Journal: Oncotarget

Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

doi: 10.18632/oncotarget.26478

Figure Lengend Snippet: (A) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment in a time dependent manner. At different time points the treated cells were collected in TRIZOL for Sphk1 and Sphk2 mRNA expression by semi quantitative RT-PCR analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. At different time points, data were represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (B) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment. Activities of sphingosine kinase 1 and sphingosine kinase 2 were determined from cell lysates following the protocol described in Materials and Methods. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (C) In a separate experiment, similarly PKCδ silenced cisplatin treated B16F10 cells were transfected with Sphk1 and Sphk2 specific siRNAs as mentioned in Materials and Methods. Cells were collected stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Data are from one of the three representative experiments.

Article Snippet: Mouse specific IRF1 small-interfering RNA (siRNA), human specific PKCδ, IRF1, Sphk2 siRNA and control siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

doi: 10.18632/oncotarget.26478

Figure Lengend Snippet: (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (B) In a separate experiment, similarly A375 cells were either transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods. The expression of PKCδ was analyzed in whole cell lysates by western blot analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (C) PKCδ silenced A375 cells were either treated with cisplatin alone or in combination with imipramine, ATK or transfected with IRF1 specific siRNAs. Cells were also treated with exogenous TNFα in absence of cisplatin. Treated cells were subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide expression was analysed by flow cytometry. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (D) PKCδ silenced A375 cells were either treated with cisplatin alone or in combination with imipramine, ATK, TNFα/TNFα-R1 neutralizing antibody or transfected with IRF1/Sphk2 specific siRNAs as mentioned in Materials and Methods. Cells were collected, stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (E) and (F) A375 cells, transfected with PKCδ siRNA followed by cisplatin treatment were cultured in hypoxic chamber as described in Materials and Methods. Cells were collected and subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide expression was analysed by flow cytometry. In a different experiment cells were stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Data are from one of the three representative experiments.

Article Snippet: Mouse specific IRF1 small-interfering RNA (siRNA), human specific PKCδ, IRF1, Sphk2 siRNA and control siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry, Cell Culture

Journal: Oncotarget

Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

doi: 10.18632/oncotarget.26478

Figure Lengend Snippet: The primer sequences

Article Snippet: Mouse specific IRF1 small-interfering RNA (siRNA), human specific PKCδ, IRF1, Sphk2 siRNA and control siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Sequencing

Journal: Journal of Translational Medicine

Article Title: In vivo efficacy of endothelial growth medium stimulated mesenchymal stem cells derived from patients with critical limb ischemia

doi: 10.1186/s12967-019-2003-3

Figure Lengend Snippet: Angiogenic effect of culture supernates. a MSCs and S-MSCs culture supernates and cell-free media (CFU-F and EGM-2) were recovered at day 28. HMEC-1 were suspended in endothelial cell growth medium MV (n = 3), in cell-free CFU-F medium (n = 3), in cell-free EGM-2 medium (n = 3), in MSCs culture supernates (obtained from 5 CLI-MSCs, n = 2 each group), in S-MSCs culture supernates (obtained from 5 CLI-S-MSCs, n = 2 each group) and incubated on Matrigel during 24 h. The extend of the network of the capillary-like tubes was appreciated at 3:30 h. b Quantification of the loops number at 3:30 h. c Quantification of total tube length (µm) at 3:30 h. x: aberrant distribution values as indicated by Box and Whiskers plots Medcalc version 7.3 software (*p < 0.05, † p < 0.01 and ‡ p < 0.001)

Article Snippet: HMEC-1 cells (8000 cells/well) were suspended in Endothelial Cell Basal medium MV (n = 3) (10 ng/mL EGF, 1 µg/mL hydrocortisone, 10 mM Glutamine, and 10% FBS) (PromoCell, Heidelberg, Germany), or in cell-free media [CFU-F (n = 3), or EGM-2 (n = 3)] or in MSCs culture supernates (obtained from 5 CLI-MSCs, n = 2 each group), or in S-MSCs culture supernates (obtained from 5 CLI-S-MSCs, n = 2 each group), laid upon Matrigel (BD Biosciences, Le Pont de Claix, France) cast in IBIDI micro wells (81,501, µ-Slide Angiogenesis, Biovalley), and allowed to form tubules for 24 h under normoxic conditions.

Techniques: Incubation, Software