Table 1.
List of qRT-primers.
Fig 1.
Expression analysis of apoptotic markers in staurosporine treated HEK293T cells: The relative expression of ERN1, MOAP1, BAK1, p53 Caspase 3, Caspase 7 and Caspase 9 mRNAs were analyzed through quantitative reverse transcription polymerase chain reaction.
RNA was isolated from staurosporine-treated HEK293T cells and expression of aforesaid genes were quantified and compared with respect to untreated cells. GAPDH was used as a housekeeping control and data plotted as a measure of relative expression. Results represent the mean ± SEM (n = 3 independent experiments) **p<0.01; ***p<0.001; ****p<0.0001; ns = non-significant.
Fig 2.
Antiapoptotic effects of hcmv-miR-UL148D in staurosporine-treated HEK293T cells: (A) hcmv-miR-UL148D mimic or inhibitor transfected HEK293T cells were treated with staurosporine followed by counterstaining with the DAPI and rhodamine phalloidin. The arrow indicates the significant changes associated with the apoptosis as decrease in the chromatin condensation and nuclear fragmentation by hcmv-miR-UL148D (image acquired at 63X). (B) From the same set of groups as in Fig A, cells were stained with propidium iodide and annexin V followed by flow cytometry analysis. The flow cytometry data represented in dot plots show decrement of apoptotic cells by hcmv-miR-UL148D. (C) The apoptotic cell ratio was calculated from the flow cytometric data using ImageJ (mean±SEM; ****, p<0.0001). (D) HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor then treated with staurosporine. Caspase 3/7 activity was measured from cell lysate using luminescence and expressed as raw luminescence units (RLU). Results represent the mean ± SEM (n = 3 independent experiments) **p<0.01; ***p<0.001; ****p<0.0001.
Fig 3.
(A) Hcmv-miR-UL148D targets ERN1 through 3’UTR binding: (A) HEK293T cells were transfected with hcmv-miR-UL148D mimic or hcmv-miR-UL148D mimic and inhibitor together. RNA was isolated and the ectopic expression level of hcmv-miR-UL148D was measured through qRT-PCR, the miRNA levels were normalized to 5s rRNA in the corresponding samples. Result represents the mean ± SEM (n = 3 independent experiments) ****, p<0.0001. (B) hcmv-miR-UL148D downregulates the ERN1 mRNA expression. HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor followed by staurosporine treatment. RNA was isolated and expression of ERN1 was quantified and compared with respect to untreated cells. GAPDH was used as a housekeeping control and data plotted as a measure of relative expression. Results represent the mean ± SEM (n = 3 independent experiments) **p<0.01; ***p<0.001. (C) Cartoonistic representation of pEZX-MT06-3’UTRWT/DEL dual-luciferase reporter vector, highlighting the binding site of hcmv-miR-UL148D. (D) hcmv-miR-UL148D targets the ERN1 3’UTR. HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor along with either pEZX-MT06-3’UTRWT-ERN1 or pEZX-MT06-3’UTRDEL-ERN1 luciferase reporter construct. Luciferase activity was measured 24 hr post transfection. Unlike the wild-type, the luciferase activity of the mutant pEZX-MT06-3’UTRDEL-ERN1 was not inhibited by hcmv-miR-UL148D.
Fig 4.
hcmv-miR-UL148D inhibits IRE1α protein level in staurosporine treated HEK293T cells.
(A) The IRE1α protein levels were analyzed in different groups of cells transfected with hcmv-miR-UL148D, hcmv-miR-UL148D and its inhibitor followed by Staurosporine treatment. The IRE1α protein and the β-actin levels were examined through western blot. The bands of the blots have been cropped with no further manipulation. (B) Relative IRE1α protein levels in different groups of cells were quantified through ImageJ software. The IRE1α levels were normalized to β-actin and plotted as fold change with respect to control. Results represent the mean ± SEM (n = 3 independent experiments) **p<0.01; ***p<0.001.
Fig 5.
Hcmv-miR-UL148D regulates XBP1 splicing and JNK phosphorylation: (A) HEK293T cells were transfected with hcmv-miR-UL148D mimic or inhibitor followed by staurosporine treatment. RNA was isolated and XBP1 mRNA spliced (sXBP1) and unspliced (uXBP1) were measured through qRT-PCR. The uXBP1 and sXBP1 expression level were normalized to that of GAPDH and fold change was calculated and plotted with respect to the untreated control. Results represent the mean ± SEM (n = 3 independent experiments) *, p<0.05, **, p<0.01. (B, C & D) Whole cell lysate was prepared from the hcmv-miR-UL148D mimic or inhibitor transfection and staurosporine treated cells. The expression of p-JNK, uXBP1 and sXBP1 were analyzed in different groups of cells through western blot and β-actin was used as a loading control. The bands of the blots have been cropped with no further manipulation. The Relative protein expression levels in different groups of cells were analyzed through ImageJ software. Results represent the mean ± SEM (n = 3 independent experiments) **, p<0.01; ***, p< 0.001; ****P<0.0001; ns = non-significant.
Fig 6.
Comparison of downregulation of ERN1 by siRNA of ERN1 and hcmv-miR-UL148D: (A) ERN1 mRNA downregulation: The relative expression levels of ERN1 mRNA after the transfection of either hcmv-miR-UL148D and siRNA of ERN1 were measured through qRT-PCR. Results were expressed as the fold change (2−ΔΔCt) (± SEM; ****, p < 0.0001; ns = non-significant). (B & C) IRE1α protein encoded by ERN1 downregulation: The IRE1α protein downregulation after transfection with either siRNA of ERN1 and hcmv-miR-UL148D were analyzed through Western blot. The relative IRE1α protein quantification was performed through ImageJ software after normalizing with β-actin. Experiments were performed in triplicates (S2 Fig), and the data from three different experiments were used for statistical analysis (±SEM; **, p < 0.01, ns = non-significant).