Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 1. Cancer-associated fibroblasts promote resistance to KRAS∗extinction in a PDAC model. (A) Crystal violet staining of iKPC1 cell colony formation assay, with Kras∗on (on dox), MRTX1133, or Kras∗off (off dox). The presence of 50% conditional medium from pCAF1, pCAF2, or CAF1 significantly increased the capacity of cell growth 4 d after treatment. (B) Measurement of cell viability using Celltiter-Glo in the same settings as in A 4 d after treatment. (C) Measurement of colony diameter of iKPC1 in Matrigel culturing assay in the same settings as in A 7 d after treatment. (D) Luciferase image of mice injected with 1 × 106 iKPC1-luc cells alone, iKPC2-luc cells alone, or iKPC1-luc or iKPC2-luc cells together with 1 × 106 pCAF1 or CAF1 cells 7 d after injection. Coinjection of iKPC and CAFs sig- nificantly increased KRAS∗-independent growth. (E) Quantification of luciferase signal from D. (F) Histology (HE, trichome, GFP, and KRASG12D staining) of tissue mass from iKPC + CAF1 injection from D. (G) Measurement of colony diameter of iKPC1 in Matrigel cul- turing assay 7 d after treatment with the indicated condition and treatment. (DMSO) Dox on with DMSO, (MRTX) dox on with 100 nM MRTX1133, (Kras∗off) dox off with DMSO, (CM) the presence of 50% CAF-CM in culture, (CM-HI) the presence of 50% heat-inactivated CAF-CM in culture. (H) Summary of the strategy for isolating CAF-secreted protein (CAF-pro). (I) Experimental settings and Western blot analysis of iKPC1 cells at the indicated time points after adding CAF-pro. (J) RTK array analysis of two iKPC cell lines after 5 min of CAF- pro or heat-inactivated CAF-pro (HI-CAF-pro) treatment. (K) Quantification of J. (L) Confirming ERBB2, ERBB3, and AKT phosphorylation in four different iKPC cell lines. Data are represented as mean ± SD for B, C, and G, and mean only for E. For B, C, E, and G, Student’s t-test was performed to calculate statistical values.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Staining, Colony Assay, Luciferase, Injection, Western Blot, Phospho-proteomics

Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 2. Paracrine Nrg1–Erbb2/3 signaling activation confers KRAS∗resistance. (A) Western blot of NRG1 protein levels in various cell lines. (B) Multiplexed RNAscope immunofluorescence staining of iKPC tumor with NRG1-specific RNA probe (white), GFP (green), PDPN (red), and DAPI (blue). (C) qPCR results of Nrg1 on flow cytometry-sorted GFP+ PDAC cells or PDPN+ CAFs from iKPC GEM tu- mors. Two independent tumors were pooled together for dissociation. (D) Measurement of colony diameter of iKPC1 in 3D Matrigel cul- turing assay with the indicated treatment for 7 d. (on) Kras∗on with dox, (off) Kras∗off without dox, (MRTX) 100 nM MRTX1133 treatment, (CAF) the presence of CAF1-pro in culture, (NRG1) the presence of 10 ng/mL rmNRG1 in culture. Data are represented as mean ± SEM; Student’s t-test. (E) Western blot of the indicated iKPC1 knockout clones Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (F) Western blot of the indicated iKPC1 ERBB3 knock- out or rescued clones K Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (G) Western blot of iKPC1 cells with 48 h of K Kras∗off and 2 h of serum starvation prior to 5-min stimulation of the indicated treatments (CAF1-pro cells were preincubated with 100 μg/mL mouse IgG or the indicated concentration of NRG1 neutraliza- tion antibody YW538.24.71 for 1 h at 37°C). (H) Western blot of AsPC1, DMSO, or 100 nM MRTX1133 treatment for 24 h and serum star- vation for 2 h with the presence of 10 μg/mL control human IgG or 10 μg/mL pertuzumab prior to 5-min stimulation of the indicated treatments (CAF-pro, 10 ng/mL rmNRG1, or CAF-sgNrg1-pro). (I) Cell viability assay with AsPC1 with the indicated treatment for 4 d. (MRTX) Treated with 100 nM MRTX1133 (with pCAF1-pro, CAF1-pro, or 10 ng/mL rmNRG1), (mIgG + hIgG) treated with 10 μg/ mL control mouse IgG and 10 μg/mL control human IgG, (anti-NRG1) the presence of 10 μg/mL YW538.24.71, (pertuzumab) the presence of 10 μg/mL pertuzumab. Data are represented as mean ± SD; two-way ANOVA. (J) Luciferase assay of iKPC1-luc cells coinjected with pCAF3 or CAF1. One day after cell implantation, luciferase signal was measured to acquire the initial value of luciferase activity, and IgG or YW538.24.71 was given at a dose of 25 mg/kg. Luciferase signal was acquired again at 7 d after implantation. (K) Quantification of luciferase signal from J. Paired multiple t-test at day 7.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Activation Assay, Western Blot, RNAscope, Immunofluorescence, Staining, Flow Cytometry, Knock-Out, Clone Assay, Concentration Assay, Control, Viability Assay, Luciferase, Activity Assay

Journal: Oncology Letters

Article Title: Decreased sirtuin 4 expression is associated with poor prognosis in patients with invasive breast cancer

doi: 10.3892/ol.2016.5021

Figure Lengend Snippet: Comparison of clinicopathological features between SIRT4 positive and negative breast cancer patients (n=409).

Article Snippet: The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), tumor protein p53 (p53) and nuclear-associated antigen Ki-67 (Ki-67) expression status was analyzed in all patients; tissue sections were incubated with antibodies to the proteins ER (catalog no. TA506414; dilution, 1:150; incubation time, 20 min; temperature, 25°C), PR (catalog no. TA802606; dilution, 1:150; incubation time, 10 min; temperature, 25°C), HER2 (catalog no. TA503443; dilution, 1:50; incubation time, 30 min; temperature, 25°C), p53 (catalog no. TA502780; dilution, 1:100; incubation time, 15 min; temperature, 25°C) and Ki-67 (catalog no. TA500265; dilution, 1:50; incubation time, 20 min; temperature, 25°C) antibodies (Origene Technologies Inc., Rockville, MD, USA).

Techniques: Comparison, Expressing

Journal: Oncology Letters

Article Title: Decreased sirtuin 4 expression is associated with poor prognosis in patients with invasive breast cancer

doi: 10.3892/ol.2016.5021

Figure Lengend Snippet: Univariate and multivariate analysis of prognostic factors in invasive breast cancer patients.

Article Snippet: The estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), tumor protein p53 (p53) and nuclear-associated antigen Ki-67 (Ki-67) expression status was analyzed in all patients; tissue sections were incubated with antibodies to the proteins ER (catalog no. TA506414; dilution, 1:150; incubation time, 20 min; temperature, 25°C), PR (catalog no. TA802606; dilution, 1:150; incubation time, 10 min; temperature, 25°C), HER2 (catalog no. TA503443; dilution, 1:50; incubation time, 30 min; temperature, 25°C), p53 (catalog no. TA502780; dilution, 1:100; incubation time, 15 min; temperature, 25°C) and Ki-67 (catalog no. TA500265; dilution, 1:50; incubation time, 20 min; temperature, 25°C) antibodies (Origene Technologies Inc., Rockville, MD, USA).

Techniques:

Journal: Endocrinology

Article Title: Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

doi: 10.1210/endocr/bqae129

Figure Lengend Snippet: Figure 5. Ovaries were collected from pregnant rats on day 12 for immunohistochemistry (IHC) (n = 3 rats per group). A, Representative photomicrographs view of corpus luteum (CL) with immunolocalization of epidermal growth factor receptor 3 (ErbB3) and steroidogenic acute regulatory protein (StAR) with Alexa Fluor 594– (red) and Alexa Fluor 488– (green) labeled secondary antibodies, respectively. The nucleus was stained with DAPI (4′,6′-diamidino-2-phenylindole) (blue). B. Representative photomicrographs view of CL with immunolocalization of ErbB2 and StAR with Alexa Fluor 594– (red) and Alexa Fluor 488– (green) labeled secondary antibodies, respectively. The nucleus was stained with DAPI (blue). C, Representative photomicrographs view of CL with immunolocalization of ErbB3 and ErbB2 with Alexa Fluor 594– (red) and Alexa Fluor 488– (green) labeled secondary antibodies, respectively. The nucleus was stained with DAPI (blue).

Article Snippet: Scientific Mouse (monoclonal) AB_2816212 http://antibodyregistry.org/AB_2816212 1:500 Total ErbB2 Anti-ErbB2 Origene Mouse (monoclonal) AB_2629050 http://antibodyregistry.org/AB_2629050 1:500 (WB) 1:50 (IHC)

Techniques: Immunohistochemistry, Labeling, Staining

Journal: Endocrinology

Article Title: Neuregulin 1 Signaling Attenuates Tumor Necrosis Factor α-Induced Female Rat Luteal Cell Death.

doi: 10.1210/endocr/bqae129

Figure Lengend Snippet: Figure 6. Effects of knockdown of endogenous neuregulin 1 (NRG1) on the exogenous treatment of tumor necrosis factor α (TNFα) on phospho(p)- and total ErbB2, ErbB3, phosphoinositide 3-kinase (PI3K), serine/threonine-protein kinase (AKT/protein kinase B), and extracellular signal-regulated kinase (ERK1/2) protein expressions in rat luteal cells (LCs). LCs were grown at 70% to 80% confluency and transiently transfected with small interfering-NRG1 (siNRG1) or scrambled RNA. After that, cells were maintained in serum-starved media and treated with TNFα at different concentrations (10 and 50 ng/mL) for 24 hours. A and D, Expression of phospho(p)- and total ErbB2, ErbB3 PI3K, AKT/PKB, and ERK1/2 protein expressions were determined by Western blot analysis. To each lane of the gel equal amounts of protein were applied. β-Actin was used as an internal control. Bar diagrams B to F represent the densitometric protein analyses in immunoblots (WBs). All bar graphs represent the mean ± SEM of results from 3 individual experiments (n = 3). Asterisks represent unpaired t test, *P less than or equal to .01; **P less than or equal to .001; ***P less than or equal to .0001; and NS, not significant.

Article Snippet: Scientific Mouse (monoclonal) AB_2816212 http://antibodyregistry.org/AB_2816212 1:500 Total ErbB2 Anti-ErbB2 Origene Mouse (monoclonal) AB_2629050 http://antibodyregistry.org/AB_2629050 1:500 (WB) 1:50 (IHC)

Techniques: Knockdown, Transfection, Expressing, Western Blot, Control