Journal: Theranostics

Article Title: Integration of Receptor Tyrosine Kinases Determines Sensitivity to PI3Kα-selective Inhibitors in Breast Cancer

doi: 10.7150/thno.17830

Figure Lengend Snippet: Hyper-activation of RAS or EGFR abrogates PI3K-regulated ERK phosphorylation. (A) T47D cells were treated with BYL719 (1 μM) or GDC0941 (1 μM) for 1 h. RAS-GTP were pulled down using GST-RAF1-RBD agarose. (B) T47D cells stably expressing empty vector or HRAS (G12V) were treated with BYL719 (1 μM) or GDC0941 (1 μM) for 1 h. (C) Cell lysate of T47D cells was applied to phospho-RTK array. (D) T47D cells stably transfected with empty vector, wild type EGFR or EGFR (L858R) were treated with BYL719 (1 μM), GDC0941 (1 μM) or BIBW2992 (1 μM) for 1 h. (E) FBS-starved T47D cells were treated with BYL719 (1 μM) or BIBW2992 (1 μM) for 1 h and then stimulated with EGF (50 ng/ml) for 15 min. Representative immunoblots for indicated proteins from at least three independent experiments are presented.

Article Snippet: Plasmids including pCMV-VSV-G (#8454), pCMV-dR8.2 dvpr (#8455), pDONR223-MEKDD (#31202), pDONR223-H-RAS V12 (#31201), pLX302 (#25896), pBabe-Puro-BRAF-V600E (#15269), EGFR L858R (#11012), EGFR WT (#11011) and pBabe-puro (#1764) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Theranostics

Article Title: Integration of Receptor Tyrosine Kinases Determines Sensitivity to PI3Kα-selective Inhibitors in Breast Cancer

doi: 10.7150/thno.17830

Figure Lengend Snippet: Hyper-activation of RAS or EGFR abrogates PI3K-regulated ERK phosphorylation. (A) T47D cells were treated with BYL719 (1 μM) or GDC0941 (1 μM) for 1 h. RAS-GTP were pulled down using GST-RAF1-RBD agarose. (B) T47D cells stably expressing empty vector or HRAS (G12V) were treated with BYL719 (1 μM) or GDC0941 (1 μM) for 1 h. (C) Cell lysate of T47D cells was applied to phospho-RTK array. (D) T47D cells stably transfected with empty vector, wild type EGFR or EGFR (L858R) were treated with BYL719 (1 μM), GDC0941 (1 μM) or BIBW2992 (1 μM) for 1 h. (E) FBS-starved T47D cells were treated with BYL719 (1 μM) or BIBW2992 (1 μM) for 1 h and then stimulated with EGF (50 ng/ml) for 15 min. Representative immunoblots for indicated proteins from at least three independent experiments are presented.

Article Snippet: Plasmids including pCMV-VSV-G (#8454), pCMV-dR8.2 dvpr (#8455), pDONR223-MEKDD (#31202), pDONR223-H-RAS V12 (#31201), pLX302 (#25896), pBabe-Puro-BRAF-V600E (#15269), EGFR L858R (#11012), EGFR WT (#11011) and pBabe-puro (#1764) were obtained from Addgene (Cambridge, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Aging (Albany NY)

Article Title: Suppression of p16 alleviates the senescence-associated secretory phenotype

doi: 10.18632/aging.202640

Figure Lengend Snippet: Knockdown of p16 decreases IL6 and CXCL8 expression in oncogene-induced senescent cells. IMR90s expressing either BRAF V600E or HRAS G12V alone or in combination with a shRNA targeting p16 (shp16 hairpin #1). An empty pBabe retroviral vector and a shRNA targeting GFP lentiviral vector were used as controls. See for an experimental timeline. ( A ) Immunoblot of BRAF and p16. Vinculin was used a loading control. ( B ) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). ( C ) Quantification of β-GAL in ( B ). ( D ) Quantification of CF in ( B ). ( E ) Immunoblot of RAS and p16. β-actin was used as loading control. ( F ) Representative images of β-GAL staining and colony formation (CF). ( G ) Quantification of β-GAL in ( F ). ( H ) Quantification of CF in ( F ). ( I , J ) IL6 and CXCL8 mRNA expression (fold change relative to control mean). Expression of target genes was normalized against multiple reference genes. Data normalized against MRPL9 are shown. n=3/group and mean±SD. 1 out of 3 experiments is shown. *p<0.05.

Article Snippet: pBabe BRAF V600E (Addgene cat#15269), pBabe HRAS G12V (Addgene cat#9051), and pBabe empty control (Addgene cat#1764) vectors were packaged into retroviral particles using the BBS/calcium chloride method as previously described in [ ]. pLKO.1-shp16 #1 (TRCN0000010482), pLKO.1-shp16 #2 (TRCN0000039751), and pLKO.1-shGFP control (Addgene, cat#30323) vectors were packaged using the ViraPower Kit (Invitrogen, cat# K497500).

Techniques: Knockdown, Expressing, shRNA, Retroviral, Plasmid Preparation, Western Blot, Control, Staining

Journal: Aging (Albany NY)

Article Title: Suppression of p16 alleviates the senescence-associated secretory phenotype

doi: 10.18632/aging.202640

Figure Lengend Snippet: Knockdown of p16 at later timepoints decreases IL6 and CXCL8 expression without bypassing oncogene-induced senescence. IMR90s expressing BRAF V600E alone or in combination with shRNAs targeting p16 (shp16 hairpin #1 and #2). An empty pBabe retroviral vector and a shRNA targeting GFP lentiviral vector were used as controls. See for an experimental timeline. ( A ) Immunoblot of BRAF and p16. β-actin was used as loading control. ( B ) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). ( C ) Quantification of β-GAL in ( B ). ( D ) Quantification of CF in ( B ). ( E ) IL6 and CXCL8 mRNA expression (fold change relative to control mean). Expression of target genes was normalized against multiple reference genes. Data normalized against PMSC4 are shown. n=3/group and mean±SD. 1 out of 3 experiments is shown. *p<0.05. ns = not significant.

Article Snippet: pBabe BRAF V600E (Addgene cat#15269), pBabe HRAS G12V (Addgene cat#9051), and pBabe empty control (Addgene cat#1764) vectors were packaged into retroviral particles using the BBS/calcium chloride method as previously described in [ ]. pLKO.1-shp16 #1 (TRCN0000010482), pLKO.1-shp16 #2 (TRCN0000039751), and pLKO.1-shGFP control (Addgene, cat#30323) vectors were packaged using the ViraPower Kit (Invitrogen, cat# K497500).

Techniques: Knockdown, Expressing, Retroviral, Plasmid Preparation, shRNA, Western Blot, Control, Staining

Journal: Aging

Article Title: Suppression of p16 alleviates the senescence-associated secretory phenotype.

doi: 10.18632/aging.202640

Figure Lengend Snippet: Figure 1. Knockdown of p16 decreases IL6 and CXCL8 expression in oncogene-induced senescent cells. IMR90s expressing either BRAFV600E or HRASG12V alone or in combination with a shRNA targeting p16 (shp16 hairpin #1). An empty pBabe retroviral vector and a shRNA targeting GFP lentiviral vector were used as controls. See Supplementary Figure 1A for an experimental timeline. (A) Immunoblot of BRAF and p16. Vinculin was used a loading control. (B) Representative images of senescence-associated β-galactosidase (β-GAL) staining and colony formation (CF). (C) Quantification of β-GAL in (B). (D) Quantification of CF in (B). (E) Immunoblot of RAS and p16. β-actin was used as loading control. (F) Representative images of β-GAL staining and colony formation (CF). (G) Quantification of β-GAL in (F). (H) Quantification of CF in (F). (I, J) IL6 and CXCL8 mRNA expression (fold change relative to control mean). Expression of target genes was normalized against multiple reference genes. Data normalized against MRPL9 are shown. n=3/group and mean±SD. 1 out of 3 experiments is shown. *p<0.05.

Article Snippet: Lentiviral and retroviral packaging and infection pBabe BRAFV600E (Addgene cat#15269), pBabe HRASG12V (Addgene cat#9051), and pBabe empty control (Addgene cat#1764) vectors were packaged into retroviral particles using the BBS/calcium chloride method as previously described in [8]. pLKO.1-shp16 #1 (TRCN0000010482), pLKO.1-shp16 #2 (TRCN0000039751), and pLKO.1-shGFP control (Addgene, cat#30323) vectors were packaged using the ViraPower Kit (Invitrogen, cat# K497500).

Techniques: Knockdown, Expressing, shRNA, Retroviral, Plasmid Preparation, Western Blot, Control, Staining