Journal: International Journal of Biological Sciences

Article Title: Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

doi: 10.7150/ijbs.69351

Figure Lengend Snippet: CircDVL1 inhibits ccRCC cell proliferation and induces apoptosis in vitro . (A) The expression of circDVL1 in several ccRCC cell lines and 293T cells were detected by RT-qPCR. (B) CircDVL1 was tolerant to RNase R. Linear RNAs β-actin (ACTB) and DVL1 were significantly decreased with RNase R, while circDVL1 was almost unaffected. Mock, absence of RNase R. (C) The abundance of circDVL1 in 786-O and ACHN cells transfected with circDVL1 and control vectors (Vector) was determined by RT-qPCR. (D) Colony formation of 786-O and ACHN transfected with circDVL1 overexpression (circDVL1-OE) or control vectors. (E) The proliferation ability of 786-O and ACHN transfected with circDVL1 overexpression or control vectors was assessed with the CCK-8 assay. (F) Ki67 expression of 786-O and ACHN transfected with circDVL1 overexpression or control vectors was assessed with immunofluorescence staining assays. Scale bars: 50 µm. (G) The percentage of apoptosis distributions cells in each stage was evaluated by flow cytometry. (H) Cell cycle profile of 786-O and ACHN transfected with circDVL1 overexpression or control vectors and quantitative analysis of results from two independent experiments are shown. (I) Western blot results of CDK6, Bcl-2, and Bax levels in 786-O and ACHN transfected with circDVL1 overexpression or control vectors, and quantitative analysis of results from two independent experiments are shown. GAPDH expression was served as a loading control. Data are exhibited as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, no significant (NS).

Article Snippet: Cell viability assay was determined with the CCK8 kit (Dojindo Laboratories, Kumamoto, Japan, Cat# CK04), as previously described .

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Over Expression, CCK-8 Assay, Immunofluorescence, Staining, Flow Cytometry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

doi: 10.7150/ijbs.69351

Figure Lengend Snippet: miR-412-3p promotes ccRCC cell proliferation, migration and invasion in vitro . (A) The expression levels of miR-412-3p in 786-O, ACHN, and 293T were detected by RT-qPCR. (B) The proliferation of 786-O and ACHN transfected with miR-412-3p mimic and miR-NC mimic was detected by the CCK-8 proliferation assay. (C) Transwell migration and invasion assays indicated that miR-412-3p mimic enhanced the migration and invasion capacities of 786-O and ACHN after transfection. (D) The colony formation ability of 786-O and ACHN transfected with miR-412-3p mimic and miR-NC. (E) Western blot results of CDK6, Bcl-2, and Bax levels in ccRCC cells lines after transfection with miR-412-3p mimic or miR-NC. (F) CCK-8 cell proliferation analysis of 786-O and ACHN cotransfected with control vectors + control mimics or circDVL1 overexpression vector + control mimics or circDVL1 overexpression vector + miR-412-3p mimics. (G) Transwell cell invasion and migration assay in treated 786-O and ACHN cells. Scale bar, 100 µm. circDVL1-OE, circDVL1 overexpression vector, negative control for overexpression. miR-NC mimic was used as the miRNA mimic negative control. Data are exhibited as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, no significant (NS).

Article Snippet: Cell viability assay was determined with the CCK8 kit (Dojindo Laboratories, Kumamoto, Japan, Cat# CK04), as previously described .

Techniques: Migration, In Vitro, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Proliferation Assay, Western Blot, Control, Over Expression, Plasmid Preparation, Negative Control

Journal: International Journal of Biological Sciences

Article Title: Circular RNA circDVL1 inhibits clear cell renal cell carcinoma progression through the miR-412-3p/PCDH7 axis

doi: 10.7150/ijbs.69351

Figure Lengend Snippet: PCDH7 is a target of miR-412-3p and suppresses ccRCC proliferation, migration and invasion. (A) Volcano plot showing downstream genes with significantly altered expression in 786-O cell overexpressing miR-412-3p and miR-NC mimics. (B) Schematic illustration exhibiting the overlap between the target mRNAs and miR-412-3p as detected by TargetScan, miRDB, and RNA-seq. (C) 786-O and ACHN were transfected with miR-412-3p or miR-NC mimics for 48 h, and the expression of downstream genes was analyzed by qPCR. (D) The sequence of WT and MUT putative miR-412-3p-binding sites in the 3'-UTR of PCDH7. (E) Relative luciferase activities were examined in 293T after transfection with PCDH7-WT or PCDH7-MUT and miR-412-3p mimic or miR-NC. (F) Western blot results of PCDH7 levels in ccRCC cell lines after transfection with miR-412-3p mimic or miR-NC. (G) PCDH7 expression in 786-O, ACHN, and 293T was analyzed by RT-qPCR. (H) The relative abundance of PCDH7 in 786-O and ACHN cells transfected with si-NC and si-PCDH7 was determined by qRT-PCR. (I) Western blot of PCDH7 levels in ccRCC cell lines after transfection si-NC and si-PCDH7. (J) Colony formation ability of 786-O and ACHN cells transfected with si-NC and si-PCDH7. (K) The proliferation ability of 786-O and ACHN transfected with si-NC and si-PCDH7 was tested by the CCK-8 assay. (L) Transwell migration and invasion assays indicated that si-PCDH7 promoted migration and invasion capacity of transfected 786-O and ACHN. Scale bar, 100 µm. si-NC, siRNA for NC. si-PCDH7, siRNA against PCDH7. miR-NC mimic was used as the miRNA mimic NC. Data are exhibited as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, no significant (NS).

Article Snippet: Cell viability assay was determined with the CCK8 kit (Dojindo Laboratories, Kumamoto, Japan, Cat# CK04), as previously described .

Techniques: Migration, Expressing, RNA Sequencing, Transfection, Sequencing, Binding Assay, Luciferase, Western Blot, Quantitative RT-PCR, CCK-8 Assay

Journal: Oncology Reports

Article Title: VNN1 overexpression in pancreatic cancer cells inhibits paraneoplastic islet function by increasing oxidative stress and inducing β‑cell dedifferentiation

doi: 10.3892/or.2023.8557

Figure Lengend Snippet: The viability and function of paraneoplastic islets are improved after suppressing VNN1-induced oxidative stress by GSH or TZD. (A and B) The extracellular and intracellular cysteamine concentrations were detected using high-performance liquid chromatography. (C) ROS contents in islets were analyzed using flow cytometry. (D) GSH concentrations in islets were detected using spectrophotometry. (E) PPARγ expression in islets was examined using western blot analysis. β-actin was used as the loading control. (F) Following islet pre-treatment with GSH or TZD, islet viability was determined using Trypan blue staining. (G) Following islet pre-treatment with GSH or TZD, cleaved caspase-3/9 levels in islets were examined using western blot analysis. β-actin was used as the loading control. (H) Following islet pre-treatment with GSH or TZD, the insulin secretion was determined using radioimmunoassay. Data are presented as the mean ± SD (n=3). Data were analyzed using one-way ANOVA followed by Tukey's post-hoc test. *P<0.05 compared with the PANC-1, CFPAC-1, PV or control group. ROS, reactive oxygen species; PPARγ, peroxisome proliferator activated receptor gamma; GSH, glutathione; TZD, thiazolidinedione; PV, PANC-1 cells with the stable overexpression of VNN1; PE, PANC-1 cells transfected with empty vector; CV, CFPAC-1 cells with the stable overexpression of VNN1; CE, CFPAC-1 cells transfected with empty vector; VNN1, Vanin-1.

Article Snippet: The supernatants were collected following centrifugation at 8,000 × g for 10 min at 4°C and GSH in the supernatants was detected using a Total Glutathione Quantification kit (cat. no. T419; Dojindo Laboratories, Inc.).

Techniques: High Performance Liquid Chromatography, Flow Cytometry, Spectrophotometry, Expressing, Western Blot, Control, Staining, RIA Assay, Over Expression, Transfection, Plasmid Preparation

Journal: International Journal of Nephrology and Renovascular Disease

Article Title: FGF23 modulates the effects of erythropoietin on gene expression in renal epithelial cells

doi: 10.2147/ijnrd.s158422

Figure Lengend Snippet: Figure 5 Expression of EpoR and Klotho protein by mouse bone marrow cells. Notes: To confirm that mouse bone marrow cells expressed EpoR and Klotho, these cells were assayed by flow cytometry. Aliquots of 106 bone marrow cells were incubated with anti-EpoR–FITC and anti-Klotho–PE antibodies, and the cells were analyzed on an FACS Aria flow cytometer (BD Biosciences, San Jose, CA, USA). (A) Erythroblast gating (P1) of bone marrow cells (FSC-A, SSC-A). (B) P1 gated, isotype controls. (C) P1 gated, EpoR- and Klotho-labeled. Bone marrow cells were obtained from the femurs and tibias of male C57BL/6J mice. PE was conjugated to an anti-human Klotho monoclonal antibody (cat. no. KO603) using a Phycoerythrin Labeling Kit- NH2 (Dojindo Molecular Technologies, Kumamoto, Japan). FITC was conjugated to an anti-mouse EpoR monoclonal antibody (cat. no. ab56310) using a Fluorescein Labeling Kit-NH2 (Dojindo Molecular Technologies). Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; FSC-A, forward scatter; SSC-A, side scatter.

Article Snippet: FITc was conjugated to an anti-mouse epoR monoclonal antibody (cat. no. ab56310) using a Fluorescein labeling Kit-Nh2 (Dojindo Molecular Technologies).

Techniques: Expressing, Flow Cytometry, Incubation, Labeling