33 research outputs found
Regulatory effect of PKCĪ“ and PLD1 on aSMase mediated ceramide generation.
<p>(A) B16F10 cells overexpressing full-length mouse PKCĪ“ (PKCĪ“OV) or empty vector (EV) were treated with Fumonisin B1(FB1) (10 uM) and Imipramine (10 uM) respectively for 1 hour. The transfected cells were washed and after 24 hour of incubation were stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody, and analyzed by flow cytometry for ceramide (FL1) expression. Representative data exhibited were from three independent experiments. (B) The transfectants of B16F10 cells overexpressing PKCĪ“ (PKCĪ“OV) or the empty vector (EV) were treated with FB1 (10 uM) and Imipramine (10 uM) for 1 hour. The transfected cells were washed and after 24 hours of incubation the whole cell lysates were subjected to SDS-PAGE and Western blot analysis to study the expression of pAKT and total AKT. Data represented here are from one of three independent experiments. (C) Similarly, B16F10 cells transfected with empty vector (EV) and full-length PKCĪ“ (PKCĪ“OV) were treated with FB1 (10 uM) and Imipramine (10 uM) as mentioned previously and expression of PLD1 was analysed by western blotting. Data represented here are from one of three independent experiments. GAPDH was used as a reference.</p
Modulatory role of PKCĪ± and PKCĪ“ on the expression of pAKT and ceramide levels.
<p>(A) B16F10 cells transfected with EV, full-length mouse PKCĪ±OV, PKCĪ“OV and siRNA oligonucleotides specific to PKCĪ± (PKCĪ± siRNA), PKCĪ“ (PKCĪ“ siRNA) or the control siRNA were collected and the whole cell lysates prepared from transfected cells and the expression of pAKT or AKT respectively were analyzed with respect to GAPDH by Western blotting. Representative blots from three independent experiments were given. (B) The transfected cells comprising empty vector (EV), overexpressed PKCĪ± (PKCĪ±OV), PKCĪ“ (PKCĪ“OV) or siRNAs corresponding to PKCĪ± (PKCĪ± siRNA), PKCĪ“ (PKCĪ“ siRNA) and control siRNA were stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody, and analyzed by flow cytometry for ceramide (FL1) expression. (C) B16F10 transfectants overexpressing full-length mouse PKCĪ± (PKCOV), PKCĪ“ (PKCĪ“OV) or empty vector (EV) were transfected with PLD1 siRNA as mentioned in materials and methods and harvested for 24 hours. The expression of pAKT and AKT was analysed in whole cell lysates by Western blotting. (D) Similarly full-length mouse PKCĪ± (PKCĪ±OV), PKCĪ“ (PKCĪ“OV) or empty vector (EV) transfected B16F10 melanoma cells treated with PLD1 siRNA and subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide (FL1) expression was analysed by flow cytometry. Data represented here are from one of three independent experiments.</p
Regulatory effect of PKCĪ± and PKCĪ“ on caspase cascade and apoptosis.
<p>(A) Whole cell lysates of B16F10 cells, transfected with empty vector (EV), full-length mouse PKCĪ± (PKCĪ±OV) and PKCĪ“ (PKCĪ“OV) plasmid as mentioned in materials and methods were probed by Western blotting to study the expression of pro Caspase 3, Caspase 3, pro Caspase 8, and Caspase 8. Representative data from three independent experiments was shown compared to GAPDH. (B) Whole cell lysates of B16F10 cells, transfected with empty vector (EV), full-length mouse PKCĪ± (PKCĪ±OV) and PKCĪ“ (PKCĪ“OV) plasmid were probed by Western blotting to study the expression of Bax and Bcl2 respectively. The ratio of Bax (proapoptotic)/Bcl2 (antiapoptotic) were represented on a log scale. (C) Similarly B16F10 cells, transfected with control vector (EV), full-length mouse PKCĪ± (PKCĪ±OV) and PKCĪ“ (PKCĪ“OV) plasmid as mentioned in materials and methods were stained with Annexin-V FITC and PI and analyzed by flow cytometry. Data represented here are from one of three independent experiments. (D) B16F10 cells, transfected with control vector (EV), full-length mouse PKCĪ± (PKCĪ±OV) and PKCĪ“ (PKCĪ“OV) plasmid as mentioned in materials and methods were stained with FITC-BrdU for tunnel assay and analysed by immunofluorescence microscopy.</p
Influence of PKCĪ± and PKCĪ“ on cell proliferation and their interaction with PLD1.
<p>(A) B16F10 cells transfected with empty vector (EV) or overexpressed full-length mouse PKCĪ± (PKCĪ±OV) or PKCĪ“ (PKCĪ“OV) isotypes as mentioned in materials and methods were harvested for 48 hrs and stained with propidium iodide to measure the DNA content by flow cytometry. (B) B16F10 cell transfected with EV, PKCĪ±OV and PKCĪ“OV isotypes were measured for incorporation of (<sup>3</sup>H) - thymidine to determine cell proliferation. (*<i>p</i><0.001). Expression of PKCĪ“OV compared to PKCĪ±OV. Results were expressed as meanĀ±SD, and are representative of three independent experiments. (C) Whole cell lysates from the stable transfectants of B16F10 cells overexpressing PKCĪ± (PKCĪ±OV), PKCĪ“ (PKCĪ“OV), or the empty vector (EV) and the siRNA oligonucleotides specific to PKCĪ± (PKCĪ± siRNA), PKCĪ“ (PKCĪ“ siRNA) or control siRNA were subjected to Western blot analysis for PLD1 expression. Representative data from three independent experiments was given compared to GAPDH.(D) In a separate set, B16F10 cells transfected with empty vector (EV), full-length mouse PKCĪ±OV and PKCĪ“OV, siRNA oligonucleotides specific to PKCĪ±, PKCĪ“ and control siRNA respectively as mentioned in materials and methods. The whole cell lysates from each transfected cells were subjected to immunoprecipitation with anti Phospholipase D1 (PLD1) antibody and the blots were probed with anti-PKCĪ± respectively. (E) Similarly, the whole cell lysates from each transfected cells were subjected to immunoprecipitation with anti PLD1 antibody and the blots were probed with anti-PKCĪ“ respectively. The blot shown is a representative of experiments performed in triplicate. GAPDH was used as a reference.</p
Differential expression of various PKC isotypes in B16F10 melanoma tumor.
<p>(A) Cell lysates from primary melanocytes and B16F10 melanoma cells were subjected to western blot analysis with anti PKCĪ±, PKCĪ², PKCĪ“, PKCĪø, PKCĪµ and PKCĪ¶ specific antibodies. GAPDH was used as a reference. Representative figure and bar diagram of densitometric analysis from three independent experiments are presented. (B) 2Ć10<sup>6</sup> primary melanocytes and B16F10 melanoma cells were separately collected in Trizol for mRNA extraction and semi quantitative RT-PCR analyses for PKCĪ±, PKCĪ², PKCĪ“, PKCĪø, PKCĪµ and PKCĪ¶ were done. Representative figure and bar diagram of densitometric analysis of target gene with respect to GAPDH was presented. (C) 2Ć10<sup>6</sup> B16F10 melanoma cells were treated with PMA (100 nM) for 1 hr. Expression of PKCĪ± and PKCĪ“ along with GAPDH in cytosolic and membrane protein fraction was analyzed by Western blot. Representative data from three independent experiments was given. (D) Cell lysates of B16F10 cells transfected with empty vector (EV), overexpressed full-length mouse PKCĪ± (PKCĪ±OV) or PKCĪ“ (PKCĪ“OV) isotypes as mentioned in materials and methods were subjected to SDS PAGE and western blot analysis to check the respective expression of PKCĪ±, PKCĪ², PKCĪ“, PKCĪµ, PKCĪø and PKCĪ¶ isotypes. Representative figure from three independent experiments and bar diagram of densitometric analysis was depicted. GAPDH was used as a reference.</p
Effect of inhibitors on plasma membrane Ca<sup>2+</sup>-ATPases during Leishmania infection.
<p>Macrophages were treated with thapsigargin (TG, 1 ĀµM) and trifluoperazine (TFP, 10 ĀµM) followed by infection with <i>L.donovani</i> in 1ā¶10 ratio. Unbound parasites were washed off after 4 hr and further incubated for 20 hr. Cells were collected, plasma membrane was isolated and Ca<sup>2+</sup>-ATPase activity was measured. (A). In a similar experiment, inhibitor treated and infected macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR were performed (B). Macrophages were seeded on 8 well chamber slides (10,000 cells/well) and treated with inhibitors followed by infection with <i>L.donovani</i> in a 1ā¶10 ratio. Cells were incubated for 24 hr, fixed with chilled methanol, and stained with Giemsa. Amastigotes were then microscopically counted. ***P<.001, *P<.05 and nsā=ānon-significant for the comparison with infected one (C). Data represented means Ā± SD from three different experiments (A and C). Result shown in B is a representative one from three different experiments.</p
Expression of SERCA3 and PMCA4 at different time periods of infection.
<p>Macrophages (2Ć10<sup>6</sup> cells) were infected with <i>L. donovani</i> (1ā¶10) and incubated at 37Ā°C for 3, 6 and 12 hr. The cells were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. PMCA4, SERCA3 and GAPDH PCR products were resolved on an agarose gel (1.5%) and quantified densitometrically using lab software as described in Methods (A). In a separate set of experiment, cell lysates were prepared from infected macrophages, followed by Western blot for PMCA4, SERCA3 and GAPDH (B). Data shown above (A and B) is a representative one experiment, performed at least three times.</p
Effects of EGTA and ionomycin on mRNA and protein expression of PKC-Ī², PKC-Ī¶, SERCA3 and PMCA4 in Leishmania-infected macrophages.
<p>Macrophages (2Ć10<sup>6</sup> cells) were infected with <i>L. donovani</i> (1ā¶10) and with 2 mM EGTA and/or 1 ĀµM Ionomycin respectively. After 4 hr, cells were washed and incubated for another 3 hr at 37Ā°C. The cells were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. PKC-Ī², PKC-Ī¶, SERCA3, PMCA4, and GAPDH PCR products were resolved on an agarose gel (1.5%) and quantified densitometrically using lab software as described in Methods (A). In a similar experiment, cell lysates were prepared from EGTA and/or ionomycin treated infected macrophages, followed by Western blot for PKC-Ī², PKC-Ī¶, SERCA3, PMCA4, and GAPDH (B). Data presented above (A and B) is a representative one of three experiments conducted under identical conditions.</p
Effect of EGTA and ionomycin on the expression of pro- and anti-inflammatory cytokines in L. donovani infected macrophages.
<p>Macrophages (2Ć10<sup>6</sup> cells) were infected with <i>L. donovani</i> (1ā¶10) and incubated at 37Ā°C in the presence of 2 mM EGTA and/or 1 ĀµM ionomycin. The cell supernatants were collected after 24 hr of infection. Cytokine assay was carried out by ELISA (A, B, C and D). Data represent means Ā± SD for 3 sets of experiments ***P<.001, *P<.05 and nsā=ānon-significant for the comparison with infected one. In a parallel experiment, EGTA or Ionomycin treated infected cells were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. IL-12, TNF-Ī±, IFN-Ī³, IL-10 and GAPDH PCR products were resolved on an agarose gel (1.5%) and quantified densitometrically using lab software as described in Methods. Data is from one representative experiment performed at least three times (E, F, G, and H).</p