Journal: Scientific Reports

Article Title: Impact of the secretome of activated pancreatic stellate cells on growth and differentiation of pancreatic tumour cells

doi: 10.1038/s41598-019-41740-x

Figure Lengend Snippet: The effect of knocking down eIF4E protein expression by small interfering RNA. Tumour cells PT45P1 and Panc-1 were transfected with 100 nM of an eIF4E-specific siRNA or a control siRNA of scrambled sequence for 48 h, followed by serum starvation overnight. Subsequently, they were grown in presence or absence of activated PSC secretome for 24 h. The effect of the various conditions on eIF4E is shown, determined by Western blotting. Also, the results of caspase-3/7 assays are shown for each column (c1 to c6). Note: the two lanes c1 and c2 are exchanged in the Western blots generated from Panc-1 cells.

Article Snippet: We used the siRNA gene silencer system (siRNA #6554) as well as a control siRNA (#6568) of Cell Signaling Technology (Danvers, USA) to perform the EIF4E gene silencing in the pancreatic cancer cell lines PT45P1, Panc-1 and Capan-1 according to the manufacturer’s protocol.

Techniques: Expressing, Small Interfering RNA, Transfection, Control, Sequencing, Western Blot, Generated

Journal: Scientific Reports

Article Title: Impact of the secretome of activated pancreatic stellate cells on growth and differentiation of pancreatic tumour cells

doi: 10.1038/s41598-019-41740-x

Figure Lengend Snippet: Transient silencing eIF4E in Capan-1 pancreatic cancer cells inhibits cell migration. Capan-1 cells were transfected with 100 nM of either eIF4E-specific siRNA or a control siRNA of scrambled sequence for 48 h. Cells were serum starved overnight and either left untreated or treated with activated PSC secretome. A gap was generated by physically scraping off cells. The gap was inspected at different time intervals of up to 48 h at the growth conditions indicated in the figure.

Article Snippet: We used the siRNA gene silencer system (siRNA #6554) as well as a control siRNA (#6568) of Cell Signaling Technology (Danvers, USA) to perform the EIF4E gene silencing in the pancreatic cancer cell lines PT45P1, Panc-1 and Capan-1 according to the manufacturer’s protocol.

Techniques: Migration, Transfection, Control, Sequencing, Generated

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 5. AMPK activator A769662 mimics the effect of metformin in MGO-stimulated cells (A) Cells were pretreated with metformin (6 mM) or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) treatment for indicated time points. AMPK phosphorylation was measured by immunoblotting. (B) Cells were 30 min pretreated with A769662 (25 μM) prior to MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. (C, D, E, F) Cells were pretreated with A769662 (25 μM) for 30 min followed by MGO (300 μg/ml) treatment for 4 h. DCFDA (C), DHE (D), mitoSOX (E), and mitoPY1 (F) were used to measure cellular ROS. (G) Cells were 30 min pretreated with compound C (10 μM) followed by MGO (300 μg/ml) stimulation for 6 h. In some experiments, ARPE-19 cells were treated with siRNA followed by stimulation with MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. AMPK expression after siRNA treatment was determined by immunoblotting. (H) Immediately after treatment with A769662 (25 μM), cells were subjected into SFe24 analyzer for OCR measurement, as described in Fig. 3D. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO; #p < 0.05, indicating the significant effects of A769662, compound C and AMPK silencing on MGO- induced responses.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 8. Metformin and A769662 reverse MGO-induced GLO1 downregulation. (A) Cells were pretreated with BBGC (10 μM), metformin (6 mM) and/or A769662 (25 μM) 30 min before MGO (100 μg/ml) stimulation. After 4 h, cell viability was determined by Annexin V-FITC/PI staining using FACS. (B) Cells were treated with siRNA to silence GLO1, then treated with MGO (100 or 300 μg/ml) for 6 h. Cell viability was determined by Annexin V-FITC/PI staining using FACS. (C–E) Cells were pretreated with metformin (6 mM) and/or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) stimulation. (C) After incubation for 1, 3, or 6 h, cell lysates were prepared for immunoblotting. (D) After incubation for 2 or 4 h, GLO1 gene expression was measured using PCR analysis. (E) After incubation for 3 or 6 h, GLO1 and GLO2 activities were determined by commercial kits according to the manufacturer’s instructions. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO. #p < 0.05, indicating the blockade effects of metformin and A769662.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Staining, Incubation, Western Blot, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Hyaluronic Acid Receptor CD44 Deficiency Is Associated with Decreased Cryptococcus neoformans Brain Infection

doi: 10.1074/jbc.m112.353375

Figure Lengend Snippet: FIGURE6.Effectofanti-CD44andanti-RHAMMsiRNAtreatmentsonasso- ciation of C. neoformans cells with mouse BMEC. A, mouse BMEC was treated with siRNA (20 pmol) individually or in combination for 5 h before the in vitro C. neoformans adhesion assay. C. neoformans CPS1 wild-type strain (B-4500FO2) and its isogenic cps1 deletion strain C559 were used in parallel. ,anti-CD44siRNAand/oranti-RHAMMsiRNA-treatedsampleasindicatedat the bottom; , control oligonucleotide (n 4). In all treatments, the MBMEC without any siRNA treatment was taken as a control for comparison, and the statistical package GraphPad Prism 5 was used to quantify the readings. Sig- nificant differences with regard to the controls: *, p 0.05; **, p 0.01). B, as a control, a Western blot was used to show the CD44 and RHAMM protein levels after siRNA treatment.

Article Snippet: All samples were examined under a fluorescence microscope at the Congressman Dixon Cellular Imaging Core Facility, Children’s Hospital Los Angeles. siRNA Treatment—Anti-CD44 siRNA (sc-35534) and antiRHAMM siRNA (sc-40182) were purchased from Santa Cruz Biotechnology, and a control oligonucleotide (sc-36869) was used in parallel.

Techniques: In Vitro, Cell Adhesion Assay, Control, Comparison, Western Blot

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.

Article Snippet: The next day, cells were transfected using Lipofectamine 2000 (Invitrogen; Carlsbad, CA) with 100 nM GDF15 siRNA, 100 nM FOXM1 siRNA, or control siRNA (Santa Cruz Biotechnology) according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.

Article Snippet: The next day, cells were transfected using Lipofectamine 2000 (Invitrogen; Carlsbad, CA) with 100 nM GDF15 siRNA, 100 nM FOXM1 siRNA, or control siRNA (Santa Cruz Biotechnology) according to the manufacturer’s protocol.

Techniques: Transfection, Control, Recombinant, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Staining

Journal: Oncotarget

Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

doi: 10.18632/oncotarget.21765

Figure Lengend Snippet: (A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.

Article Snippet: The next day, cells were transfected using Lipofectamine 2000 (Invitrogen; Carlsbad, CA) with 100 nM GDF15 siRNA, 100 nM FOXM1 siRNA, or control siRNA (Santa Cruz Biotechnology) according to the manufacturer’s protocol.

Techniques: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Knockdown, Standard Deviation

Journal: Oncology Letters

Article Title: Bufadienolides induce p53-mediated apoptosis in esophageal squamous cell carcinoma cells in vitro and in vivo

doi: 10.3892/ol.2017.7457

Figure Lengend Snippet: Involvement of p53 in bufotalin-induced apoptosis. (A) Western blot analysis of p53 and p-p53 expression following transfection with p53 siRNA and (B) the effects on cell apoptosis following treatment with bufotalin (4 µM; 24 h), as examined by propidium iodide flow cytometry analysis. Results were obtained from three independent experiments. Bars with different symbols (*, # and Δ ) are statistically different from every other group at P<0.05, and the groups with the same symbol *(control group and negative group) indicate no significant difference at P<0.05. Control siRNA was used as negative control, and no bufotalin was added in this group. p53, tumor protein p53; p-, phosphorylated; siRNA, small interfering RNA.

Article Snippet: Then, fresh serum-free DMEM medium with 10% fetal bovine serum was added to each well for 24 h with or without 2 and 4 μM bufadienolides at 4°C for 24 h. Control cells were transfected with a fluorescein-labeled non-targeted scramble control siRNA (cat. no. 78193; Cell Signaling Technology, Inc.), which allowed for the monitoring of transfection efficiency.

Techniques: Western Blot, Expressing, Transfection, Flow Cytometry, Control, Negative Control, Small Interfering RNA