Journal: Drug Discovery Today. Disease Models

Article Title: Rabbit models of heart disease

doi: 10.1016/j.ddmod.2009.02.001

Figure Lengend Snippet: Rabbit models of heart disease

Article Snippet: Transgenic rabbit models created to date include: long QT syndrome [ ], hypertrophic cardiomyopathy [ , ], G sα overexpression [ ] and phospholamban overexpression [ ].

Techniques: Blocking Assay, Ligation, In Vivo, Isolation, Transgenic Assay, Over Expression, Virus

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 3. D1-9-G129R-hPRL PRLR antagonist abrogates the PRL-mediated increase in SKBR3 cell viability. (A) Western blot of MCF7 and SKBR3 whole cell extracts probed with the anti-PRLR extracellular domain antibody, 1A2B1. (B) Western blot of nuclear extracts (10 mg/well) of SKBR3 cells starved in serum-free media for 16 hours then treated with 5 mg/mL oPRL or 5 mg/mL D1-9-G129R-hPRL PRLR antagonist (Antag) for 1 hour or left untreated, probed for p-STAT5, STAT5 (rabbit-anti-STAT5), and histone-H3 (HH3) (loading control). (C) A general schematic for treatments used in WST-1 assays including a 24-hour pretreatment, 2 hours of TOPOII poison, followed by a 48-hour recovery period. (D, E, and F) WST-1 assays using SKBR3 cells pretreated or not with 5 mg/mL oPRL 6 D1-9-G129R-hPRL for 24 hours followed by 2 hours of doxorubicin (DOX) treatment, with or without PRL or D1-9-G129R-hPRL, then a 48-hour recovery with or without PRL or D1-9-G129R-hPRL as indicated. (D) The effect of oPRL or PRLR antagonist alone. (E) The effect of DOX 6 PRL and (F) the effect of DOX + Antag 6 PRL. Graphs represents pooled experiments, n = 12. Data were analyzed with a one-way ANOVA followed by Bonferroni posttests. *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Western Blot, Control

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 4. HSP90 inhibition abrogates PRL-mediated resistance. (A, B, and C) SKBR3 or (D, E, and F) MCF7 cells were pretreated or not with 5 mg/mL oPRL, 15 nM (SKBR3) or 100 nM (MCF7) HSP90 inhibitor 17-AAG, or a combination of the two for 24 hours followed by 2 hours of doxorubicin (DOX) treatment, 6PRL or 17-AAG, and a 48-hour recovery 6 PRL. (A–C) WST-1 assays of SKBR3 (A) with PRL or 17-AAG alone, (B) with DOX 6 PRL, or (C) with DOX + 17-AAG 6 PRL. (D–F) WST-1 assays of MCF7 (D) with PRL or 17-AAG alone, (E) with DOX 6 PRL, or (F) with DOX + 17-AAG 6 PRL. Graphs represent pooled experiments for SKBR3 n = 12, and for MCF7 n = 18. (G–I) WST-1 assays of MCF7 (G) with oPRL or 800 nM BIIB021 alone, (H) with DOX 6 PRL, or (I) with DOX + BIIB021 6 PRL. n = 18. Data were analyzed with a one-way ANOVA followed by Bonferroni posttests. *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Inhibition

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 6. JAK2 inhibition abrogates the PRL increased cell viability. (A) Western blot of JAK2 inhibitor dose response. MCF7 cells were treated with twofold increasing concentrations of G6 for 12 hours. p-STAT5 levels were determined (mouse anti-p-STAT5) with total STAT5 and histone H3 (HH3) loading controls. Results are representative of two independent experimental replicates. (B) Dose-response curves of JAK2 inhibitor G6. MCF7 cells were treated with G6 at indicated time points and concentrations. The cell viability was determined with WST-1 cell viability assay. WST-1 assays of (C–E) MCF7 or (F–H) SKBR3 cells pretreated with 25 ng/mL hPRL for 24 hours or 25 mM JAK2 inhibitor G6 for 12 hours, followed by

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Inhibition, Western Blot, Viability Assay

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 7. HSP90 inhibition decreases ATM and p-ATM protein but not its mRNA. (A) MCF7 cells were treated or not with threefold increasing concentrations of 17-AAG for 24 hours followed by 2 hours of doxorubicin (DOX) treatment and recovery. Proteins (30 mg per well) were resolved on SDS-PAGE gel, and blot was probed for p-ATM, ATM, HSP90a, and GRB2. Gel is representative of two experiments. Fold difference of p-ATM with DOX treatment was calculated, in ImageJ, compared with untreated cells (set to 1). Fold difference of (B) total ATM and (C) p- ATM levels with 17-AAG and DOX treatment were calculated compared with DOX alone (set to 1). Representative blot of two experiments. (D) MCF7 cells were pretreated with oPRL (5 mg/mL) or 17-AAG for 24 hours, followed by 2 hours DOX treatment (0.2 mM). ATM expression was detected by qPCR, and the results were normalized to YWHAZ control [mean 6 standard error of the mean (SEM); n = 5]. The DDCt method was used to analyze the relative changes in gene. The letter “a” above SEM bars denotes that there were no statistically significant differences (one-way ANOVA followed by Bonferroni test, P , 0.05). (E) SKBR3 cells as above, with (F) ATM and (G) p-ATM band intensities quantified by ImageJ. Representative blot of two experiments.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: Inhibition, SDS Page, Expressing, Control

Journal: Endocrinology

Article Title: ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

doi: 10.1210/en.2017-00652

Figure Lengend Snippet: Figure 11. ATM is needed for PRL-induced increase in cell viability after DNA damage in 3D culture. Cells were pretreated with 25 ng/mL hPRL or vehicle for 24 hours followed by 2 hours of 1 mM doxorubicin (DOX) treatment (or vehicle) with or without PRL. Cells were transferred to collagen 3D culture and allowed to recover for 48 hours. Cell viability was determined with WST-1 assay, and the treatments were normalized to vehicle controls: (A) SKBR3 or (B) MCF7. For ATM knockdown experiments, (C and E) SKBR3 and (D and F) MCF7 cells were transfected with (C and D) siNT or (E and F) siATM, followed by 24 hours of 25 ng/mL hPRL and 2 hours of DOX treatment (3 mM for SKBR3, 2 mM for MCF7 cells). Cells were transferred to collagen gels to allow recovery for 96 hours (SKBR3) or 48 hours (MCF7). Bars in the graphs represent six independent experiments. Statistically significant analysis by ANOVA: *P , 0.05; **P , 0.01; ***P , 0.001.

Article Snippet: Cell culture and cell lines PRL-responsive SKBR3 (HER2 overexpressing, p53mutant) and MCF7 (estrogen receptor+, p53 wild type) human breast cancer cell lines, obtained and authenticated from American Type Culture Collection, were used within 6 months when revived from frozen storage.

Techniques: WST-1 Assay, Knockdown, Transfection

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Primers used for real-time PCR

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques:

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: CIH promoted CSC-like properties in NSCLC cells. a Microscopic observation of NSCLC cells. A549 and SPCA1 spheroids were obtained and then cultured under Nor or CIH conditions for 24 h. b qRCP analyses in triplicate of CD44, Sox2, Nanog, Oct4, and CD133 in NSCLC cells. Adherent A549 and SPCA1 were treated with 24 h-Nor or CIH exposure. GADPH expression served as an internal control. c Flow cytometry analyses of the percentage of CD44 + CD133 + cells in A549 or SPCA1 populations. Error bars represent the mean ± SEM of at least triplicate experiments. * P < 0.05, ** P < 0.01. CIH chronic intermittent hypoxia, CSC cancer stem cell

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: Cell Culture, Expressing, Flow Cytometry

Journal: Respiratory Research

Article Title: Chronic intermittent hypoxia promoted lung cancer stem cell-like properties via enhancing Bach1 expression

doi: 10.1186/s12931-021-01655-6

Figure Lengend Snippet: Knockdown of Bach1 decreased the stemness and mitochondrial ROS accumulation in CIH-treated NSCLCs. Bach1 shRNA or parental negative control (NC) was transfected into A549 or SPCA1. Then the cells were cultured under CIH conditions for 48hrs. Flow cytometry analyses of CD44 + CD133 + cells in Nor or CIH-treated A549 ( a ) or SPCA1 ( b ). c The percentage of CD44 + CD133 + cells in Nor or CIH-treated cells was measured and illustrated. Fluorescence microscopy analysis for localization of mtROS production in A549 ( d ) and SPCA1 ( e ). Nuclei was stained with Hoechst (blue), mitochondria ROS were stained with MitoSOX-red. The merged panels showed the MitoSOX-red positive cells in total cells. f The percentage of MitoSOX positive cells was measured and illustrated. All experiments were performed in triplicate, and data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 compared with the control group

Article Snippet: A549 and SPCA1 were seeded in 12-well plates at a proper concentration and cultured under Nor or CIH condition for 48 h. At the end of CIH cycles, cells were harvested, filtration and centrifugation, and FITC-labeled anti-CD44 (555478) and APC-labeled anti-CD133 (53276) (Cell Signaling Technologies, USA) were used for surface staining.

Techniques: shRNA, Negative Control, Transfection, Cell Culture, Flow Cytometry, Fluorescence, Microscopy, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: Cell grouping information

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: In silico analysis for differentially expressed genes and regulatory miRNAs related to PDAC treated with macrophages. a-c heat maps of differentially expressed genes obtained from gene expression datasets with macrophage-treated PDAC samples and common PDAC datasets. The abscissa represents the sample number, and the ordinate represents the differential expressed gene; the histogram at the left refers to gene expression cluster, and the histogram at the upper right refers to color gradation. d the intersection of the gene expression datasets analysis. The three circles in the panel represent the down-regulated genes in the three gene expression datasets, and the middle part represents the intersection of the three sets of data. e the expression of TGFBR3 in each tumor data of TCGA database. The abscissa indicates the sample type and tumor type, and the ordinate indicates the gene expression. f prediction of regulatory miRNAs of TGFBR3. The two sets of data in the panel are retrieved from the TargetScan database prediction and the study revealing miRNAs expressed in macrophage exosomes, and the middle part represents the intersection of the two sets of data

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: In Silico, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: MiR-501-3p promotes migration, invasion and tube formation of PDAC cells by down-regulating TGFBR3. a the predicted binding sites of miR-501-3p and TGFBR3 on TargetScan database. b the binding of miR-501-3p to TGFBR3 was confirmed by dual luciferase reporter gene assay. * p < 0.05 vs. mimic-NC. C. RT-qPCR detection of TGFBR3 mRNA levels in PANC-1 and BxPC-3 cells treated with miR-501-3p mimic. * p < 0.05 vs. NC mimic. d and e Western blot analysis of TGFBR3 protein in PANC-1 and BxPC-3 cells treated with miR-501-3p mimic. * p < 0.05 vs. NC mimic. f-i migration and invasion abilities of PANC-1 and BxPC-3 cells treated with miR-501-3p mimic vector, miR-501-3p mimic + oe-TGFBR3, Mp-Exo + vector, PBS + oe-TGFBR3 and Mp-Exo + oe-TGFBR3 were measured by Transwell assay (200 ×). * p < 0.05 vs. NC mimic + vector, PBS + vector. # p < 0.05 vs. NC mimic + oe-TGFBR3, PBS + oe-TGFBR3. j-m In vitro tube formation assay was used to detect HMEC-1 cell tube formation ability and quantitative analysis in response to miR-501-3p mimic vector, miR-501-3p mimic + oe-TGFBR3, Mp-Exo + vector, PBS + oe-TGFBR3 and Mp-Exo + oe-TGFBR3 (Scale bar = 100 μM). * p < 0.05 vs. NC mimic + vector, PBS + vector. # p < 0.05 vs. NC mimic + oe-TGFBR3, PBS + oe-TGFBR3. n-q Western blot analysis was used to detect migration, invasion and angiogenesis-related proteins in response to miR-501-3p mimic vector, miR-501-3p mimic + oe-TGFBR3, Mp-Exo + vector, PBS + oe-TGFBR3 and Mp-Exo + oe-TGFBR3. * p < 0.05 vs. NC mimic + vector, # p < 0.05 vs. NC mimic + oe-TGFBR3, PBS + oe-TGFBR3. R. RT-qPCR detection for mRNA expression of TGFBR3 in metastatic and non-metastatic PDAC tissues ( n = 14–42). * p < 0.05 vs. non-metastatic tissue. S and T. Western blot analysis was used to detect TGFBR3 protein in metastatic and non-metastatic PDAC tissues ( n = 14–42). * p < 0.05 vs. tissues without metastatic. The measurement data were expressed as mean ± standard deviation. The comparison between two groups was performed by independent sample t test. One-way ANOVA was employed for comparison among multiple groups, followed by Tukey’s post-hoc test. Cell experiments were repeated three times

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Migration, Binding Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transwell Assay, In Vitro, Tube Formation Assay, Expressing, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: TGF-β signaling pathway is involved in the effect of miR-501-3p on PDAC cells. a and b Western blot analysis was used to detect the TGF-β signaling pathway-related proteins in PANC-1 cells after Mp-Exo treatment. c and d Western blot analysis was applied to detect the TGF-β signaling pathway-related proteins in PANC-1 cells treated with oe-TGFBR3. e and f Western blot analysis was conducted to detect the TGF-β signaling pathway-related proteins in BxPC-3 cells after Mp-Exo treatment. g and h Western blot analysis was used to detect the TGF-β signaling pathway-related proteins in BxPC-3 cells treated with oe-TGFBR3. i and j Western blot analysis was applied to detect the TGF-β signaling pathway-related proteins in PANC-1 cells after treatment of miR-501-3p mimic, inhibitor or TGFBR3 siRNA. k and l Western blot analysis was used to detect the TGF-β signaling pathway-related proteins in BxPC-3 cells after treatment of miR-501-3p mimic, inhibitor or TGFBR3 siRNA. * p < 0.05 vs. control, vector or NC mimic. # p < 0.05 vs. NC inhibitor. & p < 0.05 vs. NC siRNA. The measurement data were expressed as mean ± standard deviation. Data between two groups were analyzed by independent sample t test. One-way ANOVA was employed for comparison among multiple groups, followed by Tukey’s post-hoc test. Cell experiments were repeated three times

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Western Blot, Plasmid Preparation, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: Inhibition of miR-501-3p in macrophage exosomes represses tumor formation and metastasis in nude mice. a-d xenograft tumors and quantitative analysis of tumor mass after Mp-Exo + miR-501-3p antagomiR treatment ( n = 7). M2 macrophages were transfected with NC antagomiR or miR-501-3p antagomiR, after which exosomes were extracted and injected into the nude mice. Tissue samples were collected after 4 weeks to detect relevant indicators. e-l HE staining analysis and quantitative analysis of liver and lung nodules after Mp-Exo + miR-501-3p antagomiR treatment (n = 7). m and n . RT-qPCR detection of the expression of miR-501-3p in subcutaneous tumor tissues and exosomes from the serum of nude mice in response to Mp-Exo + miR-501-3p antagomiR. o and p RT-qPCR detection of expression of tumor cell stemness-related genes and THFBR3 in mouse subcutaneous tumor tissues in response to Mp-Exo + miR-501-3p antagomiR. q-t Western blot analysis of migration, invasion and angiogenesis-related proteins as well as TGFBR3 protein in PANC-1 and BxPC-3 cells treated with Mp-Exo + miR-501-3p antagomiR. * p < 0.05 vs. Mp-Exo + NC antagomiR. The measurement data were expressed as mean ± standard deviation. Data between two groups were analyzed by independent sample t test. One-way ANOVA was employed for comparison among multiple groups. The data at different time points were compared using repeated measures ANOVA, followed by Tukey’s post-hoc test

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Inhibition, Transfection, Injection, Staining, Quantitative RT-PCR, Expressing, Western Blot, Migration, Standard Deviation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Macrophage-derived exosomal microRNA-501-3p promotes progression of pancreatic ductal adenocarcinoma through the TGFBR3-mediated TGF-β signaling pathway

doi: 10.1186/s13046-019-1313-x

Figure Lengend Snippet: Schematic representation of macrophage-derived exosomal miR-501-3p in PDAC and the involvement of TGFBR3-mediated TGF-β signaling pathway. M2 macrophages deliver miR-501-3p through exosomes in PDAC cells, thereby down-regulating TGFBR3 expression, and ultimately accelerate the development of PDAC via the TGF-β signaling pathway, corresponding to enhanced PDAC cell invasion and migration

Article Snippet: The miR-501-3p mimic, miR-501-3p inhibitor, and TGFBR3 siRNA (there were three siRNAs to be assessed) at a concentration of 50 nM and TGFBR3 overexpressing adenoviruses were all purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Derivative Assay, Expressing, Migration