Fig 1: RNA-seq Analysis Identified MIAT Targets in Human Th17 Cells. (A) Venn diagrams on the top show the overlap of the number of differentially regulated genes (FDR <0.05 and LFC >1.5) when comparing LNA1- or LNA4-transfected samples to the negative control LNA (NC). The numbers in red indicate common and unique upregulated genes, and numbers in blue indicate common and unique downregulated genes upon MIAT silencing. Heatmap representation of the expression of the top 50 DE genes similarly regulated (see results section for details) upon MIAT silencing at 24 (left) and 72 h (right) in Th17 cells. NC, LNA1 and LNA4 columns represent the gene expression (RPKM) in respective samples. FC columns represent the fold-change in gene expression upon MIAT silencing. (B) TaqMan qPCR of PRKCA expression analysis in MIAT silenced Th17 cells with two different LNAs at 24 h, where each dot represents an independent experiment. Students’ paired two-tailed t-test was used to determine the significance. *, p <0.05. (C) Western blot expression analysis of PKCa proteins in MIAT-silenced 24-h Th17 cells. A representative result (lower panel) from three independent experiments for PKCa is shown along with ß-actin for loading control. WB quantification was performed using ImageJ, from three independent experiments (upper panel), and significance was calculated using Students’ t-test (two-tailed, paired). *, p <0.01. The data were plotted as arbitrary units (AU). (A-C) The experiments were performed on three pools of CD4+ T cells, where each pool contained cells purified from umbilical cord blood collected from 3-5 individuals. (D) TaqMan qPCR of PRKCA expression analysis showing knockdown efficiency of the siRNA at RNA level at 24 h and 72 h. Students’ paired two-tailed t-test was used to determine the significance. **, p <0.01; ****, p <0.0001. (E) Western blot expression analysis of PKCa proteins showing knockdown efficiency of the siRNA at protein level at 24 h and 72 h The quantification was performed using ImageJ, from three independent experiments, each having two biological replicates. The intensities were normalized to ß-actin. The significance was calculated using Students’ t-test (two-tailed, paired). ****, p <0.0001. The data were plotted as arbitrary units (AU). (F) TaqMan qPCR of IL17A expression from three independent experiments, each having two biological replicates. The Ct values were normalized to EF1A. The significance was calculated using Students’ t-test (two-tailed, paired). **, p <0.01. (G) IL-17A secretion as measured by ELISA. The data was obtained from three independent experiments, each having two biological replicates. The data has been normalized to number of cells in the culture-well. The significance was calculated using Students’ t-test (two-tailed, unpaired). ****, p <0.0001. (D-G) The experiments were performed on six pools of CD4+ T cells, where each pool contained cells purified from umbilical cord blood collected from 3-5 individuals. (H) IL-17A secretion as measured by ELISA. The scheme for this experiment has been shown in Figure S7A . In the first nucleofection cells were transfected with NC or MIAT targeting LNA, whereas in the second nucleofection the cells were either mock transfected or they were transfected with in vitro transcribed GFP or PRKCA RNA. The experiments were performed on three pools of CD4+ T cells, where each pool contained cells purified from umbilical cord blood collected from 3-5 individuals. The significance was calculated using Students’ t-test (two-tailed, paired). **, p <0.01; *, p <0.05; ns denotes not significant.