Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A activates the Wnt/β-catenin pathway by stabilizing β-catenin. A-B , MYC, CCND1 and nuclear β-catenin levels in NSCLC cells as indicated were analyzed by western blotting. C , CTNNB1 transcript levels in NSCLC cells as indicated was analyzed by qRT-PCR. ns, not significant. D , Subcellular localization of SETD1A and β-catenin in NSCLC cells was analyzed by confocal laser scanning microscope. The subcellular distribution of SETD1A and β-catenin was quantified by Image J software. E , The interaction between SETD1A and β-catenin in PC9 cells was analyzed by CoIP assay. F - G , β-catenin stability in the SETD1A knockdown and negative control group PC9 cells was analyzed by CHX chase assay. H , Ubiquitination of β-catenin in PC9 cells was analyzed by CoIP assay following SETD1A knockdown. I , Two-step co-immunoprecipitation of the complex containing SETD1A, β-catenin and PKA is shown. HEK293T cells were cotransfected with the Flag-tagged SETD1A plasmid and HA-tagged β-catenin plasmid. The first immunoprecipitation was performed using anti-FLAG M2 beads. The complex was eluted using 3 × Flag peptide followed by the second step of co-immunoprecipitation with anti-HA antibody. Then the protein samples were analyzed by western blotting with anti-Flag, anti-HA and anti- anti-PKAα cat antibody. Data are shown as means ± SD.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Western Blot, Quantitative RT-PCR, Laser-Scanning Microscopy, Software, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A binds to β-catenin via its SET domain, A , Schematic representation of SETD1A mutants. B , HEK293T cells were cotransfected with the indicated plasmids of Flag-tagged SETD1A mutants and HA-tagged full length β-catenin. Cell lysates were immunoprecipitated with anti-Flag antibody. C , The total, cytoplasmic and nuclear β-catenin levels were detected by western blot analysis following transfection with the empty vector, wild-type SETD1A plasmid and ΔSET plasmid. D , Sphere formation ability was determined following transfection with the empty vector, wild-type SETD1A plasmid and ΔSET plasmid. Scale bar, 100 μm. E , Cisplatin sensitivity was detected by the CCK-8 assay following transfection with the empty vector, wild type SETD1A plasmid and ΔSET plasmid. Data are shown as means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A activates NEAT1 and EZH2 transcription to activate the Wnt/β-catenin pathway. A-B , The correlation between SETD1A and NEAT1, EZH2 was analyzed using GEPIA online tool. R, Pearson’s correlation coefficient. C , Schematic showing the ChIP-PCR detection site in the NEAT1 and EZH2 promoters. D , The enrichment of SETD1A and H3K4me3 in the NEAT1 and EZH2 promoters in A549 cells was detected by ChIP-PCR assay. E , The relative enrichment of SETD1A and H3K4me3 in the NEAT1 and EZH2 promoters was detected by ChIP-qPCR assay following SETD1A knockdown in A549 cells. F , The promoter activity was analyzed by luciferase activity assay in A549 cells. G - H , NEAT1 and EZH2 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A knockdown. I , EZH2 protein levels in NSCLC cells as indicated were detected by western blotting following SETD1A knockdown. J-K , NEAT1 and EZH2 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A overexpression. L , EZH2 protein levels in NSCLC cells were detected by western blotting following SETD1A overexpression. Data are shown as means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Activity Assay, Luciferase, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: The SETD1A/β-catenin axis promotes NSCLC progression in vivo. A , A photograph of the tumors collected from nude mice after 28 days ( n = 5). B , The tumor volumes were measured at the indicated time points. C , The tumor weights were measured after xenograft resection. D , Immunohistochemical staining of Ki67, EZH2 and β-catenin in the xenograft specimens. Scale bar, 50 μm. E , The transcript levels of NEAT1, EZH2 and CTNNB1 in the xenograft tissues were detected by qRT-PCR. F , Tumor initiation was monitored for 8 weeks and the tumor initiation frequency was calculated using the ELDA. Data are shown as means ± SD. ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: In Vivo, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A is a direct target of the Wnt/β-catenin pathway. A , Schematic showing the putative TCF/LEF binding site in the TCF7L2/TCF4 enriched region. Red line, putative TCF/LEF binding site predicted by PROMO online tool; green region, TCF7L2/TCF4 enriched region derived from ENCODE database. B , The enrichment of β-catenin and TCF4 in the SETD1A promoter region was detected by ChIP-PCR assay. C , The enrichment of β-catenin and TCF4 in the SETD1A promoter region was detected by ChIP-qPCR assay. D , The promoter activity of SETD1A gene in NSCLC cells was analyzed by luciferase activity assay. ns, not significant. E , SETD1A transcript levels in NSCLC cell as indicated were detected by qRT-PCR. H, SETD1A protein levels in NSCLC cells as indicated were detected by western blotting. Data are shown as the means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Binding Assay, Derivative Assay, Activity Assay, Luciferase, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: Schematic diagram of the SETD1A/Wnt/β-catenin positive feedback loop in this study. In NSCLC cells, SETD1A activates the Wnt/β-catenin pathway through two mechanisms as follows: i, SETD1A interacts with and stabilizes β-catenin protein; ii, SETD1A promotes the transcription of NEAT1 and EZH2. In turn, the Wnt/β-catenin pathway activates SETD1A transcription, thus forming a positive feedback loop to coordinately promote NSCLC progression

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Effective elimination of chronic lymphocytic leukemia cells in the stromal microenvironment by a novel drug combination strategy using redox-mediated mechanisms

doi: 10.3892/mmr.2015.4364

Figure Lengend Snippet: Effect of SAHA on levels of GSH-associated enzyme and GSH in CLL cells co-cultured with stromal cells. (A) SAHA increased the expression of Nrf2 in CLL cells. CLL cells were treated with 2 µ M SAHA for 20 h, and cell lysates were assayed for Nrf2 using western blot analysis. Representative western blot results from three samples from patients with CLL are shown. The right panel shows the quantification of Nrf2 band density of eight CLL samples, with β-actin expression as an internal control (mean ± standard deviation; * P<0.05; Ctrl, control cells without treatment; S, SAHA treatment). (B) SAHA induced the translocation of Nrf2 between the cytosol and nucleus. SAHA (2 µ M) was added to the CLL cells for 20 h, and the cells were cytospun and immunostained with Nrf2 antibodies, and observed using a confocal laser scanning microscope. The nuclei were stained with 4,6-diamidino-2-phenylindole. (C) Upregulation of mRNA expression of GCLC following SAHA treatment. CLL cells were treated with 2 µ M SAHA for 22 h and the GCLC mRNA expression was examined using reverse transcription-quantitative polymerase chain reaction. (D) SAHA increases the expression of GCLC in CLL cells. CLL cells were treated with 2 µ M SAHA for 24 h, and the cell lysates were then assayed for the expression levels of GCLC by western blot analysis. The upper panel shows the representative western blot results from samples of four patients with CLL. The lower panel shows the quantification of GCLC band density of eight CLL samples, with β-actin as the internal control (mean ± standard deviation; ** P<0.01. (E) Treatment with SAHA enhanced stromal-mediated GSH upregulation in CLL cells. The CLL cells were treated with 2 µ M SAHA for 48 h in the presence or absence of HS5 cells. In another treatment group, CLL and HS5 cells in co-culture were incubated with cystine transporter inhibitor S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Values are presented as the mean ± standard deviation of three independent experiments using three CLL samples. (F) Sensitization of CLL cells to SAHA by inhibiting the cystine transporter with S-4-CPG. CLL and HS5 cells in co-culture were incubated with S-4-CPG (500 µ M) for 24 h, then exposed to SAHA (2 µ M) for 48 h. Cell viability was analyzed using an Annexin V/PI assay. Representative dot plots are shown. CLL, chronic lymphocytic leukemia; SAHA, suberoylanilide hydroxamic acid; GSH, glutathione; PI, propidium iodide; Nrf2, nuclear factor-E2-related factor 2; CPG, carboxyphenylglycine Ctrl, untreated control; S, SAHA treatment.

Article Snippet: The fixed CLL cells were incubated with rabbit anti-human polyclonal Nrf2 antibody (1:50; cat no. sc-13032; Santa Cruz Biotechnology, Inc.), at 4°C overnight, followed by incubation with Alexa-Fluor-594 goat anti-rabbit polyclonal antibody (1:400; cat. no. A11594; Molecular Probes at room temperature for 1 h. Finally, the slides were washed with phosphate-buffered saline, mounted and counterstained with mounting medium supplemented with DAPI prior to examination with a Nikon Eclipse TE2000 confocal microscope and analysis with Nikon EZ-C1 3.80 software (Nikon Corporation, Tokyo, Japan).

Techniques: Cell Culture, Expressing, Western Blot, Control, Standard Deviation, Translocation Assay, Laser-Scanning Microscopy, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Co-Culture Assay, Incubation

Journal: The FASEB Journal

Article Title: Shining a Light on Skeletal Muscle Regeneration: Red Photobiomodulation Boosts Myoblast Differentiation In Vitro

doi: 10.1096/fj.202502477R

Figure Lengend Snippet: Analyses of mitochondria and of peroxisome proliferator‐activated receptor‐gamma coactivator (PGC)‐1α expression. Myoblasts were cultured in proliferation medium (PM) or exposed or not (untreated) to PBM with 4 J/cm 2 energy density and then cultured in differentiation medium (DM) for 48 h. (A–C) Representative confocal laser scanning microscopy (CLSM)fluorescence images of myoblasts labeled with MitoTracker to mark mitochondria (red) and immunostained for PGC‐1α expression (green). Scale bar: 20 μm. (D, E) Densitometric analyses of the fluorescence intensity (F.I.) of mitochondria and PGC‐1α respectively, expressed in a.u. (arbitrary units). (F) WB analysis of PGC‐1α in the indicated conditions: representative blot and bar charts showing the densitometric analysis of the bands normalized to α‐tubulin. Molecular weight of full length of PGC‐1α, 115 kDa, molecular weight of N‐terminal truncated NT‐PGC‐1α: 37 kDa. a.u.: arbitrary units. Data are mean ± SD. ** p < 0.01, *** p < 0.001 vs. untreated PM; ° p < 0.05 vs. untreated DM 48 h (One‐way ANOVA with post hoc Tukey).

Article Snippet: After permeabilization with cold acetone for 3 min, fixed cells were blocked with 0.5% bovine serum albumin (BSA; Sigma Aldrich) and 3% glycerol in phosphate‐buffered saline (PBS) for 20 min and then incubated overnight at 4°C in a humidified chamber, with the following primary antibodies: rabbit polyclonal anti‐MyoD (M‐318) (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc‐760, RRID:AB_2148870), mouse monoclonal anti‐myogenin (F5D) (1:50; Santa Cruz, Cat# sc‐12732, RRID:AB_627980), mouse monoclonal anti‐peroxisome proliferator‐activated receptor‐gamma coactivator (PGC)‐1α (1:100; Santa Cruz, Cat# sc‐518025, RRID:AB_2890187).

Techniques: Expressing, Cell Culture, Confocal Laser Scanning Microscopy, Fluorescence, Labeling, Molecular Weight

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: Immunostaining of the EGFR on Hek 293T cells (left) and the MDA MD 468 cells (right). Images were taken via confocal laser scanning microscopy.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Immunostaining, Confocal Laser Scanning Microscopy

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Schematic representation of the experimental findings on NR selectivity: the differential binding profile between the targeted cell line (MDA MD 468) and the non-targeted cell line (Hek 293T) with the targeted NRs (NRs_18Ab and NRs_18Apt). For the MDA MD 468 cells, binding events with the targeted NRs are characterized by very long trajectories, indicating specific binding, while the binding trajectories with the Hek 293T are typically much shorter, indicating non-specific binding. b) Representative image of NRs_18Ab trajectories at 60 minutes after their incubation with MDA MD 468 and Hek 293T cells. Cell contours are indicated by the dotted line. Color bar indicates the diffusion coefficients (ranging from 0 to 4 µm 2 /s). c) Scatter plot of all the binding events for non-functionalized NRs, NR_18Ab and NR_18Apt (i.e. all the trajectories with D ≤ 1), plotted against their respective trajectory length (y-axis). d) Bar plot displaying the differential specific binding percentage between MDA MD 468 and Hek 293T for the different NR designs (NR_18Ab and NR_18Apt) at 3 distinct time points (10 min, 30 min and 60 min). Results are shown as the mean +-standard error of the mean. n = 3 biological replicates (per biological replicate, the trajectories of 5 different movies were combined, i.e. 5 technical replicates) *: significant difference between groups with p ≤ 0.05, ** : significant difference between groups with p ≤ 0.01, *** : significant difference between groups with p ≤ 0.001.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Incubation, Diffusion-based Assay

Journal: bioRxiv

Article Title: Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

doi: 10.1101/2024.12.23.628980

Figure Lengend Snippet: a) Scheme of the binding kinetics between targeted NRs (NR_18Ab and NR_18Apt) and cells (left panel). The corresponding formula of the binding kinetics is displayed in the right panel. b) Comparison of the total number of specific binding events between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 and Hek 293T cells. The amount of binding events can be directly related to k on . c) Example of the exponential decay fitting for the binding time of NR functionalized with 18 antibodies in MDA MD 468 cells, where dotted line represents the fitted courve and the corresponding equation is displayed. d) τ B values obtained via an exponential decay fitting of all the binding events for each NR design and cell type. This value is inversely proportional to k off . A comparison was made between NRs with 8 or 18 binding ligands (antibody or aptamer) in both MDA MD 468 or Hek 293T. Results are shown as mean fitted value, where error bars represent the standard error of the fitting for each NR design in the different cell lines.

Article Snippet: The MDA-MB-468 (ATCC cat. HTB-132) and Hek 293T (ATCC cat. CRL-3519) cell lines were cultured in Thermo ScientificTM NuncTM Cell Culture Treated Flasks with Filter Caps.

Techniques: Binding Assay, Comparison

Journal: Genes & Diseases

Article Title: Down-regulation of microRNA-23a promotes pancreatic ductal adenocarcinoma initiation and progression by up-regulation of FOXM1 expression

doi: 10.1016/j.gendis.2023.101203

Figure Lengend Snippet: Correlation between miR-23a and FOXM1 expression in human pancreatic ductal adenocarcinoma (PDAC) tissues. (A) Total mRNA including miRNA was extracted from distinct cohorts of pancreatic tumor samples with varied levels of FOXM1 expression (negative/weak, moderate, and strong) through laser-assisted microdissection ( n = 10). (B, C) The miR-23a expression levels were quantified using quantitative PCR in triplicate. Notably, an inverse correlation was observed between FOXM1 protein expression and miR-23a-3p ( n = 30; r = −0.5400, P = 0.0021) and -5p ( n = 30; r = −0.6374, P = 0002) expression. (D) Decreased level of both miR-23a-3p and -5p was apparent in human pancreatic cancer cell lines. (E, F) The levels of miR-23a-3p and -5p in high FOXM1 mRNA expressing human pancreatic cells were lower than those in low FOXM1 mRNA expressing cells. An inverse correlation was also observed between FOXM1 mRNA expression and miR-23a-3p expression.

Article Snippet: All the miRNAs were reverse transcribed into the first strand of cDNA by A-tailing using miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, CAT# KR211-02), and were further amplified using miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN, CAT# FP411-02) with designed forward primer of miRNAs and universal reverse primer in the kit.

Techniques: Expressing, Laser Capture Microdissection, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Down-regulation of microRNA-23a promotes pancreatic ductal adenocarcinoma initiation and progression by up-regulation of FOXM1 expression

doi: 10.1016/j.gendis.2023.101203

Figure Lengend Snippet: Decreased miR-23a expression in three different mouse models of ADM in vivo . (A, B) In vivo ADM induction using three mouse models generated as described in the Materials and Methods section, including PDL, CAE, and KC mouse models of pancreatic cancer. FoxM1 and miR-23a expression were determined with quantitative PCR in triplicate. The induction of ADM was accompanied by decreased expression of both miR-23a-3p and miR-23a-5p in KC, PDL, and CAE mouse models ( n = 5). Note that the increased expression of FoxM1 correlated with a significantly decreased expression of miR-23a-3p and miR-23a-5p. (C) Mice were treated with caerulein for two days and miR-23a expression was determined with quantitative PCR 2, 5, and 8 days after the treatment. Caerulein treatment decreased the expression of both miR-23a-3p and miR-23a-5p, while the decreased level of miR-23a-3p and -5p recovered after caerulein treatment was stopped for eight days. PDL, pancreatic ductal ligation; CAE, caerulein treatment; KC, Pdx1-Cre; LSL-Kras G12D/+ . ADM, acinar-to-ductal metaplasia.

Article Snippet: All the miRNAs were reverse transcribed into the first strand of cDNA by A-tailing using miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, CAT# KR211-02), and were further amplified using miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN, CAT# FP411-02) with designed forward primer of miRNAs and universal reverse primer in the kit.

Techniques: Expressing, In Vivo, Generated, Real-time Polymerase Chain Reaction, Ligation

Journal: Genes & Diseases

Article Title: Down-regulation of microRNA-23a promotes pancreatic ductal adenocarcinoma initiation and progression by up-regulation of FOXM1 expression

doi: 10.1016/j.gendis.2023.101203

Figure Lengend Snippet: Induction of ADM decreased the expression of miR-23a in primary acinar cells and acinar cells in vitro . (A) Primary acinar cells were treated with TGF-α for three days, immunofluorescence staining was performed using specific antibodies against CK19 and amylase. Note that the induction of ADM was accompanied by an increased level of CK19 and decreased expression of amylase. (B) Primary acinar cells, MPC-83, and 266-6 cells were treated with or without 50 ng/mL TGF-α for three days. miR-23a-3p and miR-23a-5p levels were determined with quantitative PCR in triplicate. Note that induction of ADM correlated with a significantly decreased expression of miR-23a-3p and miR-23a-5p in primary acinar cells and MPC-83 and 266-6 pancreatic cells. ADM, acinar-to-ductal metaplasia.

Article Snippet: All the miRNAs were reverse transcribed into the first strand of cDNA by A-tailing using miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, CAT# KR211-02), and were further amplified using miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN, CAT# FP411-02) with designed forward primer of miRNAs and universal reverse primer in the kit.

Techniques: Expressing, In Vitro, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction

Journal: Genes & Diseases

Article Title: Down-regulation of microRNA-23a promotes pancreatic ductal adenocarcinoma initiation and progression by up-regulation of FOXM1 expression

doi: 10.1016/j.gendis.2023.101203

Figure Lengend Snippet: miR-23a down-regulated the expression of FOXM1 in both mouse pancreatic cells and human pancreatic cancer cells. (A – C) CFPAC-1 and MIA PaCa-2 human PDAC cells and MPC-83 mouse pancreatic cells were treated with either miR-23a-3p or miR-23a-5p for 48 h, and FOXM1 protein and mRNA expression were determined with western blot analyses and quantitative PCR in triplicate. (D – F) CFPAC-1 and MIA PaCa-2 human PDAC cells and MPC-83 mouse pancreatic cells were treated with inhibitors of either miR-23a-3p or miR-23a-5p for 48 h and FOXM1 protein and mRNA expression were determined with western blot analyses and quantitative PCR. Note that miR-23a-3p and miR-23a-5p inhibited the expression of FOXM1 protein and mRNA in CFPAC-1 and MIA PaCa-2 human pancreatic cancer cells and MPC-83 mouse pancreatic cells, while the inhibitors of miR-23a-3p and miR-23a-5p increased the expression of FOXM1 protein and mRNA in CFPAC-1 and MIA PaCa-2 human pancreatic cancer cells and MPC-83 mouse pancreatic cell.

Article Snippet: All the miRNAs were reverse transcribed into the first strand of cDNA by A-tailing using miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, CAT# KR211-02), and were further amplified using miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN, CAT# FP411-02) with designed forward primer of miRNAs and universal reverse primer in the kit.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction