Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Intracellular localization of berberine and effects on viability in HDF, U343 and MIA PaCa-2 cells. ( a ) Confocal images of berberine distribution in HDF, U343 and MIA PaCa-2 cells. Cells were photographed 1 hour after treatment with berberine (10 μM, 50 μM or 150 μM). Black arrows point out nuclei. Scale bars represent 5 μm. ( b ) Reduction of cell viability after 48 hours of treatments with 0.4 μM, 2 μM, 10 μM, 50 μM berberine in HDF, U343 and MIA PaCa-2 cells. Graph columns represent mean of viable cells ± S.D. normalized versus control group (DMSO). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Control

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine localizes in mitochondria and affects mitochondrial function. ( a ) Berberine was visualized by confocal microscopy in mitochondria of HDF, U343 and MIA PaCa-2 cells after 48 hours of exposure to 10 μM or 50 μM berberine. Merge columns represent overlapping of the berberine green signal with the TMRM red signal. DMSO-treated cells, used as a control, lack green fluorescence. Differential interference contrast (DIC) highlighted the cell morphology. Scale bars indicate 5 μm. ( b ) Citrate synthase activity was measured in the three cell lines after treatments in the presence or absence of berberine as described in Methods. U = Units of enzymatic activity. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Confocal Microscopy, Control, Fluorescence, Activity Assay

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine alters the cell cycle and the transcriptional profile of cell cycle regulators. ( a ) Representative images of cell cycle distribution of HDF, U343 and MIA PaCa-2 cells. Cell cycle distribution was assayed by flow cytometry, following treatment with 10 μM or 50 μM berberine for 48 hours; subG1 (yellow bars), G1 (blue bars), S (green bars), G2 (red bars). Upper right insets represent the zoomed in subG1 population relative to each graph. Cell number (count) is reported in y-axis; fluorescence intensity (DsRed/PE) is reported in x-axis. ( b ) Quantification of cell cycle distribution; ( c ) The transcriptional profile of P53 , P21 , P16 was analyzed by Real Time RT-PCR after 1 hour or 48 hours berberine treatments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine induces cell senescence in HDF, U343 and MIA PaCa-2 cells. ( a ) Brightfield images of SA β-gal-positive HDF, U343 and MIA PaCa-2 cells after berberine treatments. Treatments: 10 μM or 50 μM berberine or DMSO for 48 hours. Scale bars indicate 50μm. ( b ) The graph shows the number of SA β-gal-positive cells normalized to the control (DMSO). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Control

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Effects of berberine on caspase-3 activity. ( a ) HDF, U343 and MIA PaCa-2 cells were incubated for 48 hours with 50 μM berberine and analyzed for caspase-3 activity monitored for 5 hours. Caspase-3 activity was measured as absorbance variation/hour/mg protein. Ber = berberine. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Berberine induces autophagy in U343 and MIA PaCa-2 cells, but not in HDF. ( a ) Relative mRNA expression is reported in y-axis. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. ( b ) Immunofluorescence experiments. Cells were treated with berberine or DMSO for 48 hours and immunohistochemistry was performed with LC3 antibody (red fluorescence), as described in Methods. Nuclei are visualized by Hoechst staining. ( c ) Cells were treated as in ( b ) and proteins were subjected to electrophoresis and western blotting as described in Methods. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Expressing, Immunofluorescence, Immunohistochemistry, Fluorescence, Staining, Electrophoresis, Western Blot

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Wound healing and cell invasion assays. ( a ) Representative brightfield images of the scratches (marked by white lines) in MIA PaCa-2 cells at 0, 24 and 48 hours, after treatments with berberine or DMSO. ( b ) Representative brightfield images of the scratches in U343 cells at 0, 24, 48 and 72 hours, after treatments with berberine or DMSO. ( c , d ) The values reported in the graphs represent the mean distance taken at each time from the wound edges (normalized to the DMSO group) ±S.D. Ber = berberine. ( e ) Impaired invasion of berberine-treated MIA PaCa-2 in transwell assay. ( f ) The graph reports the relative invasive score (±S.D.) corresponding to each berberine concentration, compared to DMSO. ( g ) The transcriptional profile of DAP1 and CXCR4 was analyzed by Real Time RT-PCR. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Transwell Assay, Concentration Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cell-specific pattern of berberine pleiotropic effects on different human cell lines

doi: 10.1038/s41598-018-28952-3

Figure Lengend Snippet: Effects of berberine on the transcriptional profile of DNMT1, DNMT3A, DNMT3B and MGMT in HDF, U343 and MIA PaCa-2 cells. Cells were treated with berberine for 1 hour or 48 hours and Real Time RT-PCR was performed as described in Methods. The treatments with berberine or DMSO alone are in x-axis. The values reported in graphs represent the mean ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: We used the human glioblastoma U343 GBM cells (U-343 MGa, CLS Cell Lines Service), the pancreatic carcinoma MIA PaCa-2 cells and normal primary dermal fibroblasts HDF (American Type Culture Collection ATCC, USA).

Techniques: Quantitative RT-PCR

Journal: Cell Discovery

Article Title: ASO-based PKM splice-switching therapy increases anti-CTLA-4 antibody efficacy in pancreatic ductal adenocarcinoma

doi: 10.1038/s41421-026-00882-9

Figure Lengend Snippet: a IC 50 of ASO1-TMO. Three classical PDAC cell lines (SUIT-2, PANC-1, and AsPC-1), two basal-like PDAC cell lines (Hs 766 T and BxPC-3), and the MIA PaCa-2 cell line were used for IC 50 determination. Cells were transfected with Lipofectamine and varying concentrations (0, 25, 50, 100, and 200 nM) of ASO1-TMO on Day 0, 10 3 cells were plated on a 96-well plate on Day 1, and cell viability was monitored by a colorimetric assay on Day 3. b Radioactive RT-PCR showing the extent of PKM splice switching after MIA PaCa-2 cells were transfected with the indicated concentrations of ASO1-TMO for 2 days (left). ImageJ was used to quantify the expression of the PKM2, PKM1, and PKMds isoforms (right). c Radioactive RT-PCR results showing the extent of PKM splice switching after BxPC-3 cells were transfected with various concentrations of ASO1-TMO as described in b for 2 days (left). Isoform quantification in b (right). d Western blot analysis showing the extent of isoform switching after MIA PaCa-2 or BxPC-3 cells were transfected with 50 nM ASO for 3 days. NTC, no-treatment control. e Viability of MIA PaCa-2 cells at each time point. Cells were transfected with 50 nM ASO on Day 0, 0.5 × 10 3 cells were plated on a 96-well plate on Day 1, and the OD450 was monitored daily by a colorimetric assay. f PK activity was measured on Day 3 after transfection with 50 nM ASO. g Lactic acid levels were measured on Day 3 after transfection with 50 nM ASO. Statistical analysis: unpaired two-sided t test ( a , b , c , f , g ); two-way ANOVA ( e ).

Article Snippet: MIA PaCa-2 (ATCC, CRL-1420), BxPC-3 (ATCC, CRL-1687), PK1 (RCB1972), Hs 766 T (ATCC, HTB-134), SUIT-2 (JCRB1094), PANC-1 (ATCC, CRL-1469), and AsPC-1 (ATCC, CRL-1682) cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Transfection, Colorimetric Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Activity Assay

Journal: Cell Discovery

Article Title: ASO-based PKM splice-switching therapy increases anti-CTLA-4 antibody efficacy in pancreatic ductal adenocarcinoma

doi: 10.1038/s41421-026-00882-9

Figure Lengend Snippet: a Schematic diagram of the animal experiment. ASO1-TMO and ASO2-TMO target the human PKM exon 10 regions (red and blue, respectively) that we previously targeted with MOE ASOs . ASO2-TMO targets an SRSF3 binding site that is conserved between humans and mice (yellow). b Whole-animal live imaging of luciferase-expressing MIA PaCa-2 cells transplanted into the pancreas. Luminescence images of the transplanted mice on the indicated days are shown with a color scale in photons/sec/cm 2 /steradian. A total of 14 mice were randomized to each treatment group. c Quantification of the luciferase signal in ( b ). Statistical analysis: two-way ANOVA. d Images of representative tumors from animals treated as indicated on Day 23. The duodenum, pancreas, tumor, and spleen are shown. Dashed lines delineate the tumor. Scale bar, 1 cm. e Quantification of tumor weight on Day 23 (after the duodenum, normal pancreas, and spleen were removed). f ASO1-TMO and ASO2-TMO induce PKM splice switching in pancreatic tumors, as determined by radioactive RT-PCR of RNA from pancreatic tumor samples from tumor-bearing mice on Day 23. g Western blot analysis of PKM1, PKM2, and Vinculin in orthotopic tumor samples from mice treated with SCR-TMO, ASO1-TMO, or ASO2-TMO. h Representative H&E and IHC images of a tumor, showing the expression of PKM1 and PKM2. i Representative IF staining images of tumor sections from mice treated with saline, SCR-TMO, ASO1-TMO, or ASO2-TMO showing the expression of PKM2 (green), the proliferation marker Ki67 (red), and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 50 µm. Statistical analysis: unpaired two-sided t -test ( e , f ); two-way ANOVA ( c ).

Article Snippet: MIA PaCa-2 (ATCC, CRL-1420), BxPC-3 (ATCC, CRL-1687), PK1 (RCB1972), Hs 766 T (ATCC, HTB-134), SUIT-2 (JCRB1094), PANC-1 (ATCC, CRL-1469), and AsPC-1 (ATCC, CRL-1682) cells were cultured in Roswell Park Memorial Institute medium supplemented with 10% FBS and penicillin/streptomycin.

Techniques: Binding Assay, Imaging, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Saline, Marker

Journal: Oncotarget

Article Title: MicroRNA-1 down-regulates proliferation and migration of breast cancer stem cells by inhibiting the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: 293T cells were transfected with luciferase reporter plasmids containing the 3UTR sequences of the fizzled 4, 5, 7, or TNKS2 and miR-1 or control miR-1NC and the effect of miR-1 on luciferase activity was determined A. The luciferase activity in the control cells that had been transfected with miR-1NC were considered as 100%. CD44+CD24− CSC from MCF-7 and SKBR3 cells were sorted and transfected with, or without, miR-1NC, miR-1inhibitor, or miR-1mimic for 24 or 48 h. The relative levels of fizzled 7, TNKS2 and c-Myc to control GAPDH were determined by Western blot B. In addition, the relative levels of cytoplasmic and nuclear β-catenin to GAPDH or lumen B1 in each group of cells were determined by Western blot C. Moreover, MCF-7/CSC and SKBR3/CSC were transfected with miR-1mimic, miR-1NC or miR-1inhibitor for 24 h. The cells in each group were transfected with TopFlash and pRL-TK for 48 h. The relative levels of β-catenin-dependent firefly luciferase activity in different groups of CSCs were determined by the dual luciferase assay D. The relative levels of β-catenin-dependent luciferase activity in the control cells that had been transfected with miR-1NC, TopFlash and pRL-TK were designated as 1. Finally, the relative levels of Oct4 and Nanog expression in each group of cells were determined by Western blot E. Data are representative images and expressed as the mean ± SD of each group of cells from three separate experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The primary antibodies included goat anti-Frizzled 7 (sc-31061), TNKS2 (SC-22854), mouse anti-c-Myc (sc-40) and rabbit anti-GAPDH (sc-25778, Santa Cruz Biotechnology, Santa Cruz, USA) and negative controls of rabbit or mouse IgG.

Techniques: Transfection, Luciferase, Control, Activity Assay, Western Blot, Expressing