Journal: BMC Cancer

Article Title: Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion

doi: 10.1186/s12885-019-5527-2

Figure Lengend Snippet: 37 lung cancer cell lines

Article Snippet: 19 , NCI-H460 , LC , KRAS Q61H, PIK3CA E545K 1 , ATCC , HTB-177 , 10%FBS-RPMI1640.

Techniques: Mutagenesis, Amplification

Journal: Molecular Cancer Research

Article Title: AKT1 E17K Inhibits Cancer Cell Migration by Abrogating β-Catenin Signaling

doi: 10.1158/1541-7786.mcr-20-0623

Figure Lengend Snippet: Figure 4. AKT2, but not AKT1, increases the transcriptional activity of b-catenin on the ZEB1 promoter, and decreases E-cadherin expression. A, p53ko/E17K cells were infected with a CRISPR/Cas9 lentivirus targeting AKT1 and p53ko cells were infected with a CRISPR/Cas9 lentivirus targeting AKT2. Cells lysates were then subjected to ChIP using a b-catenin antibody. Top: Western blot of whole cell lysates from the indicated p53ko/E17K cells and p53ko cells before and after AKT1 knockdown (AKT1KD) or AKT2 knockdown (AKT2KD) as specified. Bottom: Relative fold of ChIP pull-down of the ZEB1 promoter (b-catenin fold enrichment) was quantified by real-time qPCR, normalized by both input DNA and IgG values. Error bars, mean SEM (n ¼ 3). P values were determined by one-way ANOVA followed by Tukey post hoc test. , P < 0.0001. B, Western blots of lysates from MCF-10A p53ko/E17K or MCF7 PIK3CA WT cells expressing HA-tagged Myr-AKT2 and FLAG-tagged Myr-AKT1 alone and in combination. C, Western blot of lysates from p53ko cells: Left: infected with PTEN CRISPR/Cas9 lentivirus; Right: infected with PIK3CA E545K lentivirus. Cell lysate of parental MCF-10A cells was used as an E-cadherin–positive control.

Article Snippet: PIK3CA E545K–mutant cDNA (Cat#RC400348), purchased from OriGene, was inserted into the AsiS I and Mlu I sites of plasmid Lenti-C-Myc-DDK-IRES-Neo (Cat#PS100081, OriGene), to generate PIK3CA-E545K lentiviral expression vector.

Techniques: Activity Assay, Expressing, Infection, CRISPR, Western Blot, Knockdown, Positive Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

doi: 10.1111/jcmm.14879

Figure Lengend Snippet: Lovastatin caused p53 activation in MCF‐7 cells. A, MCF‐7 cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The phosphorylation or acetylation status of p53 (MW 53 kD) was determined by immunoblotting. Each column represents the mean ± SEM of seven independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P < .05, compared with the control group). B, Cells were transiently transfected with PG13‐luc (p53‐luc) or p21 promoter reporter construct (p21‐pro‐luc) plus renilla‐luc for 24 h followed by the treatment with lovastatin at 30 μmol/L for another 24 h. Reporter assay was performed as described in the ‘ ’ section. Each column represents the mean ± SEM of five independent experiments performed in duplicate (Statistically significant differences were determined using the Mann‐Whitney test. * P < .05, compared with the control group). (C) Cells were treated with vehicle or lovastatin at 30 μmol/L for the indicated periods. A ChIP assay was performed as described in the ‘ ’ section. Typical traces representative of five independent experiments with similar results are shown

Article Snippet: Construct of PG13‐luc with p53 binding sites (p53‐luc, Addgene plasmid #16642) and p21/WAF1 promoter luciferase construct (p21 pro‐luc, Addgene plasmid # 16451) as described previously were kindly provided by Dr Bert Vogelstein.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Transfection, Construct, Reporter Assay, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

doi: 10.1111/jcmm.14879

Figure Lengend Snippet: p38MAPK contributes to lovastatin‐induced p53 activation, p21 elevation and survivin reduction in MCF‐7 cells. A, Cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The extent of p38MAPK phosphorylation (MW 38 kD) was examined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P < .05, compared with the control group). Cells were pre‐treated with p38MAPK inhibitor III (p38i) at 1 μmol/L for 30 min. After treatment, cells were stimulated with lovastatin at 30 μmol/L for another 24 h. Protein levels of p21 (B) or survivin (C) were determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P < .05, compared with the vehicle‐treated control group; # P < .05, compared with the group treated with lovastatin alone). D, Cells were pre‐treated with p38MAPK inhibitor III (p38i) at 1 μmol/L for 30 min. After treatment, cells were stimulated with lovastatin at 30 μmol/L for another 1 h. The extent of p53 phosphorylation was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P < .05, compared with the vehicle‐treated control group; # P < .05, compared with the group treated with lovastatin alone). (E) Cells were transiently transfected with PG13‐luc (p53‐luc) plus renilla‐luc for 24 h. After transfection, cells were pre‐treated with p38MAPK inhibitor III (p38i) at 1 μmol/L for 30 min followed by the stimulant with lovastatin (30 μmol/L) for another 24 h. Reporter assay was performed as described in the ‘ ’ section. Each column represents the mean ± SEM of five independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P < .05, compared with the vehicle‐treated control group; # P < .05, compared with the group treated with lovastatin alone)

Article Snippet: Construct of PG13‐luc with p53 binding sites (p53‐luc, Addgene plasmid #16642) and p21/WAF1 promoter luciferase construct (p21 pro‐luc, Addgene plasmid # 16451) as described previously were kindly provided by Dr Bert Vogelstein.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, MANN-WHITNEY, Transfection, Reporter Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

doi: 10.1111/jcmm.14879

Figure Lengend Snippet: AMPK mediates lovastatin‐induced p38MAPK and p53 phosphorylation in MCF‐7 cells. Cells were treated with vehicle or lovastatin at 30 μmol/L for indicated periods. The extent of LKB1 (MW 54 kD) (A) or AMPK (MW 62 kD) (B) phosphorylation was determined by immunoblotting. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Kruskal‐Wallis test. * P < .05, compared with the control group). Cells were transfected with pcDNA or AMPK‐DN for 48 h. After transfection, cells were treated with vehicle or lovastatin (30 μmol/L) for another 1 h. The extent of p38MAPK (C) or p53 (D) phosphorylation was determined by immunoblotting. Each column represents the mean ± SEM of four independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P < .05, compared with the vehicle‐treated control group; # P < .05, compared with the group treated with lovastatin alone)

Article Snippet: Construct of PG13‐luc with p53 binding sites (p53‐luc, Addgene plasmid #16642) and p21/WAF1 promoter luciferase construct (p21 pro‐luc, Addgene plasmid # 16451) as described previously were kindly provided by Dr Bert Vogelstein.

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

doi: 10.1111/jcmm.14879

Figure Lengend Snippet: LKB1 contributes to lovastatin‐induced AMPK, p38MAPK and p53 phosphorylation in MCF‐7 cells. A, Cells were transfected with negative control siRNA or LKB1 siRNA for 48 h. After transfection, cells were treated with vehicle or lovastatin (30 μmol/L) for another 1 h. The extent of LKB1 and phosphorylation status of AMPK, p38MAPK or p53 was determined by immunoblotting. The compiled results of AMPK (B), p38MAPK (C) and p53 (D) phosphorylations are shown. Each column represents the mean ± SEM of six independent experiments (Statistically significant differences were determined using the Mann‐Whitney test. * P < .05, compared with the vehicle‐treated control group; # P < .05, compared with the group treated with lovastatin alone). (E) After transfection as described in (A), cells were treated with vehicle or lovastatin (30 μmol/L) for another 24 h. The percentage of propidium iodide‐stained cells in subG1, G0/G1, S and G2/M phases was analysed by flow cytometry. Each column represents the mean ± SEM of eight independent experiments (Statistically significant differences were determined using one‐way ANOVA, with Tukey's post hoc test. * P < .05, compared with the negative control siRNA‐transfected group; # P < .05, compared with the negative control siRNA‐transfected group in the presence of lovastatin). F, Schematic summary of the signalling pathway involved in lovastatin‐induced MCF‐7 breast cancer cell death

Article Snippet: Construct of PG13‐luc with p53 binding sites (p53‐luc, Addgene plasmid #16642) and p21/WAF1 promoter luciferase construct (p21 pro‐luc, Addgene plasmid # 16451) as described previously were kindly provided by Dr Bert Vogelstein.

Techniques: Phospho-proteomics, Transfection, Negative Control, Western Blot, MANN-WHITNEY, Control, Staining, Flow Cytometry