Journal: Communications Biology

Article Title: Isoform specific anti-TGFβ therapy enhances antitumor efficacy in mouse models of cancer

doi: 10.1038/s42003-021-02773-z

Figure Lengend Snippet: B16 tumors from mice were harvested 11 days following tumor implantation. After creating single-cell suspensions, tumors underwent processing and staining for flow cytometry analysis as described in “Methods”. a Breakdown of the immune infiltrate in B16 tumors of mice harvested 11 days after tumor implantation. b Representative histograms displaying mean fluorescence intensity (MFI) of either TGFβ1 (left panel) or TGFβ3 (right panel) on specific tumor-infiltrating immune cells as detected by flow cytometry. The light gray peak represents each cell type’s fluorescence minus one (FMO) and was used to determine positive expression, indicated by the colored peak. c Representative graph illustrating the relative expression of TGFβ1 (top graph) or TGFβ3 (bottom graph) by various lymphocytic and myeloid cell types in the tumor microenvironment 11 days after tumor implantation. Data ( n = 5 mice/group) are displayed as MFI \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SD. Data are representative of three independent experiments. MFI is measured in arbitrary units and is a variable used to measure relative expression levels of staining antibodies, in this case of TGFβ1 and TGFβ3 protein expression, on tumor-infiltrating immune cells. FMO controls were derived by staining the immune cells with all the fluorophores minus one fluorophore, in this case, the fluorophore (Alexa Fluor 647) that was conjugated to TGFβ1 and TGFβ3. The pattern of intracellular TGFβ1 and TGFβ3 expression is similar to the surface staining pattern demonstrated in Supplementary Fig. .

Article Snippet: Gene-specific oligonucleotide target probes against non-homologous regions of TGFβ1 and TGFβ3 were created and pre-optimized by the manufacturer (ThermoFisher Scientific).

Techniques: Tumor Implantation, Staining, Flow Cytometry, Fluorescence, Expressing, Derivative Assay

Journal: Communications Biology

Article Title: Isoform specific anti-TGFβ therapy enhances antitumor efficacy in mouse models of cancer

doi: 10.1038/s42003-021-02773-z

Figure Lengend Snippet: a Therapy regimen beginning 11 days post tumor implantation with 250,000 B16 cells. Mice were treated with 8 doses of anti-TGFβ therapy given every other day via intraperitoneal injection at 200 µg/mouse ( n = 10 mice/group). b Individual tumor growth curves for control and treated groups in B16 melanoma. Data are representative of three independent experiments. c Tumor size (measured as surface area in mm 2 ) of untreated animals and animals treated with anti-TGFβ1, anti-TGFβ3, and 1D11 (pan-TGFβ inhibition) in B16 melanoma (left) ( d ) and CT26 colon tumors (right). Data is displayed as ± standard error the mean (SEM). Statistics were calculated using 2-way ANOVA 24 days post tumor implantation for B16 and 23 days post tumor implantation for CT26. Only statistically significant differences among untreated and treated groups are shown. * p < 0.05; *** p < 0.0005; **** p < 0.0001.

Article Snippet: Gene-specific oligonucleotide target probes against non-homologous regions of TGFβ1 and TGFβ3 were created and pre-optimized by the manufacturer (ThermoFisher Scientific).

Techniques: Tumor Implantation, Injection, Inhibition

Journal: PLoS ONE

Article Title: Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0032783

Figure Lengend Snippet: Summary of YAP1 immunostaining in pancreatic tumors and adjacent normal samples.

Article Snippet: YAP1 expression silencing was achieved with two YAP1 siRNA duplex oligonucleotides targeting two different regions of YAP1 transcript (Qiagen, Valencia, CA) at a final concentration of 20 nM using the siLentfect transfection reagent (Bio-Rad) according to the manufacturer's protocol.

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0032783

Figure Lengend Snippet: Nuclear YAP1 expression level and its correlation with histopathological parameters in pancreatic cancer.

Article Snippet: YAP1 expression silencing was achieved with two YAP1 siRNA duplex oligonucleotides targeting two different regions of YAP1 transcript (Qiagen, Valencia, CA) at a final concentration of 20 nM using the siLentfect transfection reagent (Bio-Rad) according to the manufacturer's protocol.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0032783

Figure Lengend Snippet: A) Western blotting analysis of YAP1 protein expression in a panel of eight pancreatic cancer and two immortalized (non-transformed) pancreatic cell lines. B–E) Immunohistochemical staining for YAP1 in pancreatic cancer xenografts: B) AsPC-1; C) BxPC-3; D) MIA PaCa-2; and E) PANC-1.

Article Snippet: YAP1 expression silencing was achieved with two YAP1 siRNA duplex oligonucleotides targeting two different regions of YAP1 transcript (Qiagen, Valencia, CA) at a final concentration of 20 nM using the siLentfect transfection reagent (Bio-Rad) according to the manufacturer's protocol.

Techniques: Western Blot, Expressing, Transformation Assay, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0032783

Figure Lengend Snippet: As cell density increases, the pancreatic cancer cell lines, BxPC-3 and PANC-1, response to the deactivation of YAP1 as a transcription cofactor by cytosolic sequestration is less substantial than HPDE6. Cells were extracted at increasing cell densities after 48 hours of initial seeding. PARP serves as the loading control for the nuclear fraction. α-tubulin serves as the loading control for the whole cell lysates and cytosolic fraction.

Article Snippet: YAP1 expression silencing was achieved with two YAP1 siRNA duplex oligonucleotides targeting two different regions of YAP1 transcript (Qiagen, Valencia, CA) at a final concentration of 20 nM using the siLentfect transfection reagent (Bio-Rad) according to the manufacturer's protocol.

Techniques:

Journal: PLoS ONE

Article Title: Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0032783

Figure Lengend Snippet: The cell growth curves of A) BxPC-3 and B) PANC-1 transfected with YAP1 siRNA oligonucleotides. The independent two-sample t test (two-tailed) was utilized to calculate the statistical significance at the 96-hour time point compared to Neg siRNA. * denotes p<0.05, and ** denotes p<0.01. C) The Caspase-Glo 3/7 Assay (Promega) was used to determine the level of apoptosis in pancreatic cancer cells transfected with YAP1 siRNA oligonucleotides after 72 hours. ** denotes an independent two-sample t test (two-tailed) p-value of <0.01 when compared to the negative siRNA control (Neg siRNA).

Article Snippet: YAP1 expression silencing was achieved with two YAP1 siRNA duplex oligonucleotides targeting two different regions of YAP1 transcript (Qiagen, Valencia, CA) at a final concentration of 20 nM using the siLentfect transfection reagent (Bio-Rad) according to the manufacturer's protocol.

Techniques: Transfection, Two Tailed Test, Caspase-Glo Assay

Journal: PLoS ONE

Article Title: Down-Regulation of Yes Associated Protein 1 Expression Reduces Cell Proliferation and Clonogenicity of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0032783

Figure Lengend Snippet: A) Percentage of colonies relative to the Cells Only control on soft agar after 21 days of growth from initial seeding. p-values were calculated by comparing the YAP1 siRNA (YAP1_1 and YAP1_2) to the Neg siRNA (independent two tailed t test, two-tailed). B) qPCR analysis of YAP1 mRNA expression after 48 hours of siRNA transfection. C) Western blotting analysis of YAP1 expression after siRNA-treatment for 72 hrs. The samples for each cell line were separated and analyzed on the same blot.* denotes p<0.05, and ** denotes p<0.01.

Article Snippet: YAP1 expression silencing was achieved with two YAP1 siRNA duplex oligonucleotides targeting two different regions of YAP1 transcript (Qiagen, Valencia, CA) at a final concentration of 20 nM using the siLentfect transfection reagent (Bio-Rad) according to the manufacturer's protocol.

Techniques: Two Tailed Test, Expressing, Transfection, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

doi: 10.1111/jcmm.14809

Figure Lengend Snippet: Suv39h1 is an essential regulator of the migratory and proliferative behaviours of isolated VSMCs. Isolated VSMCs were treated with NG, MN or HG. The endogenous expression of Suv39h1, H3K9me3, complement C3, p‐ERK1/2, ERK1/2, p‐p38 and p38 were detected by Western blot and quantified from four independent experiments (A, n = 4). VSMCs were infected with either Ad‐Suv39h1 vs Ad‐Null or LV‐Suv39h1 vs LV‐NC under HG stimulation. Migration and proliferation were detected by transwell migration assay (B, n = 5) and CCK‐8 kit (C, n = 6). The expressions of molecules, including H3K9me3, complement C3, p‐ERK1/2, ERKE1/2, p‐p38, p38 and PCNA (D, n = 4), were detected by Western blot and quantified as the ratio to internal control protein GAPDH (relative protein level). ▲ P < .05, as compared to the NG group. * P < .05, as compared to the Ad‐Null group; # P < .05, as compared to the LV‐NC group

Article Snippet: To down‐regulate Suv39h1 expression, four shRNA oligonucleotides targeting different regions of the Suv39h1 gene were designed, synthesized and cloned into the lentiviral plasmid GV118 from GeneChem as described previously.

Techniques: Isolation, Expressing, Western Blot, Infection, Migration, Transwell Migration Assay, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

doi: 10.1111/jcmm.14809

Figure Lengend Snippet: Suv39h1 is the key regulator of complement C3, p15 and p16 induced by HG. The mRNA expressions of p15 and p16 were tested by RT‐PCR after transfection of Ad‐Suv39h1, Ad‐Null, LV‐Suv39h1 or LV‐NC (A‐D, n = 3). H3K9me3 levels at the promoters of complement C3, p15 and p16 in VSMCs were quantified by Chip‐PCR (E, n = 3). Moreover, Suv39h1 critically regulates the expression of complement C3 in diabetic vascular injury in vivo . Rats were fed with HFD for 4 weeks and received a single intraperitoneal injection of STZ at 40 mg/kg followed by resumption of HFD for a further 2 weeks. Carotid artery balloon injury model was established at 2 weeks after STZ injection. On day 7 after establishing the carotid artery balloon injury model and infecting the injured vessels locally with normal saline (NS; F, G), Ad‐Suv39h1 vs Ad‐Null or LV‐Suv39h1 vs LV‐NC, the injured vessels were excised, and the steady‐state mRNA levels of Suv39h1 and complement C3 were detected by RT‐PCR (from H‐K, n = 3). Three carotid arteries were pooled in each separate experiment. Nor‐rat: non‐diabetic rats undergoing carotid artery balloon injury and local inoculation of NS; DM‐rat: diabetic rats undergoing carotid artery balloon injury and local inoculation of NS. ▲ P < .05, as compared to the Nor‐rat group; * P < .05, as compared to the Ad‐Null group; # P < .05, as compared to the LV‐NC group

Article Snippet: To down‐regulate Suv39h1 expression, four shRNA oligonucleotides targeting different regions of the Suv39h1 gene were designed, synthesized and cloned into the lentiviral plasmid GV118 from GeneChem as described previously.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing, In Vivo, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

doi: 10.1111/jcmm.14809

Figure Lengend Snippet: Suv39h1 promotes neointima formation, but inhibits the re‐endothelialization of injured vessels in diabetic rats. A, On day 28 after establishing the carotid artery balloon injury model and infecting the injured vessels locally with Ad‐Suv39h1 vs Ad‐Null or with LV‐Suv39h1 vs LV‐NC, the injured vessels were isolated and stained with HE to measure medial area (MA) and intimal area (IA), as well as to calculate the I/M ratio. Representative images from each group are shown at the top and the quantification of IA and I/M ratio at the bottom. The proliferative activity (B) and re‐endothelialization (C) within the neointima were detected by immunohistochemical staining of PCNA and CD31, respectively. The representative image from each group is shown on the left (scale bar: 100 µm), and quantification of the average percentage of PCNA + cells from five random fields or the rate of CD31 + coverage cells is displayed on the right. The red arrows indicate PCNA + cells. The black arrows indicate CD31 + endothelium. Representative Masson's trichrome‐stained photomicrographs of carotid arteries from different groups were also performed (scale bar: 50 µm, D). Collagen was stained blue. * P < .05, as compared to the Ad‐Null group; # P < .05, as compared to the LV‐NC group (n = 6)

Article Snippet: To down‐regulate Suv39h1 expression, four shRNA oligonucleotides targeting different regions of the Suv39h1 gene were designed, synthesized and cloned into the lentiviral plasmid GV118 from GeneChem as described previously.

Techniques: Isolation, Staining, Activity Assay, Immunohistochemical staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Down‐regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats

doi: 10.1111/jcmm.14809

Figure Lengend Snippet: Altering Suv39h1 expression leads to altered genes controlling different biological processes in diabetic injured vessels. A, B, Heat map depicting the gene expression profiles in injured carotid artery of diabetic rats following local inoculation of either Ad‐Suv39h1 (as compared to Ad‐Null, left panel) or LV‐Suv39h1 (as compared to LV‐NC, right panel). Owing to the limited volume, the injured artery segments pooled from three individual mice were integrated into one sample. Three samples from each group were analysed by microarray analysis. GO analysis revealed the distribution of differentially expressed genes by altering vascular Suv39h1 among different biological processes (C, D) and different molecular functions (E, F)

Article Snippet: To down‐regulate Suv39h1 expression, four shRNA oligonucleotides targeting different regions of the Suv39h1 gene were designed, synthesized and cloned into the lentiviral plasmid GV118 from GeneChem as described previously.

Techniques: Expressing, Microarray