Journal: Molecular cancer therapeutics

Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

doi: 10.1158/1535-7163.MCT-18-1319

Figure Lengend Snippet: Summary of RNA-Seq results: a. individual gene expression by cell line (FC = fold change versus control expressed as the base 2 logarithm), n=2 biological replicates per cell line; b. Geneset enrichment analysis results of RNA-Seq data; genesets are from the Broad Institute mSigdb (size = number of genes in geneset, NES = Normalized Enrichment Score; FDR = False Discovery Rate).

Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

Techniques: Gene Expression, Control

Journal: Molecular cancer therapeutics

Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

doi: 10.1158/1535-7163.MCT-18-1319

Figure Lengend Snippet: Molecular effects of selinexor treatment on HGG cells: a. NGFR expression by qPCR following vehicle or selinexor treatment (16h) at indicated dose levels (n=3); b. total NGFR protein expression following vehicle or selinexor treatment; c. cell surface NGFR protein expression; d. plot of NGFR cell surface expression versus selinexor IC50 in DMG cell lines; e. nuclear IκB-α protein expression following vehicle or selinexor treatment; f. nuclear NF-κB (p65) protein expression following vehicle or selinexor treatment; g. ratio of nuclear NF-κB/IκB-α from panels e and f; h. nuclear p-NF-κB (S536) protein expression following vehicle or selinexor treatment; i. ratio of nuclear p-NF-κB (S536)/NF-κB from panels f and h. Panels c – f are quantification of immunofluorescence images following 16h selinexor treatment at 5xIC50 dose by cell line (n = number of cells quantified *p<0.05, **p<0.01, ***p<0.001; p-values are versus control levels).

Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

Techniques: Expressing, Immunofluorescence, Control

Journal: Molecular cancer therapeutics

Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

doi: 10.1158/1535-7163.MCT-18-1319

Figure Lengend Snippet: In vitro effects of selinexor treatment: total NGFR, nuclear IκB-α and NF-κB (p65) from immunofluorescence images of mouse PDX tumor samples (n = number of cells quantified *p<0.05, **p<0.01, ***p<0.001; p-values are versus control levels).

Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

Techniques: In Vitro, Immunofluorescence, Control

Journal: Molecular cancer therapeutics

Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

doi: 10.1158/1535-7163.MCT-18-1319

Figure Lengend Snippet: Molecular and proliferative effects of NGFR KD: a. NGFR gene expression levels in DIPG6 and SF7761 shNull (empty vector control) and shNGFR (NGFR KD) cells (n=3); b. ratio of RELA/NFKBIA gene expression in SF7761 shNull and shNGFR cells (n=3); c. nuclear IκB-α and NF-κB (p65) protein levels by immunofluorescence and NF-κB (p65)/IκB-α in SF7761 shNull and shNGFR cells (n=number of cells quantified); d. week over week expansion rate for DIPG6 and SF7761 shNull and shNGFR (n=4 for all samples).

Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

Techniques: Gene Expression, Plasmid Preparation, Control, Immunofluorescence

Journal: Molecular cancer therapeutics

Article Title: Exportin 1 inhibition induces nerve growth factor receptor expression to inhibit the NF-κB pathway in preclinical models of pediatric high-grade glioma

doi: 10.1158/1535-7163.MCT-18-1319

Figure Lengend Snippet: Effects of selinexor treatment on NGFR KD and NGFR overexpressing cells: a. NGFR gene expression in SF7761 shNull and shNGFR KD cells (n=3) following selinexor treatment for 16h at indicated dose levels; b. selinexor IC50 levels in SF7761 shNull and shNGFR cells (n=3); c. apoptosis levels by caspase 3/7 luminescence in SF7761 shNull and shNGFR cells at 24h and 48h time points (n=3); d. log10 of relative transcriptionally active NF-κB by ELISA in SF7761 shNull and shNGFR cells following selinexor treatment for 16h at 1μM dose level (n=3); e. relative gene expression levels of NGFR, NFKBIA and RELA by qPCR (n=3) and protein expression levels of total NGFR and nuclear NF-κB and IκB-α in cells transfected with empty vector or NGFR cDNA; f. selinexor IC50 levels in DIPG4 wild type or cells transfected with IκB-α super repressor (n=3); g. selinexor IC50 levels in DIPG4 shNull, shNGFR and shNGFR cells transfected with IκB-α super repressor (n=3).

Article Snippet: Primary antibodies and concentrations were: NGFR (Santa Cruz sc-8317, 1:200), IκB-α (Santa Cruz sc-371, 1:200), NF-κB (p65) (Cell Signaling #6956, 1:400), phosphorylated-NF-κB (S536) (Cell Signaling #3033, 1:200).

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: SHP2 inhibits CRC cell proliferation and migration. ( A ) SHP2 knockdown by siSHP2#1 and #2 markedly increased the proliferation of HCT116 and SW480 cells. ( B ) Colony formation assays were conducted to estimate the growth rate of HCT116 and SW480 cells. SHP2 knockdown increased the colony numbers compared with the control group. Representative pictures of colonies (left) and quantification of colony numbers (right) are shown. ( C ) Transwell assay was performed to access the effect of SHP2 on cell migration by siRNA mediated knockdown. Representative pictures of cells (left) and quantification of cell numbers (right) are shown. ( D ) SW480 cells were transfected with pJ-SHP2 plasmid to overexpress SHP2 and the cell proliferation was accessed by MTT assay. ( E ) Colony formation assay was done by overexpression of SHP2 in SW480 cells. SHP2 overexpression decreased the colony numbers compared with the control group. Representative pictures of colonies (left) and quantification of colony numbers (right) are shown. ( F ) Transwell assay was performed to access the effect of SHP2 on cell migration by overexpressing SHP2 in SW480 cells. Representative pictures of cells (left) and quantification of cell numbers (right) are shown. Overexpression of SHP2 in HCT116 and SW480 cells rescued SHP2 knockdown induced cell proliferation ( G ), colony formation ( H ) and cell migration ( I ). ( J and K ) PHPS1 improved HCT116 and SW480 cell proliferation during 3 days in a time- and dose-dependent manner. ( L ) Cells were treated with PHPS1, and colony formation was measured after two weeks by crystal violet staining. PHPS1 increased the number of CRC cell colonies. (M)Transwell assay showing blockade of SHP2 phosphatase activity by PHPS1 improved CRC cell migratory ability. NC, non-silencing control siRNA. Values represent mean ± SEM (n = 3–4), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Migration, Knockdown, Control, Transwell Assay, Transfection, Plasmid Preparation, MTT Assay, Colony Assay, Over Expression, Staining, Activity Assay

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: SHP2 suppressing role in CRC is mediated by STAT3 dephosphorylation. ( A ) STAT3 phosphorylation was increased in CRC cells knocked down for SHP2 by siRNA#1 and #2. ( B ) SHP2 overexpression reduced STAT3 phosphorylation in SW480. ( C ) PHPS1 enhanced the levels of pSTAT3 in 10 mg/L time-dependently. ( D ) Nuclear distribution of pSTAT3 was enhanced after SHP2 knockdown (×400). ( E ) Cryptotanshinone significantly reversed CRC cell proliferation induced by PHPS1. Values represent mean ± SEM (n = 3), **P ≤ 0.01, compared with Vehicle group. ( F ) SW480 was more sensitive to IL-6-induced proliferation after SHP2 knockdown, which was rescued by SHP2 overexpression; meanwhile overexpression SHP2 inhibited IL-6 induced SW480 proliferation. Values represent mean ± SEM (n = 3) # P ≤ 0.05, **P ≤ 0.01, compared with NC + pJ3 group. Whole blots are shown in Supplementary Fig. .

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Over Expression, Knockdown

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Association between the combination of SHP2/nuclear STAT3 and survival in patients with CRC. ( A ) Kaplan–Meier analysis showed that Patients with high SHP2 and low nuclear STAT3 present better DSS (left panel) and DFS (right panel) than patients with low SHP2 and high nuclear STAT3. ( B ) Kaplan-Meier analysis showed that Patients with high SHP2 and low nuclear STAT3 present better DSS (left panel) and DFS (right panel) than patients with low SHP2 or high nuclear STAT3. ( C ) Kaplan-Meier analysis showed that patients with high SHP2 or low nuclear STAT3 present better DFS (right panel) but not DSS (left panel) than patients with low SHP2 and high nuclear STAT3. P values were determined using log-rank test.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques:

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Univariate Cox regression analysis for clinical parameters and the combinations of SHP2 and nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Multivariate Cox regression analysis for clinical parameters and the combinations of high SHP2 and low nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant

Journal: Scientific Reports

Article Title: SHP2 associates with nuclear localization of STAT3: significance in progression and prognosis of colorectal cancer

doi: 10.1038/s41598-017-17604-7

Figure Lengend Snippet: Multivariate Cox regression analysis of DFS for clinical parameters and the combinations of low SHP2 and high nuclear STAT3.

Article Snippet: Wild-type SHP2 expressing plasmid pJ3 (pJ3-SHP2 WT) was a gift from Ben Neel (Addgene plasmid # 8317) .

Techniques: Adjuvant