Journal: International Journal of Biological Sciences

Article Title: DHA Sensor GPR120 in Host Defense Exhibits the Dual Characteristics of Regulating Dendritic Cell Function and Skewing the Balance of Th17/Tregs

doi: 10.7150/ijbs.39551

Figure Lengend Snippet: MyD88 and NF-κB facilitate FA metabolism and inhibit FFAR expression. (A) Flow-cytometric analysis of the proportion of CD86, CD80, and MHCII in CD11c-positive cells that are stimulated by RV, JEV, LPS, and Poly (I:C) for 24 h, and then incubated with ST2825 or PDTC for 12 hpi. (B) Analysis of DC viability after treatment with ST2825 and PDTC using trypan blue. (C) Changes in mRNA expression in DC stimulated with RV, RV and ST2825, and LPS. After 48 h of treatment, the miRNA expression level was measured by RNA deep sequencing and compared with those for the mock-treated control. The color scale is based on log 2 changes in expression. (D) Quantitative RT-PCR (qRT-PCR) analyses of the fatty acid transporter protein expression levels in DC stimulated with RV and LPS. (E) Quantitative RT-PCR (qRT-PCR) analyses of the fatty acid receptor proteins.

Article Snippet: ST2825 and PDTC were purchased from Apexbio (Houston, TX, USA) and dissolved in dimethylsulfoxide (DMSO).

Techniques: Expressing, Incubation, Sequencing, Control, Quantitative RT-PCR

Journal: Oncology Reports

Article Title: TNF-α promotes the malignant transformation of intestinal stem cells through the NF-κB and Wnt/β-catenin signaling pathways

doi: 10.3892/or.2020.7631

Figure Lengend Snippet: Effects of inhibitors of the NF-κB and Wnt/β-catenin pathways on their activities in TNF-α-induced NCM460s cells. NCM460s cells were respectively incubated with 1, 10, 100 µM of (A) PDTC or (B) IWP-2 without or with 1 ng/ml TNF-α for 3 h. Western blotting was used to analyze the protein expression of genes related with NF-κB and Wnt/β-catenin signaling in the total cell extracts and the nuclear/cytoplasmic extracts. TNF, tumor necrosis factor; NCM460s, NCM460 spheroid; PDTC, pyrrolidine dithiocarbamate; IKKα/β, IκB kinase complex α/β; GSK, glycogen synthase kinase.

Article Snippet: The NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; cat. no. M4005) and Wnt inhibitor IWP-2 (cat. no. M2237) were purchased from Abmole (Abmole Bioscience, Inc.).

Techniques: Incubation, Western Blot, Expressing

Journal: Biomolecules & Therapeutics

Article Title: Platelet-Derived Growth Factor Receptor-α Subunit Targeting Suppresses Metastasis in Advanced Thyroid Cancer In Vitro and In Vivo

doi: 10.4062/biomolther.2020.205

Figure Lengend Snippet: CRISPR/Cas9-based PDGFRA gene editing in SW579 cells. (A) Schematic representation of the human PDGFRA DNA locus and two protospacer sequences (blue underline) of CRISPR/Cas9 gene editing. The protospacer adjacent motif (PAM, red underline) is the motif required for Cas9 nuclease activity. The arrowhead on the protospacer sequence indicates the expected cleavage site. Scrambled (SC) sgRNA and two PDGFRA sgRNAs containing lentivirus were independently delivered to SW579 cells. After transduction, DNA from virus-infected cells was purified and subjected to Sanger sequencing of PDGFRA exon 2 and exon 3. (B) and (C) show the wild-type PDGFRA sequences of SW579 cells. (D) PDGFRA sgRNA_1 produced a multiplex DNA sequence mixture around the expected Cas9 cleavage point in a pool of gene-edited cells, whereas (E) PDGFRA sgRNA_2 retained the wild-type DNA sequence. Using TIDE algorithm analysis, it was shown that (F) PDGFRA sgRNA_1 virus-transfected SW579 cells obtain an aberrant sequence signal in the scrambled mixture (green vs black), indicating that PDGFRA sgRNA_1 had a significantly high editing efficiency (indels, insertions and deletions) (G), whereas PDGFRA sgRNA_2 cells showed no indels presence in Sanger sequencing. (H) The pie chart demonstrates PDGFRA sgRNA_1 infection with different efficiency rates caused different gene indels in the whole cell population. The gene editing efficiency of PDGFRA sgRNA_1 was 95.6% (pink color), and the two most common -1 and other indels were presented at rates of 41% (green color) and 28.9% (brown color), respectively. (I) The PDGFRA gene in SW579 cells was further analyzed by RGEN-RFLP assay to quantify the gene editing efficiency. The agarose image of PDGFRA gene cleavage with specific PDGFRA sgRNA_1 and PDGFRA sgRNA_2 and Cas9 additions shows the indel percentage in the gene editing pool in vitro . The fragments of cleaved DNA are highlighted with an asterisk.

Article Snippet: Human thyroid squamous cell carcinoma SW579 cell line was purchased by Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: CRISPR, Activity Assay, Sequencing, Transduction, Virus, Infection, Purification, Produced, Multiplex Assay, Transfection, RFLP Assay, In Vitro

Journal: Biomolecules & Therapeutics

Article Title: Platelet-Derived Growth Factor Receptor-α Subunit Targeting Suppresses Metastasis in Advanced Thyroid Cancer In Vitro and In Vivo

doi: 10.4062/biomolther.2020.205

Figure Lengend Snippet: The alteration of EMT markers and invasion ability in PDGFRA gene-edited cells. (A) Western blotting of PDGFRA protein expression in wild-type (WT), scrambled (SC), PDGFRA sgRNA_1 and PDGFRA sgRNA_2 virus-transfected SW579 cells. (B) Western blotting of EMT markers in SC- and PDGFRA sgRNA_1 virus-transfected SW579 cells. The EMT markers included vimentin, E-cadherin, N-cadherin and slug proteins, whereas GAPDH served as an internal loading control. (C) The Matrigel-based Transwell assay was used to assess the cancer invasion abilities of SC- and PDGFRA sgRNA_1 virus-transfected SW579 cells. Three days after plating, the cells that migrated through the Transwell membrane were washed, fixed and stained with 0.5% crystal violet. Images of migrated SC- and PDGFRA sgRNA_1 virus-transfected SW579 cells were taken at 100× microscope magnification and the cells were counted. Scale bar=100 µm. Data are presented as the mean and standard error of the number of invading cells from microarray analysis. Off-target investigation of the PDGFRA -targeted CRISPR/Cas9 system. (E) The CRISPR Design website was used to predict off-target candidate genes for PDGFRA sgRNA_1 viruses. Similarities from off-target candidate genes in the human DNA sequence are presented as dots, and mismatch sites are indicated by nucleotide substitution. (F) Sanger sequencing of SW579 cells infected with the SC or PDGFRA sgRNA_1 virus was used to determine the potential indel presence.

Article Snippet: Human thyroid squamous cell carcinoma SW579 cell line was purchased by Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Western Blot, Expressing, Virus, Transfection, Control, Transwell Assay, Membrane, Staining, Microscopy, Microarray, CRISPR, Sequencing, Infection

Journal: Biomolecules & Therapeutics

Article Title: Platelet-Derived Growth Factor Receptor-α Subunit Targeting Suppresses Metastasis in Advanced Thyroid Cancer In Vitro and In Vivo

doi: 10.4062/biomolther.2020.205

Figure Lengend Snippet: PDGFRA gene editing decreased spontaneous pulmonary metastasis in an animal model. Female NOD-SCID mice were divided into 2 groups and SC- or PDGFRA sgRNA_1 virus-transfected SW579 cells were subcutaneously injected into the mammary fat pad. (A) Mouse body weights and (B) xenograft volumes were measured every week during tumor development. The mice were sacrificed 10 weeks after tumor implantation. (C) The lung tissue slides from the two groups were stained for IF with vimentin (green) or Hoechst (red). Scale bar=50 μm. (D) Spontaneous pulmonary metastatic foci were counted. The values are presented as the mean and standard error. Data were analyzed with Student’s t-test; all p -values are two-sided. p -values less than 0.05 are indicated as *, whereas p -values less than 0.01 are indicated as **. (E) Xenografts from both the SC and PDGFRA sgRNA_1 groups were examined for PDGFRA and EMT marker expression. The EMT markers included vimentin, E-cadherin, N-cadherin and slug, and β-actin and GAPDH served as internal loading controls. The two randomly selected duplicate lung tissues and xenografts from each group are presented as #1 and #2 in the IF staining and protein analysis.

Article Snippet: Human thyroid squamous cell carcinoma SW579 cell line was purchased by Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Animal Model, Virus, Transfection, Injection, Tumor Implantation, Staining, Marker, Expressing

Journal: Biomolecules & Therapeutics

Article Title: Platelet-Derived Growth Factor Receptor-α Subunit Targeting Suppresses Metastasis in Advanced Thyroid Cancer In Vitro and In Vivo

doi: 10.4062/biomolther.2020.205

Figure Lengend Snippet: Imatinib suppressed cell growth signals, induced cell cycle protein regulation and cell apoptosis in SW579 cells. (A) The IC50 values of DMSO control- or imatinib-treated SW579 cells were determined using MTT assays after treatment for 24 (green color) and 48 h (red color). Imatinib significantly (B) induced P21, P27, P53 and cleaved PARP (c-PARP) protein expressions and (C) inhibited AKR and ERK phosphorylations (p-AKT and p-ERK) in a dose-dependent manner, as indicated by western blot analysis. The (D) p-AKT and (E) p-ERK suppression ratios were measured in triplicate experiments and normalized by their total AKT (t-AKT) and ERK (t-ERK) protein expressions. The protein expression was qualified by ImageJ software. (F) The LIVE/DEAD cell viability assay was performed by DMSO control and 1 and 10 μM imatinib treatment of SW579 cells for 24 h. Cells were subjected to viability assays to identify live (green) and dead (red) cells. (G) Cell death was quantified by counting dead cells in a 50× microscope magnification image and analyzed for significance. Scale bar=250 µm. (H) The NIADS detected imatinib-induced cell apoptosis in SW579 cells. SW579 cells stably expressing the NIADS probe were exposed to either DMSO or 1 and 10 µM imatinib for 12 h, and luciferase activity was measured. The cells were exposed to luciferin at a concentration of 1.5 μg/mL before bioluminescence detection. The intense luciferase signal indicates apoptotic signals from the NIADS. The bar figure illustrates the percent mean and standard error of the photons in the treatment group compared to the DMSO group. Data were analyzed with Student’s t-test; all p -values are two-sided. All p-values less than 0.01 are indicated as **.

Article Snippet: Human thyroid squamous cell carcinoma SW579 cell line was purchased by Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan.

Techniques: Control, Western Blot, Expressing, Software, Viability Assay, Microscopy, Stable Transfection, Luciferase, Activity Assay, Concentration Assay

Journal: Free radical biology & medicine

Article Title: Resolvin D1 blocks H 2 O 2 -mediated inhibitory crosstalk between SHP2 and PP2A and suppresses endothelial-monocyte interactions

doi: 10.1016/j.freeradbiomed.2018.01.034

Figure Lengend Snippet: A & C. Quiescent HUVECs were treated with and without LPS (500 ng/ml) for the indicated time periods and equal amount of protein from each condition was analyzed by Western blotting for the indicated proteins using their specific antibodies. B & D. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml) for the indicated time periods and equal amounts of proteins from each condition were analyzed by Western blotting for the indicated proteins using their specific antibodies. E, F, H & I. Quiescent HUVEC monolayer was treated with and without LPS (500 ng/ml) in the presence and absence of RvD1 (200 ng/ml), PDTC (50 μM), QNZ (10 μM) or neutralizing ICAM1 or VCAM1 antibodies (2 μg/dish) for 2 hrs. After the treatments, the HUVEC monolayer was washed and BCECF-AM-labeled THP1 cells were seeded onto the monolayer and incubated for 1 hr for adhesion (E & H) and overnight for transmigration (F & I) assays. G. Quiescent HUVECs were treated with and without LPS (500 ng/ml) in the presence and absence of PDTC (50 μM) or QNZ (10 μM) for 1 hr and equal amount of protein from each condition was analyzed by Western blotting for the indicated proteins using their specific antibodies. The bar graphs represent quantitative analysis of three experiments. The values are expressed as Means ± SD. *, p < 0.05 vs control; **, p < 0.05 vs LPS.

Article Snippet: Pyrrolidinedithiocarbamic acid (PDTC) (20713), Pertussis toxin (19546), QNZ (10006734) and Resolvin D1 (10012554) were purchased from Cayman Chemical Company (Ann Arbor, MI).

Techniques: Western Blot, Labeling, Incubation, Transmigration Assay, Control