, ** p<0.01, *** p<0.001, compared with the saline group (A, C, D, E, L) or the CS group (F, G, H) or the 0 group (K). Statistical methods: ANOVA followed by the Dunnett's test (A, C, D, E, K, L) or the Mann Whitney test (F, G, H). The data of B, I, J are from one experiment, respectively, that represent 3 independent experiments.

Article Snippet: TW1 CRISPR/Cas9 KO plasmids, Abs of RASAL1 (B-2), STAT3 (F-2), pSTAT3 (23G5), KRas (F234), CD11b (44; AF488), HRP-His tag (H-3), Ly6G Ab (RB6-8C5; AF594), CD45 (35-Z6, AF790), CD11c (3.9, AF680), CD3 (PS1, AF700), CD19 (B-1, AF594), EpCAM (C-10, AF647) and Farnesyl thiosalicylic acid (FTS) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Activation Assay, Expressing, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Binding Assay, Recombinant, Saline, MANN-WHITNEY

Journal: Theranostics

Article Title: Twist1 contributes to developing and sustaining corticosteroid resistance in ulcerative colitis

doi: 10.7150/thno.62256

Figure Lengend Snippet: Inhibition of TW1 restores CS in the colon mucosa . A murine CR-UC model was developed by treating BALB/c mice with DSS and hypoxia. A, representative histology images show colon tissue structures. DSS/hypoxia: Mice were treated with DSS and hypoxia. Dex: Mice were treated with dexamethasone (5 mg/kg, i.p.) daily for 7 days (day 8-day 14). TW1KO: Mice with TW1-knockout neutrophils. Harmine: Mice were treated with harmine (10 mg/kg, i.p.) daily for 7 days (day 8-day 14). B, the bars show CD45 + cell counts (by FACS) in LPMCs isolated from the colon tissues. C, the curves show the body weight changes in the experimental period. D-I, the bars show inflammatory cytokines in the colon tissue protein extracts (by ELISA). J-K, neutrophils were isolated from the colon tissues and stimulated by PMA (J) or LPS (K) in the culture. The bars show NE levels in the culture supernatant. L, the gated FACS plots show neutrophil counts in LPMCs isolated from the colon tissues. M, the bars show summarized neutrophil counts from 6 mice per group. *, p<0.01 (ANOVA followed by the Dunnett's test), compared with group a. The group labels of subpanel B-M are the same as those in subpanel A.

Article Snippet: TW1 CRISPR/Cas9 KO plasmids, Abs of RASAL1 (B-2), STAT3 (F-2), pSTAT3 (23G5), KRas (F234), CD11b (44; AF488), HRP-His tag (H-3), Ly6G Ab (RB6-8C5; AF594), CD45 (35-Z6, AF790), CD11c (3.9, AF680), CD3 (PS1, AF700), CD19 (B-1, AF594), EpCAM (C-10, AF647) and Farnesyl thiosalicylic acid (FTS) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

Techniques: Inhibition, Knock-Out, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Summary of the cancer types and frequencies of the genetic alterations of RASAL1 in 33 types of cancer. (A) Cancer types studied from TCGA database and their full names and corresponding abbreviations. The value in the parentheses represents the number of cases. (B) Presentation of the frequencies of genetic alterations of RASAL1 . For each cancer type, yellow bar represents the collective frequency of mutation and copy number loss of RASAL1 , the blue bar represents the frequency of copy number loss, and the green bar represents the frequency of mutations. The first group, labeled as “Total,” shows the overall frequencies of genetic alterations of RASAL1 among the 33 types of cancer of the entire cohort of the patients. (C) Genes whose mutations showed concurrence with RASAL1 alterations. Shown are 11 genes whose mutations frequencies were > 5% and concurred with RASAL1 alterations in all cancer samples. The width of the line linking each gene pair is calculated by the −log ( P ) ( P is the adjusted P value calculated for the strength of concurrence between the pair of genes by fisher exact test). The more significant the adjusted P value is, the wider the line linking the pair of genes is. The abbreviations of the cancer names are as defined in Panel A and Table .

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Mutagenesis, Labeling

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Association between RASAL1 alterations and the PI3K/AKT pathway activation and poor clinical outcomes of cancer. (A) Association between RASAL1 alterations and AKT phosphorylation (two‐sided Student's t ‐test). Shown are the results in breast cancer, prostate adenocarcinoma and thymoma. The thick black bar in the middle represents the quartile range and the thin black line extending from it represents the 95% confidence interval. (B) Association between RASAL1 alterations and AKT phosphorylation in patients without PTEN and TP53 mutations in breast cancer and lung adenocarcinoma (two‐sided Student's t ‐test). (C) Association between RASAL1 alterations and disease‐specific mortality on the analyses of all patients or patients without PTEN and TP53 alterations (chi‐squared test). (D) Effects of RASAL1 alterations on Kaplan–Meier disease‐specific survival curves on the analysis of all patients. (E) Effects of RASAL1 alterations on Kaplan–Meier disease‐specific survival curves on the analysis of patients without PTEN and TP53 alterations. (F) Effects of RASAL1 alterations on disease progression rates on the analyses of all patients or patients without PTEN and TP53 alterations (chi‐squared test). (G) Effects of RASAL1 alterations on Kaplan–Meier disease progression‐free survival curves on the analysis of all patients. (H) Effects of RASAL1 alterations on Kaplan–Meier disease progression‐free survival curves on the analysis of patients without PTEN and TP53 alterations. Definitions of genotypes: no RASAL1 , no RASAL1 alterations; RASAL1 , there were RASAL1 alterations; no PTEN , no PTEN alterations; no TP53 , no TP53 alterations; no PTEN & TP53 , no PTEN and TP53 alterations; no RASAL1 _no PTEN _no TP53 , no alterations in RASAL1 , PTEN and TP53 ; RASAL1 _no PTEN _no TP53 , there were RASAL1 alterations, but no PTEN and TP5 3 alterations. The name abbreviations for various cancer types are as defined in Fig. and Table .

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Activation Assay, Phospho-proteomics, Biomarker Discovery

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Cooperation of concurrent RASAL1 and PTEN alterations in synergistically activating the PI3K pathway (AKT phosphorylation) and promoting aggressive clinical outcomes. (A) Cooperative effects of concurrent RASAL1 and PTEN alterations on AKT phosphorylation in breast cancer, cervical cancer, and low‐grade glioma patients without TP53 mutations (two‐sided Student's t ‐test). The thick black bar in the middle represents the quartile range and the thin black line extending from it represents the 95% confidence interval. (B) Cooperative effects of concurrent RASAL1 and PTEN alterations on disease‐specific mortality rates and disease progression rates on the analyses of all cancer patients (chi‐squared test). (C) Cooperative effects of concurrent RASAL1 and PTEN alterations on Kaplan–Meier disease‐specific survival curves in patients with breast cancer or cervical cancer patients without TP53 mutations. (D) Cooperative effects of concurrent RASAL1 and PTEN alterations on Kaplan–Meier disease‐specific survival curves and disease progression‐free survival curves on the analyses of all cancer patients. (E) Status of ER, PR, and HER2 expression with various genotypes of RASAL1 , PTEN , and TP53 alterations in breast cancer (chi‐squared test). (F) Distribution of breast cancer subtypes among various genotypes of RASAL1 , PTEN and TP53 alterations (chi‐squared test). (G) Kaplan–Meier disease‐specific survival curves and disease progression‐free survival curves with various genotypes of RASAL1 , PTEN , and TP53 alterations on the analyses of all cancer patients. (H) Kaplan–Meier disease‐specific survival curves in various genotypes of RASAL1 , PTEN and TP53 alterations in patients with chromophobe renal cell carcinoma or uterine corpus endometrial carcinoma. The definitions of various genotypes of RASAL1 , PTEN , and TP53 alterations are as defined in Fig. .

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Phospho-proteomics, Biomarker Discovery, Expressing

Journal: Molecular Oncology

Article Title: The genetic duet of concurrent RASAL1 and PTEN alterations promotes cancer aggressiveness by cooperatively activating the PI3K – AKT pathway

doi: 10.1002/1878-0261.13701

Figure Lengend Snippet: Genetic knockout mice show the tumor suppressor role of RASAL1 and the cooperation between RASAL1 and PTEN defects in driving oncogenesis and cancer aggressiveness, as found in humans. (A) Generation of the global Rasal1 ‐knockout mouse model and colony development. (B) Illustration of the location of two candidate gRNAs within Exon 2 of mRasal1 gene. (C) All mice ( n = 150) were genotyped for the Rasal1 gene using PCR sequencing. Representative PCR amplification patterns demonstrate a single upper band (398 bp) in wild‐type Rasal1 +/+ mice, a single lower band (351 bp) in nullizygous Rasal1 −/− mice, and both bands in hemizygous Rasal1 +/− mice. (D) Western blotting analyses ( n = 9) of the expression levels of several signaling proteins in various Rasal1 genotypes. (E) Pie chart summary ( n = 40, 74, 15 and 21, respectively) of the frequencies of pathological conditions in various knockout genotype mice. (F) Representative microscopic images of pathological conditions (thyroid hyperplasia ( n = 17), thyroid cancer ( n = 11), metastatic thyroid cancer in lungs ( n = 6), and uterine adenocarcinoma ( n = 2)) that developed in knockout mice. The scale bars represent 50 μm. (G) Schematic illustration of the mechanistic model in which concurrent RASAL1 and PTEN alterations can cooperatively activate the PI3K pathway to promote oncogenesis and cancer aggressiveness. In this mechanism, the defect of RASAL1 results in retention of RAS‐GTP, thus maintaining constitutive RAS activation; this initiates the signaling of the PI3K‐AKT pathway, which cannot be terminated in the presence of PTEN defects.

Article Snippet: A Rasal1 knockout (KO) mouse model was created using CRISPR‐CAS technology (Applied StemCell, Inc.).

Techniques: Knock-Out, Sequencing, Amplification, Western Blot, Expressing, Activation Assay