Fig 1.
Analysis of hsa-miRNA expression profile by dual-color-based array of human miRNAs from total RNA extracted from Jurkat-Tat101, Jurkat-Tat72 and control cells.
The miRNAs differentially expressed in Jurkat-Tat101 cells (A) and in Jurkat-Tat72 (B) are shown in fold-change and heat maps. Data shown are referred to control cells. Changes in miRNAs expression were considered statistically significant with fold changes >1.5 or <1.5 and p-values <0.05 in at least three probes. p-values and FDR were calculated by using Limma method.
Fig 2.
Analysis by qRT-PCR of hsa-miRNA differentially expressed in Jurkat-Tat72 and Jurkat-Tat101 cells. miRNAs (A) and pre-miRNAs (B) were analyzed by qRT-PCR in total RNA extracted from Jurkat-Tat101, Jurkat-Tat72 cells and control cells.
β-actin was used as housekeeping gene. Relative expression referred to control cells is expressed as fold change. Mean and standard error of the mean (SEM) of at least three independent experiments is shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (*, ** or *** for p<0.05, p<0.01 or p<0.001, respectively).
Fig 3.
Analysis of miRNA expression in PBMCs with transient transfection of Tat101 and in Jurkat cells transfected with a Tat-defective HIV-1 strain.
(A) Resting PBMCs were transiently transfected with pCMV-Tat101 expression vector or with pcDNA3 as negative control. Intracellular expression and nuclear subcellular localization of Tat were confirmed by immunofluorescence using a monoclonal antibody against Tat and a secondary antibody conjugated to Alexa 546. DAPI was used for nuclear staining. (B) These PBMCs were also co-transfected with pLTR-LUC expression vector as control for Tat-mediated transcriptional activation. Synthesis of Renilla (RLUs) was measured 18 hours post-transfection. Data shown are media and SEM from three independent experiments. (C) Expression levels of miRNAs were analyzed by qPCR in total RNA extracted from the transfected PBMCs. β-actin was used as housekeeping gene. A representative experiment from three independent experiments is shown. Relative expression referred to control cells is expressed as fold change. (D) Jurkat cells were co-transfected with Tat-defective HIV-1 vector pNL4.3-TatM1I and pCMV-Tat101 or pcDNA3 as negative control. Gag/p24 (pg/ml) levels were quantified as measurement of HIV-1 replication. (E) Expression of hsa-miRNAs were measured by qRT-PCR in total RNA extracted from these transfected Jurkat cells. β-actin was used as housekeeping gene. Relative expression referred to control cells is expressed as fold change. Data shown are media and SEM from three independent experiments. Statistical significance was calculated by Mann-Whitney test (* for p<0.05, *** for p<0.005).
Fig 4.
Treatment of Jurkat-Tat101 cells with doxycycline reduced the expression of hsa-miR-21, -222, -29a, and -1290.
(A) Jurkat-Tat101 cells were co-transfected with pLTR-LUC and pEGFP, as control of transfection efficiency. Cells were treated or not with doxycycline 1μg/ml immediately after transfection. After incubation for 18 hours, the expression of luciferase (left graph) and EGFP (right graph) was analyzed by chemiluminescence and flow cytometry, respectively. Data shown are media and SEM from three independent experiments. Statistical significance was calculated by two-tailed unpaired t-test (**** for p<0.0001). (B) Expression of hsa-miR-21, -222, -29a, and -1290 was analyzed by qRT-PCR in Jurkat-Tat101 cells treated or not with doxycycline for 18 hours. Media and SEM from three independent experiments are shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (**** for p<0.0001).
Fig 5.
Analysis of the expression of targets for hsa-miRNAs deregulated in Jurkat-Tat101 cells.
Expression of mRNA encoding for PTEN, PDCD4 and CDKN1B was analyzed by qRT-PCR using β-actin as housekeeping gene. Media and SEM from at least three independent experiments are shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (*, ** or *** for p<0.05, p<0.01 or p<0.001, respectively).
Fig 6.
Analysis of PTEN-AKT-FOXO3a signaling pathway in Jurkat-Tat101.
(A) Scheme of PTEN-AKT-FOXO3a signaling pathway. (B) The expression of PTEN, pAKT S473 and total AKT was analyzed by immunoblotting in cytosolic protein extracts from Jurkat-Tat72, Jurkat-Tat101 and control cells. (C) The expression of pFOXO3aS253 and total FOXO3a was analyzed by immunoblotting in nuclear and cytosolic protein extracts from Jurkat-Tat72, Jurkat-Tat101 and control cells. β-actin was used as loading control. (D) Analysis of the rate of apoptosis in Jurkat-Tat cells in comparison with control cells. Apoptosis was measured by chemiluminescence after treatment with FasL for 4 hours. Media and SEM from at least three independent experiments are shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (* or ** for p<0.05 or p<0.01, respectively).
Fig 7.
Analysis of PTEN-AKT-FOXO3a signaling pathway in Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 cells.
Expression of hsa-miR-21 and hsa-miR-222 was measured in Jurkat-hsa-miR-21 (A) and Jurkat-hsa-miR-222 (B), respectively, by qRT-PCR. (C) The expression of PTEN, pAKTS473 and total AKT was analyzed by immunoblotting in cytosolic protein extracts from Jurkat-hsa-miR-21, Jurkat-hsa-miR-222 and Jurkat-hsa-miR-Null as control cells. (D) Analysis of the rate of apoptosis in Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 in comparison with Jurkat-hsa-miR-Null control cells. Apoptosis was measured by chemiluminescence after treatment with FasL for 4 hours. Media and SEM from at least three independent experiments are shown. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (*, ** or *** for p<0.05, p<0.01 or p<0.001, respectively).
Fig 8.
Analysis of cell cycle progression and cell morphology of Jurkat-Tat101 and Jurkat-Tat72 cells.
(A) Analysis of the percentage of cells presented in each stage of the cell cycle. Jurkat-Tat101 and Jurkat-Tat72 cells were serum depleted and then serum stimulated for 48 hours. Fluorescence from cells stained with propidium iodide was analyzed by flow cytometry in FL2 channel. The percentage of cells gathered in each phase of the cell cycle is indicated in the histograms. Only cells stimulated with serum for 48 hours are shown. (B) Fold of percentage of cells in each stage of the cell cycle that were serum depleted (t = 0) and then serum stimulated for 48 hours (t = 48h). (C) Analysis by immunofluorescence of giant cells with multiple nuclei or multi-lobed nuclei observed in the Jurkat-Tat101 population versus control cells. Cell diameter was measured using LAS AF software (Leica). Average percentage is represented in the bar diagrams with SEM. Statistical significance was calculated by two-way ANOVA with Bonferroni post-test analysis (* or ** for p<0.05 or p<0.01, respectively).
Fig 9.
Analysis of cell cycle progression and cell morphology of Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 cells.
(A) Analysis of the percentage of cells presented in each stage of the cell cycle. Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 cells were serum depleted and then serum stimulated for 48 hours. Fluorescence from cells stained with propidium ioide was analyzed by flow cytometry in FL2 channel. The percentage of cells gathered in each phase of the cell cycle is indicated in the histrograms. Only cells stimulated with serum for 48 hours are shown. (B) Fold of percentage of cells in each stage of the cell cycle that were serum stimulated for 48 hours. (C) Analysis by immunofluorescence of giant cells with multiple nuclei or multi-lobed nuclei observed in Jurkat-hsa-miR-21 and Jurkat-hsa-miR-222 versus control cells. Cell diameter was measured using LAS AF software (Leica).