Journal: Cells

Article Title: MiR-34b Regulates Muscle Growth and Development by Targeting SYISL

doi: 10.3390/cells14050379

Figure Lengend Snippet: All primers used in RT-qPCR.

Article Snippet: Antibodies that were used included MyoG (sc-12732; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), MyHC (sc-376157; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (sc-4777; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), CDK6 (BA1513; 1:300; BOSTER, Wuhan, China), Ki67 (ab16667; 1:1000; Abcam, Cambridge, United Kingdom), p21 (BM4382; 1:200; BOSTER, Wuhan, China), and a secondary antibody (1:3000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Sequencing

Journal: Cells

Article Title: MiR-34b Regulates Muscle Growth and Development by Targeting SYISL

doi: 10.3390/cells14050379

Figure Lengend Snippet: Effects of miR-34b on genes related to myoblast proliferation. ( A ) The inhibiting effect of miR-34b inhibitor is remarkable. Data were presented as mean ± SDs, n = 3. * p < 0.05. ( B ) RT-qPCR of proliferated C2C12 myoblasts showed that CDK6 and ki67 levels are significantly increased and p21 level is significantly decreased in miR-34b knockdown ( miR-34b inhibitor) group compared with the negative control (miR-NC) group. Data were presented as mean ± SDs, n = 3. * p < 0.05. ( C ) Western blotting of proliferated C2C12 myoblasts showed that CDK6 and ki67 protein levels are significantly increased and p21 protein level is significantly decreased in miR-34b knockdown ( miR-34b inhibitor) group compared with the negative control (miR-NC) group. Data were presented as mean ± SDs, n = 3. * p < 0.05. ( D ) Representative photograph of EdU staining in proliferating C2C12 myoblasts. Quantification of three independent experiments showed that cell proliferation is promoted after inhibiting miR-34b . Data were presented as mean ± SDs, n = 3 * p < 0.05. ( E ) The overexpression effect of miR-34b mimics is remarkable. Data were presented as mean ± SDs, n = 3. *** p < 0.001 ( F ) RT-qPCR of proliferated C2C12 myoblasts shows that CDK6 and ki67 levels are significantly decreased and p21 level is significantly increased in miR-34b overexpression ( miR-34b mimics) group compared with the negative control (miR-NC) group. Data were presented as mean ± SDs, n = 3. * p < 0.05. ( G ) Western blotting of proliferated C2C12 myoblasts showed that CDK6 and ki67 protein levels are significantly decreased and p21 protein level is significantly increased in miR-34b overexpression ( miR-34b mimics) group compared with the negative control (miR-NC) group. Data were presented as mean ± SDs, n = 3. * p < 0.05. ( H ) Representative photograph of EdU staining in proliferating C2C12 myoblasts. Quantification of three independent experiments showed that cell proliferation is inhibited after miR-34b overexpression. Data were presented as mean ± SDs, n = 3. * p < 0.05.

Article Snippet: Antibodies that were used included MyoG (sc-12732; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), MyHC (sc-376157; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (sc-4777; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), CDK6 (BA1513; 1:300; BOSTER, Wuhan, China), Ki67 (ab16667; 1:1000; Abcam, Cambridge, United Kingdom), p21 (BM4382; 1:200; BOSTER, Wuhan, China), and a secondary antibody (1:3000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Knockdown, Negative Control, Western Blot, Staining, Over Expression

Journal: Cells

Article Title: MiR-34b Regulates Muscle Growth and Development by Targeting SYISL

doi: 10.3390/cells14050379

Figure Lengend Snippet: MiR-34b regulates myoblast proliferation and differentiation by targeting SYISL . ( A ) RT-qPCR of proliferated C2C12 myoblasts showed that p21 level is significantly decreased in the SYISL overexpression vector of miR-34b binding site mutation and miR-34b mimics co-transmutation treatment group (pcDNA3.1-mut SYISL + miR-34b mimics) compared with the wild-type SYISL overexpression vector and miR-34b mimics co-transmutation treatment group (pcDNA3.1- SYISL + miR-34b mimics). Data were presented as mean ± SDs, n = 3. ns p ≥ 0.05, * p < 0.05. ( B ) RT-qPCR of differentiated C2C12 myoblasts showed that MyoG level is significantly decreased in the SYISL overexpression vector of miR-34b binding site mutation and miR-34b mimics co-transmutation treatment group (pcDNA3.1-mut SYISL + miR-34b mimics) compared with the wild-type SYISL overexpression vector and miR-34b mimics co-transmutation treatment group (pcDNA3.1- SYISL + miR-34b mimics). Data were presented as mean ± SDs, n = 3. ns p ≥ 0.05, * p < 0.05. ( C ) Western blotting of proliferated C2C12 myoblasts showed that p21 protein level is decreased in the SYISL overexpression vector of miR-34b binding site mutation and miR-34b mimics co-transmutation treatment group (pcDNA3.1-mut SYISL + miR-34b mimics) compared with the wild-type SYISL overexpression vector and miR-34b mimics co-transmutation treatment group (pcDNA3.1- SYISL + miR-34b mimics). Data were presented as mean ± SDs, n = 3. ns p ≥ 0.05, ** p < 0.01. ( D ) Western blotting of differentiated C2C12 myoblasts shows that MyoG protein level is decreased in the SYISL overexpression vector of miR-34b binding site mutation and miR-34b mimics co-transmutation treatment group (pcDNA3.1-mut SYISL + miR-34b mimics) compared with the wild-type SYISL overexpression vector and miR-34b mimics co-transmutation treatment group (pcDNA3.1- SYISL + miR-34b mimics). Data were presented as mean ± SDs, ns p ≥ 0.05, n = 3. *** p < 0.001. ( E ) Representative images of EdU staining of the proliferation in mouse myogenic progenitor and quantification of three independent experiments showed that cell proliferation was inhibited after miR-34b overexpression (WT + miR-34b mimics) in wild-type cells compared to the negative control (WT + miR-NC). After SYISL knockout, there was no significant difference in cell proliferation after miR-34b overexpression (KO + miR-34b mimics) compared with negative control (KO + miR-NC). Scale bars, 50 μm. Data were presented as mean ± SDs, ns p ≥ 0.05, n = 3. * p < 0.05. ( F ) Representative images of immunofluorescence staining in differentiated mouse myogenic progenitor and MyHC quantification of three independent experiments showed that overexpression of miR-34b (WT + miR-34b mimics) promoted cell differentiation compared with negative control (WT + miR-NC). After SYISL knockout, there was no significant difference in cell differentiation after miR-34b overexpression (KO + miR-34b mimics) compared with negative control (KO+miR-NC). Scale bars, 50 μm. Data were presented as mean ± SDs, n = 3. ns p ≥ 0.05, * p < 0.05.

Article Snippet: Antibodies that were used included MyoG (sc-12732; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), MyHC (sc-376157; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (sc-4777; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), CDK6 (BA1513; 1:300; BOSTER, Wuhan, China), Ki67 (ab16667; 1:1000; Abcam, Cambridge, United Kingdom), p21 (BM4382; 1:200; BOSTER, Wuhan, China), and a secondary antibody (1:3000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Over Expression, Plasmid Preparation, Binding Assay, Mutagenesis, Western Blot, Staining, Negative Control, Knock-Out, Immunofluorescence, Cell Differentiation

Journal: BioMed Research International

Article Title: Blockage of TGF- α Induced by Spherical Silica Nanoparticles Inhibits Epithelial-Mesenchymal Transition and Proliferation of Human Lung Epithelial Cells

doi: 10.1155/2019/8231267

Figure Lengend Snippet: Effects of TGF- α and its neutralizing antibodies on the EMT of BEAS-2B cells and tumor growth. (a) The effects of TGF- α (0.4 ng/mL) and anti-TGF- α (1.6 ng/mL) on the proliferation of BEAS-2B cells, control group treated only by BPDE. (b) The colony formation rate of BEAS-2B cells. (c) The growth rate curve of xenografted tumors. (d, e) Ki67, PCNA, and proteins involved EMT in tumor tissue were detected by western blotting. (f) The apoptosis of the tumor tissue was detected using the TUNEL assay, and the markers of EMT in tumor tissue were detected by immunohistochemistry in tumor tissues.

Article Snippet: The separated protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane after blocking with defatted milk at 37°C for 2 hours and incubated at 4°C overnight with anti-PCNA antibody (2586, Cell Signaling Technology, USA) and ki67 antibody (BM438, Boster, China); antibodies for proteins involved in EMT used the same as IHC method, goat anti-mouse and rabbit (ab205719, ab205718) as second antibody.

Techniques: Control, Western Blot, TUNEL Assay, Immunohistochemistry

Journal: Molecular Cancer

Article Title: LncRNA PVT1 promotes gemcitabine resistance of pancreatic cancer via activating Wnt/β-catenin and autophagy pathway through modulating the miR-619-5p/Pygo2 and miR-619-5p/ATG14 axes

doi: 10.1186/s12943-020-01237-y

Figure Lengend Snippet: PVT1 promotes pancreatic cancer cell resistance to gemcitabine in vitro and in vivo . a After nocodazole treatment for 12 h, the expression level of PVT1 in PANC-1 and SW1990 human pancreatic cancer cell lines and the PANC-1/Gem and SW1990/Gem gemcitabine resistant cell lines was examined by real-time qPCR. b and c PANC-1 cells were treated with gemcitabine at different concentrations and for different durations as indicated. Then, the expression levels of PVT1 were determined. d-g PANC-1/Gem and SW1990/Gem cells with stable PVT1knockdown and PANC-1 and SW1990 cells with stable PVT1 expression were treated with gemcitabine at different concentrations for 48 h, and cell viability was then measured by MTT assay. h PANC-1 cells stably expressing PVT1 were treated with gemcitabine (1 μM) for different durations as indicated, and cell viability was measured by MTT assay. i and j Apoptotic cells among PVT1 overexpressing PANC-1 and ASPC-1 cells treated with gemcitabine were analyzed by TUNEL assay, the number of TUNEL-positive cells was quantified. Scale bars: 100 μm. k and l Representative photographs of tumor-bearing mice in different groups and tumors excised from the mice were shown. m Growth curve showing changes in tumor volume in mice from different groups; growth was assessed every 5 days beginning from the injection and during gemcitabine (50 mg/kg) treatment. n Weight of the tumors excised from mice in each group. o and p Representative H&E staining images and immunohistochemical images of Ki67 in excised tumor tissues. Scale bars: 100 μm. Data were represented as mean ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Commercially available antibodies and dilutions used are as follows: anti-β-catenin (Proteintech, 51,067–2-AP; 1:1000 dilution), anti-CyclinD1 (Proteintech, 60,186–1-Ig; 1:1000 dilution), anti-C-myc (Proteintech, 60,178–1-Ig; 1:1000 dilution), anti-Axin2 (Proteintech, 20,540–1-AP; 1:1000 dilution), anti-Ki67 (BOSTER, BM4381; 1:1000 dilution), anti-GAPDH (Proteintech, 60,004–1-Ig; 1:1000 dilution), anti-LC3 (Cell Signaling Technology, 4108; 1:1000 dilution), anti-p62/SQSTM1 (Proteintech, 18,420–1-AP; 1:1000 dilution), anti-Mouse HA (EMD Millipore, M180–3; 1:1000 dilution), anti-Rabbit HA (Proteintech, 51,064–2-AP; 1:1000 dilution), anti-Pygo2 (Santa Cruz, sc-390,506, 1:1000 dilution), anti-P-Glycoprotein/P-gp (Proteintech, 22,336–1-AP; 1:1000 dilution), anti-Ago2 (Proteintech, 10,686–1-AP; 1:1000 dilution), anti-ATG14/Barkor (N-Terminal) (Proteintech, 19,491–1-AP; 1:1000 dilution), anti-Beclin1/BECN1 (Proteintech, 11,306–1-AP; 1:1000 dilution), anti-PIK3C3/VPS34 (Proteintech, 12,452–1-AP; 1:1000 dilution). anti-ATG7 (Proteintech, 67,341–1-Ig; 1:1000 dilution). anti-ATG9 (Proteintech, 26,276–1-AP; 1:1000 dilution). anti-PIK3C3/VPS34 (Proteintech, 12,452–1-AP; 1:1000 dilution). anti-ULK1 (Boster, BM5170; 1:500 dilution).

Techniques: In Vitro, In Vivo, Expressing, MTT Assay, Stable Transfection, TUNEL Assay, Injection, Staining, Immunohistochemical staining