Journal: Adipocyte

Article Title: Staufen1 unwinds the secondary structure and facilitates the translation of fatty acid binding protein 4 mRNA during adipogenesis

doi: 10.1080/21623945.2021.1948165

Figure Lengend Snippet: Expression of STAU1, C/EBPα, PPARγ , and FABP4 after downregulation of STAU1 . A-D: qPCR quantification of STAU1, C/EBPα, PPARγ , and FABP4 mRNA levels on days 2, 4, and 6, as compared to the levels at day 0. E: Western blot was used to assess the levels of STAU1, C/EBPα, PPARγ, and FABP4 on days 2, 4, and 6; these levels were compared with those of negative control (NC). F-I: Quantification of STAU1, C/EBPα, PPARγ, and FABP4 expression levels assessed via western blotting on days 2, 4, and 6 and compared to the levels at day 0. The results are presented as the mean ± standard deviation (* P < 0.05, compared with NC)

Article Snippet: Two siRNAs targeting STAU1 were synthesized by Sangon Biotech according to a previous study, siRNA1: 5′-r (CAACUGUACUACCUUUCCA) d (TT)-3′; siRNA2: 5′-r (AACGGUAACUGCCAUGAUA) d (TT)-3′ [ ].

Techniques: Expressing, Western Blot, Negative Control, Standard Deviation

Journal: Adipocyte

Article Title: Staufen1 unwinds the secondary structure and facilitates the translation of fatty acid binding protein 4 mRNA during adipogenesis

doi: 10.1080/21623945.2021.1948165

Figure Lengend Snippet: Binding of STAU1 to the CDS of FABP4 . A: Results obtained using RT-PCR show the expression of FABP4 mRNA precipitated by IgG or STAU1 antibody at 0- and 4-days post-induction. Western blot showing specific IPs of STAU1 at 0- and 4-days post-induction. B: The schematic shows PAR-CLIP primers designed for the different positions. C: Secondary structure of FABP4 mRNA. Different putative SBS are indicated by different numbers. D: Results obtained via qPCR show the percentage of STAU1 binding to FABP4 mRNA at different positions as compared with input FABP4 mRNA. The results are presented as the mean ± standard deviation

Article Snippet: Two siRNAs targeting STAU1 were synthesized by Sangon Biotech according to a previous study, siRNA1: 5′-r (CAACUGUACUACCUUUCCA) d (TT)-3′; siRNA2: 5′-r (AACGGUAACUGCCAUGAUA) d (TT)-3′ [ ].

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

Journal: Adipocyte

Article Title: Staufen1 unwinds the secondary structure and facilitates the translation of fatty acid binding protein 4 mRNA during adipogenesis

doi: 10.1080/21623945.2021.1948165

Figure Lengend Snippet: STAU1 upregulates the expression of FABP4 protein. A. Polysome profiling of 3T3-L1 cells on day 4 post-induction, obtained by the continuous reading of absorbance at 254 nm after downregulation of STAU1. B. Levels of FABP4 mRNA were measured by qRT-PCR using 15 sucrose gradient fractions obtained after downregulation of STAU1 and on day 4 post-induction. C. 3T3-L1 cells were treated with 0.1 μM okadaic acid on day 4 post-induction; then, the UPF1 and FABP4 mRNA expressions were quantified by RT-qPCR. DMSO-treated 3T3-L1 cells were used as controls. D-E: Western blot showing the expression of STAU1 and FABP4 protein in OA-treated, DMSO-treated, and untreated 3T3-L1 cells

Article Snippet: Two siRNAs targeting STAU1 were synthesized by Sangon Biotech according to a previous study, siRNA1: 5′-r (CAACUGUACUACCUUUCCA) d (TT)-3′; siRNA2: 5′-r (AACGGUAACUGCCAUGAUA) d (TT)-3′ [ ].

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Adipocyte

Article Title: Staufen1 unwinds the secondary structure and facilitates the translation of fatty acid binding protein 4 mRNA during adipogenesis

doi: 10.1080/21623945.2021.1948165

Figure Lengend Snippet: STAU1 regulates adipogenesis. A. Representative images of oil red O-stained 3T3-L1 cells at days 0, 1, 2, and 3. Scale bar, 50 µm. B. Lipid accumulation was assessed at 495 nm in 3T3-L1 cells stained with oil red O and de-stained with isopropyl alcohol (n = 3). C. Glycerol content in the cell culture medium (n = 3). F. FFA content in the cell culture medium (n = 3). FFA: free fatty acid. Values are expressed as means ± SEM vs . control group, * P < 0.05

Article Snippet: Two siRNAs targeting STAU1 were synthesized by Sangon Biotech according to a previous study, siRNA1: 5′-r (CAACUGUACUACCUUUCCA) d (TT)-3′; siRNA2: 5′-r (AACGGUAACUGCCAUGAUA) d (TT)-3′ [ ].

Techniques: Staining, Cell Culture, Control

Journal: Adipocyte

Article Title: Staufen1 unwinds the secondary structure and facilitates the translation of fatty acid binding protein 4 mRNA during adipogenesis

doi: 10.1080/21623945.2021.1948165

Figure Lengend Snippet: Effect of STAU1 knockdown on body weight and lipid metabolism in diet-induced obese mice. Gene expression analysis was performed using adipose tissue from mice fed standard chow (SC) and high-fat diet (HFD). The real-time PCR analysis of STAU1 (a) and FABP4 (b) mRNA expression relative to that of wild type SC-fed mice (3–5 animals per group). C: Bodyweight per mouse of groups fed HFD or SC diet and treated with AAV-control or AAV- shSTAU1 . D: Western blot showing STAU1 and FABP4 expression in wild type SC- and HFD-fed mice after downregulation of STAU1. E-F: Quantification of STAU1 and FABP4 (f) protein levels relative to that of wild-type SC-fed mice. G: Nuclear magnetic resonance (NMR) analysis of body fat composition presented as percentage of body weight. *Represents a significant difference between wild-type group and AAV-control group or AAV shSTAU1 group of SC-fed mice. #Represents a significant difference between wild-type group and AAV-control group or AAV shSTAU1 group of HFD-fed mice

Article Snippet: Two siRNAs targeting STAU1 were synthesized by Sangon Biotech according to a previous study, siRNA1: 5′-r (CAACUGUACUACCUUUCCA) d (TT)-3′; siRNA2: 5′-r (AACGGUAACUGCCAUGAUA) d (TT)-3′ [ ].

Techniques: Knockdown, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, Nuclear Magnetic Resonance

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: Restriction enzyme digestion of DGAT1-shRNA vector by SacI. A fragment about 1000 bp was cut out as expected. Lane M: D2000 molecular weight marker; lane 1 and 2: vectors digested by SacI.

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: shRNA, Plasmid Preparation, Molecular Weight, Marker

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: Green fluorescence from BMECs transfected with the DGAT1 RNAi vector and the GFP empty vector. Twenty-four hours after transfection with the DGAT1 RNAi vector and the GFP empty vector, the expression of GFP was observed under a fluorescence microscope. (A) Cells transfected with the DGAT1 RNAi vector. (B) Cells transfected with the GFP empty vector.

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: Fluorescence, Transfection, Plasmid Preparation, Expressing, Microscopy

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: The stranded curve of plasmid for real time RT-PCR for DGAT1. Absolute quantitation with real time RT-PCR was employed to investigate the effect of DGAT1 RNAi on mRNA expression. From the equation of stranded curve (Ct = −2.591 logC0 + 30.25), the R 2 was 0.997. The value of R 2 indicated that the stranded curve could be used in the next step.

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: Plasmid Preparation, Quantitative RT-PCR, Quantitation Assay, Expressing

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: The melting curve of DGAT1 RT-PCR. The melting curve of DGAT1 RT-PCR showed that the reaction condition was qualified.

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: DGAT1 mRNA expression in mammary epithelial cell after shRNA transfection. The expression of DGAT1 was detected by real-time PCR. The copy number of positive cells (cells trans-fected with DGAT1 RNAi vector) was 84.21 ± 4.39, and the copy number of negative cells (cells transfected with GFP empty vector) was 142.91 ± 4.11. The interference efficiency was 41.6%.

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: Expressing, shRNA, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: DGAT1 expression in mammary epithelial cell detected by Western blot analysis. Western blot analysis indicated that protein expression in test cells was downregulated, compared with normal cells as a control (β-actin was used as an internal control). (A) β-Actin expression in normal mammary epithelial cells. (B) β-Actin expression in positive cells (cells transfected with DGAT1 RNAi vector). (C) DGAT1 expression in normal mammary epithelial cells. (D) DGAT1 expression in positive cells (cells transfected with DGAT1 RNAi vector).

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Gene Expression

Article Title: RNA Interference-Mediated Knockdown of DGAT1 Decreases Triglyceride Content of Bovine Mammary Epithelial Cell Line

doi:

Figure Lengend Snippet: Comparison of the TG content in positive and control cells. The TG contents of positive cells and the negative control cells were analyzed with a TG kit and compared. The difference was statistically significant (p < 0.09). 1, 2, 3: the TG contents in the control cells (cells transfected with GFP empty vector) and 4, 5, 6: the TG content in positive cells (cells transfected with DGAT1 RNAi vector).

Article Snippet: A 21-mer siRNA oligonucleotide targeting the coding sequence of DGAT1 was designed and synthesized by Genepharma Corporation (Shanghai, China).

Techniques: Comparison, Control, Negative Control, Transfection, Plasmid Preparation

Journal: Oncology Letters

Article Title: PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

doi: 10.3892/ol.2018.8075

Figure Lengend Snippet: Generation of PLAC1-specific TCR-engineered CD8+ T cells. (A) Schematic illustration of the lentiviral vector encoding an anti-PLAC1 TCR expression cassette. TCRα- and TCRβ-chains were linked with a 2A peptide sequence. (B) CD8+ T cells, selected from peripheral blood mononuclear cells with a negative selection procedure using magnetic beads, were analyzed using flow cytometry for CD3 and CD8 expression via FITC-labeled mAb against human CD8 and PerCP-Cy5.5-labeled mAb against human CD3. (C) CD8+ T cells transduced with PLAC1-specific TCR or negative control or untransduced were stained with PE-labeled PLAC1-multimer (PE-multimer) and FITC-labeled CD8 mAb (CD8-FITC). The results are representative of three independent experiments. PLAC1, placenta-specific 1; TCR, T cell receptor; CD8+ T cell, cytotoxic T cell; CD, cluster of differentiation; FITC, fluorescein isothiocyanate; PE, phycoerythrin; mAb, monoclonal antibody.

Article Snippet: One pair of siRNA oligonucleotides corresponding to the target sequence for human PLAC1 (CACCTACCGTGTTACTGAA) were designed and synthesized by RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China).

Techniques: Plasmid Preparation, Expressing, Sequencing, Selection, Magnetic Beads, Flow Cytometry, Labeling, Transduction, Negative Control, Staining

Journal: Oncology Letters

Article Title: PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

doi: 10.3892/ol.2018.8075

Figure Lengend Snippet: Identification of PLAC1 and HLA-A2 serotype-positive breast cancer cell lines. (A) Immunofluorescence staining with rabbit anti-PLAC1 primary antibody or a rabbit IgG and PE-conjugated secondary antibody in the human non-metastatic breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDAMB-231. The scale bar indicates 50 µm. (B) MCF-7 and MDAMB-231 cells were analyzed using flow cytometry for HLA-A2 expression using the PE-labeled HLA-A2 antibody and PE-labeled isotype control. The results are representative of three independent experiments. PLAC1, placenta-specific 1; HLA, human leukocyte antigen; PE, phycoerythrin; IgG, immunoglobulin G.

Article Snippet: One pair of siRNA oligonucleotides corresponding to the target sequence for human PLAC1 (CACCTACCGTGTTACTGAA) were designed and synthesized by RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Expressing, Labeling, Control

Journal: Oncology Letters

Article Title: PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

doi: 10.3892/ol.2018.8075

Figure Lengend Snippet: Evaluation of the function of PLAC1 TCR-engineered CD8+ T cells. Human leukocyte antigen-A2-restricted and PLAC1-specific TCR-, NC-transduced or untransduced CD8+ T cells were co-cultured for 4 h with 1×104 MCF-7 and MDAMB-231 cells respectively at a range of E:T ratios (5:1, 10:1 and 20:1). Concentration of (A) IFN-γ and (B) TNF-α secreted into the culture medium were measured using an enzyme-linked immunosorbent assay. (C) TCR- or NC-transduced CD8+ T cells were co-cultured for 4 h with 1×104 MCF-7 and MDAMB-231 cells. Cytolysis was determined using a lactate dehydrogenase activity assay. Subsequent to background subtraction, the percentage lysis was calculated by 100% × [(experimental release-effector spontaneous release-target spontaneous release)/(target maximum release-target spontaneous release)]. *P<0.05 and #P<0.01 with comparisons shown by lines. PLAC1, placenta-specific 1; TCR, T cell receptor; CD8+ T cell, cytotoxic T cell; NC, negative control; E:T, effector cell to target cell; IFN-γ, interferon γ; TNF-α, tumor necrosis factor α.

Article Snippet: One pair of siRNA oligonucleotides corresponding to the target sequence for human PLAC1 (CACCTACCGTGTTACTGAA) were designed and synthesized by RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China).

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Lysis, Negative Control

Journal: Oncology Letters

Article Title: PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

doi: 10.3892/ol.2018.8075

Figure Lengend Snippet: PLAC1 TCR-engineered CD8+ T cells specifically recognize and kill breast cancer cells. (A) Immunofluorescence staining with rabbit anti-PLAC1 primary antibody or a rabbit IgG and PE-conjugated secondary antibody in the human breast cancer cell lines T47D. Scale bar indicates 50 µm. The results are representative of three independent experiments. (B) T47D cells were analyzed using flow cytometry for HLA-A2 expression using the PE-labeled HLA-A2 antibody and PE-labeled isotype control. The results are representative of three independent experiments. (C) Lysis of T47D breast cancer cells by the engineered CD8+ T cells was determined using a LDH activity assay. Data are expressed as the means ± SD of three independent experiments. (D) MCF-7 cells were transfected with PLAC1 siRNA or NC siRNA and then analyzed using western blot analysis for PLAC1 expression following normalization to β-actin. The results are representative of three independent experiments. Bars represent the relative protein level as compared with the MCF-7 cells alone group. #P<0.01 vs. MCF-7 + NC siRNA group. (E) Lysis of MCF-7-NC siRNA and MCF-7-PLAC1 siRNA cells by the engineered CD8+ T cells was determined using a LDH activity assay. Data are expressed as the means ± SD of three independent experiments. *P<0.05 and #P<0.01 with comparisons shown by lines. PLAC1, placenta-specific 1; PE, phycoerythrin; IgG, immunoglobulin G; E:T, effector cell to target cell; TCR, T cell receptor; NC, negative control; siRNA, small interfering RNA; CD8+ T cell, cytotoxic T cell; HLA, human leukocyte antigen; SD, standard deviation; LDH, lactate dehydrogenase.

Article Snippet: One pair of siRNA oligonucleotides corresponding to the target sequence for human PLAC1 (CACCTACCGTGTTACTGAA) were designed and synthesized by RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China).

Techniques: Immunofluorescence, Staining, Flow Cytometry, Expressing, Labeling, Control, Lysis, Activity Assay, Transfection, Western Blot, Negative Control, Small Interfering RNA, Standard Deviation

Journal: Oncology Letters

Article Title: PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

doi: 10.3892/ol.2018.8075

Figure Lengend Snippet: TCR-transduced CD8+ T cells inhibit tumor growth in vivo. MCF-7 cells (5×106) were inoculated subcutaneously into 15 BALB/c-nu mice to establish a subcutaneous transplant tumor model. When tumors reached a mean volume of 150 mm3, the mice underwent intravenous tail vein transplantation with either normal saline, 1×107 PLAC1 TCR-transduced CD8+ T cells or NC-transduced CD8+ T cells twice with a 1-week interval. The tumors were measured once/week using electronic calipers. The longest length and width were recorded, and the tumor volume was calculated according to the formula (π/6) (length) × (width)2. *P<0.05 compared with the TCR (PLAC1) group, with comparisons shown by lines. TCR, T cell receptor; CD8+ T cells, cytotoxic T cells; PLAC1, placenta-specific 1; NC, negative control.

Article Snippet: One pair of siRNA oligonucleotides corresponding to the target sequence for human PLAC1 (CACCTACCGTGTTACTGAA) were designed and synthesized by RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China).

Techniques: In Vivo, Transplantation Assay, Saline, Negative Control

Journal: The Biochemical journal

Article Title: An interplay between the p38 MAPK pathway and AUBPs regulates c - fos mRNA stability during mitogenic stimulation

doi: 10.1042/BJ20141100

Figure Lengend Snippet: (A) HeLa TetOff cells were transfected with the reporter constructs Luc–fos, Luc–fosΔARE and Luc–β-globin along with vectors that were either empty or expressed murine HA-tagged HuR. After addition of tetracycline, mRNA decay assays were run. The Western blot on the right shows expression of murine WT HA–HuR. (B) siRNA depletion of HuR in NIH3T3 cells transfected with HuR siRNA or scrambled siRNA. Starved cells were stimulated with PDGF for 30 min, after that period cells were lysed and levels of expression of endogenous HuR and Fos proteins were visualized in Western blots. (C) A mRNA decay assay was run on extracts from cells transfected with either empty plasmid or expressing WT or a triple mutant of human HuR (T118A, S202A, S221A). The panel on the right validates expression as seen on a Western blot. (D) As in (C) but co-transfecting the reporter with vectors that express the FLAG-tagged PP2A inhibitors and HuR-binding proteins pp32 and APRIL. (E) As in (C) but in cells transfected with plasmids expressing EGFP–KSRP. Treatment with SB203580 was performed as indicated in the upper panel. In the middle panel, cells were co-transfected with the reporter and also with vectors that express EGFP–KSRP or HA–HuR. A Western blot against GFP is shown in the lower panel. (F) Endothall, a PP2A and PP1 inhibitor, was also tested at 50 μM final concentration. HeLa TetOff cells transfected with the Luc–fos reporter were incubated with endothall and/or tetracycline for different times as indicated and mRNA present in the extract was tested by RT-qPCR (real-time qPCR). In Western blots, NT corresponds to non-transfected cells. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: NIH3T3 were transfected with HiPerfect (Qiagen) with a siRNA oligonucleotide-targeting HuR sequence (Qiagen) and AllStars oligonucleotide as negative control (Qiagen).

Techniques: Transfection, Construct, Western Blot, Expressing, Mrna Decay Assay, Plasmid Preparation, Mutagenesis, Binding Assay, Concentration Assay, Incubation, Quantitative RT-PCR

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: Expression and survival curve of 8 members of CENP family. A The expression heatmap of eight members (CENPA, CENPE, CENPF, CENPH, CENPI, CENPK, CENPM, CENPU) of CENP family in TCGA ccRCC. Left: 535 ccRCC tissues; right: 72 cancer-adjacent tissues. B – I The OS and DFS curve of the eight CENP family members. In each analysis, all patients were sorted in ascending order based on corresponding gene expression, then they were divided into two groups with the same sample size. The OS and DFS of patients in two groups were visualized by Kaplan–Meier plot. OS: overall survival; DFS: disease free survival

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Expressing, Gene Expression

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of CENPA mRNA level and patient overall survival (OS)

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques:

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: CENPA was closely related to clinical traits and overexpressed in ccRCC tissues and cells. Public datasets showed CENPA overexpression in ccRCC compared to normal tissues. A In TCGA-KIRC cohort, 533 ccRCC samples showed higher CENPA expression than 72 normal samples. For tissues gathered from the same patients, CENPA overexpressed in cancer tissues compared to cancer-adjacent tissues in B TCGA-KIRC dataset (72 pairs of samples), C GSE40435 dataset (101 pairs of samples), D GSE66272 dataset (26 pairs of samples) and E Jones Renal dataset (23 pairs of samples). The expression of CENPA elevated with various clinicopathological factors in public datasets, including F , J T stage (528 samples in TCGA-KIRC and 26 samples in GSE66272), G AJCC clinical stage (527 samples in TCGA-KIRC), and H , I , K G grade (522 samples in TCGA-KIRC, 101 samples in GSE40435 and 26 samples in GSE66272). L The ROC curve of CENPA expression (AUC = 0.9651; p < 0.0001) in TCGA-KIRC cohort. Our own cohort validated CENPA overexpression in ccRCC through M qRT-PCR assays (42 pairs), N immunoblotting tests (12 pairs), and O immunohistochemical analyses (2 pairs). CENPA overexpressed in renal cancer cell lines (786-O, A498, ACHN, Caki-1 and OSRC-2) compared to normal renal cell line (HK-2) via P qRT-PCR and Q immunoblotting tests. Relative *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars indicate mean ± SD. AUC: areas under the curve

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: CENPA promoted the proliferation, migration and invasion of ccRCC cells. CENPA A , B mRNA (n = 3 per group) and C protein levels were verified by qRT-PCR in A498 and Caki-1 cells with transient CENPA knockdown or overexpression. D – F Cell viability of A498 and Caki-1 cells after depleting or overexpressing CENPA was calculated using CCK-8 assays for four days (n = 6 per group). H The cell migration and invasion ability of transfected A498 and Caki-1 cells were evaluated using transwell assays (Magnification: 200×, n = 3 per group). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars indicate mean ± SD

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Migration, Quantitative RT-PCR, Knockdown, Over Expression, CCK-8 Assay, Transfection

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: CENPA promoted the proliferation and migration of ccRCC cells. A , B The colony formation ability of transfected A498 and Caki-1 cells was evaluated by colony numbers for 12 days after seeding 1000 cells in the culture dish. The assays were independently conducted in triplicate. C – F The migration ability of transfected ccRCC cells was evaluated by wound healing assays. The cells were wounded by a 10-μl pipet when reaching 100% confluence. The images were taken 24 h or 36 h later. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars indicate mean ± SD

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Migration, Transfection

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: CENPA accelerated the cell cycle and activated the Wnt signaling pathway. A The GSEA results of CENPA using TCGA-KIRC expression dataset. 533 tumoral samples was divided into two groups based on CENPA level. Genes expression patterns of two groups was different in cell cycles and Wnt pathway. B The expression of WNT5A is positively correlated to the expression of CENPA in TCGA-KIRC ccRCC tissues. C , D The expression of cyclin D1(CCND1) and β-catenin (CTNNB1) were down-regulated or upregulated in ccRCC cells with knockdown or overexpression of CENPA. E Western blot revealed the expression of CTNNB1 in the cytoplasm and nucleus. β-actin and Lamin B1 were used as internal references in the cytoplasm and nucleus, respectively. F – G Cell cycle distribution was analyzed by PI staining in Caki-1 cells after transfection by Si-CENPA or CENPA plasmid for 48 h. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars indicate mean ± SD

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Expressing, Knockdown, Over Expression, Western Blot, Staining, Transfection, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: The Wnt/β-catenin pathway is involved in CENPA-mediated proliferation and metastasis. A A498 and Caki-1 cells with CENPA overexpression were treated with Si-CTNNB1 for 48 h. Then, expression of CTNNB1 was measured by western blotting. B , C A498 and Caki-1 cells with CENPA overexpression or knockdown were treated with Wnt-pathway inhibitor XAV-939 (10 μmol/L) or agonist CHIR-99012 (10 μmol/L) for 24 h. Then, western blotting determined that the drugs were effective to Wnt pathway. D – F Cell viability was assessed in A498 and Caki-1 cells with CENPA overexpression and Wnt pathway inhibition/excitation via CCK-8 assays (n = 4 per group). G , H Caki-1 cells with overexpression or knockdown of CENPA were treated with Si-CTNNB1 for 48 h or CHIR-99021 (10 μmol/L) for 24 h as indicated and subjected to migration assay and invasion assay (magnification: 200×, n = 3 per group). NC: negative control; n.s: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars indicate mean ± SD

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Over Expression, Expressing, Western Blot, Knockdown, Inhibition, CCK-8 Assay, Migration, Invasion Assay, Negative Control

Journal: Journal of Translational Medicine

Article Title: CENPA promotes clear cell renal cell carcinoma progression and metastasis via Wnt/β-catenin signaling pathway

doi: 10.1186/s12967-021-03087-8

Figure Lengend Snippet: Correlation between CENPA mRNA expression and clinicopathological parameters of ccRCC patients

Article Snippet: Plasmids overexpressing CENPA, siRNA targeting CENPA (si-CENPA) oligonucleotide sequences and their corresponding negative controls were constructed in Vigene Biosciences (Shandong, China).

Techniques: Expressing