Identifying Glioblastoma stem cell-specific and conserved cancer/testis LncRNA.

A. The Scatterplot depicts the differentially regulated protein-coding and lncRNA. The X-axis shows a differentially regulated gene (log2 fold change) in GBM vs. normal (TCGA patient cohort), and Y-axis represents a differentially regulated gene (log2 fold change) in the GSC Vs. DGC dataset (GSE54791). Significant genes with log2 fold change > 1 were represented by red dots. B, C & D. The expression in log2 fold change of FAM95B1 was shown in TCGA and two patient cohorts of the CGGA dataset (CGGA_693 & CGGA_325). Glioviz was used to obtain the gene expression matrix, and a t.test was performed using GraphPad Prism v6. E. Plot depicts log2 fold change of FAM95B1 in our patient cohort (normal, n=3 and GBM, n=80). F. Expression of FAM95B1 in GSC vs. NSC dataset (GSE92459). G. Expression of FAM95B1 in GSC vs. DGC dataset (GSE54791). H. Relative expression of FAM95B1 was quantified by qRT-PCR in different Glioblastoma cell lines and immortal astrocytes (IHA). I, Relative expression of FAM95B1 was measured in seven GSCs and its corresponding DGCs using the qRT-PCR method. J. Relative expression (RPKM) of FAM95B1 across different cancer types in the TCGA Pan-cancer cohort. K. Expression in TPM of FAM95B1 amongst the GTEX normal bulk tissue RNA-seq dataset. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

Glioblastoma cell proliferation and chemosensitivity altered by PITAR silencing.

A. Plot depicts subcellular fractionation of U87 cells followed by quantification using qRT-PCR. MALAT1 is a positive control for nuclear gene expression, and GAPDH is a positive control for cytoplasmic gene expression. B. The RNAScope images of PITAR (red) and the DAPI (nucleus, blue) counterstain in U-87 cells. C. The knockdown efficiency of siRNAs (siPITAR#1, siPITAR#2, and siPITAR#3) against PITAR was measured by qRT-PCR. D. the cell proliferation was measured by MTT assay upon PITAR knockdown in U87 cells. E. the viable cell count was measured using a viable cell counter following the Trypan blue method. F. Colony formation assay was performed upon PITAR knockdown compared to siNT in U87 cells. G. Cell cycle analysis was performed in PITAR-silenced U87 cells. H. the time-dependent apoptotic cell death was measured by Annexin V/PI staining in PITAR-silenced U87 cells. I. the chemosensitivity upon PITAR silencing was measured by MTT assay against Adriamycin and Temozolomide in U87 cells compared to control cells. J. the viable cell count of human astrocytes (NHA) was measured upon PITAR silencing. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

Identification of PITAR targets.

A. Genomic track for PITAR derived from ChIRP-RNA sequencing using Odd, Even, and LacZ antisense probe. B. PITAR Pulldown by ChIRP assay was quantified using qRT-PCR. C. The Venn diagram represents the association of four datasets (ChIRP enriched genes, PITAR positive correlated genes from TCGA, GBM vs. normal upregulated genes from TCGA, and GSC vs. DGC upregulated genes from GSE54791). D. The selected fifteen genes from the Venn diagram are plotted in the scatter plot, and an arrow marked TRIM28 as a selected target. E. The volcano plot depicts up-regulated (n=420) and down-regulated (n=526) genes upon PITAR knockdown compared to siNT. The gene expression matrix between siPITAR and siNT was used to construct a volcano plot to visualize differentially expressed genes. F. Gene set enrichment analysis (GSEA) of differentially regulated genes was performed based on PITAR expression level at log2fold >0.58 and p<0.05. G. The GSEA plots depict the enrichment of p53 up and down target genes results derived from PITAR silencing in U87 cells. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

PITAR regulates the expression of TRIM28 by physical interaction with TRIM28.

A. qPCR of PITAR and TRIM28 in ChIRP-RNA interactome samples. Probes for Odd and LacZ were used to pull down endogenous PITAR and interacting TRIM28 mRNA from U87 cells. B. Schematic represents the predicted RNA–RNA interaction between PITAR and the 3′ UTR of TRIM28. C. RNAScope images of co-localized signals of PITAR (red) and TRIM28 (green) in U-87 cells. The panel shows the 3D reconstructed cell images (Imaris) of the merged 2D image. Yellow dots shown by white arrows depict co-localized PITAR (red) and TRIM28 (green). Magnified co-localized puncta were shown in the inset at the upper right corners, indicated by a white dotted box. The panel shows the RNAScope images of the localization of PITAR (red), TRIM28 (green), and PDE2A (green/red). PDE2A RNA was used as a negative control. The indicative scale bar on the images is 50µm. D. Schematic of vector plasmid construct of PITAR OE, ΔPITAR OE, TRIM28 3′ UTR, and ΔTRIM28 3′ UTR. E. PITAR interaction with the TRIM28 3′ UTR was measured using an in vitro RNA–RNA interaction assay and compared to a panel of control RNAs (PITAR antisense, Fluc control, Bead control. The binding affinity was quantified by qRT-PCR analysis of the TRIM28. Data were normalized to the PITAR-AS control. F. The Luc activity of TRIM28 3′ UTR was measured after the ectopic expression of PITAR and ΔPITAR in U87 cells using a luciferase reporter assay. G. Luciferase assay was performed in PITAR silenced U87 cells co-transfected with VC and PITAR OE vector. H. The Firefly luciferase activity was measured in U87 cells containing a deleted PITAR binding site of TRIM28 3’UTR and co-transfected with VC and PITAR OE. I. Relative expression of TRIM28 in PITAR-silenced U87 cells was measured by qPCR. J. The TRIM28 protein expression was measured by immunoblotting. K. TRIM28 transcript was measured at indicated time points post Actinomycin D (5 μg/ml) treatment in siNT and siPITAR-transfected U87 cells by qPCR. The log2 ratio of the remaining TRIM28 was plotted using linear regression after normalizing to the 0th hour of the respective condition. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

PITAR regulates wild-type p53 protein levels via TRIM28-mediated ubiquitination.

A. Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR-silenced U87 cells compared to siNT. B. The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in PITAR-silenced U87 cells compared to siNT. C & D. Cells were transfected with pcDNA-PITAR (PITAR OE)/vector control plasmid (pcDNA) and harvested 48h post-transfection for qRT-PCR (PITAR, TRIM28, TP53, and CDKN1A) or immunoblotting with indicated antibodies (TRIM28, p53, and p21). GAPDH served as the control. E. The Immunoblotting Half-life of p53 protein in PITAR-silencing (siPITAR) and control (siNT) U87 cells was measured, and Immunoblotting assays were used to detect p53 in U87 cells with the treatment of cycloheximide (CHX; 50 μg/mL). The relative expression of the remaining TRIM28 was plotted using linear regression after normalizing to the 0th hour of the respective condition. F. The endogenous level of p53 ubiquitination was measured in pcDNA-PITAR (PITAR OE)/vector plasmid (pcDNA) U87 cells by p53 immunoprecipitation followed by immunoblotting with the indicated antibodies in the presence and absence of MG132. G. The PG13-Luc activity was measured in PITAR-silenced/siNT U87 cells, which were further transfected with TRIM28 overexpression/Vector control plasmid. H. The relative expression of CDKN1A was measured by qPCR in PITAR-silenced/siNT U87 cells, which were further transfected with TRIM28 overexpression/Vector control plasmid. I. The protein expression of TRIM28, p53, and CDKN1A was measured by immunoblotting in PITAR-silenced/siNT U87 cells, which were further transfected with TRIM28 overexpression/Vector control plasmid. J. The relative expression of CDKN1A was measured in the presence and absence of adriamycin by qPCR in PITAR OE/VC U87 cells. K. The protein expression of TRIM28, p53, ac-p53, and CDKN1A was measured in the presence and absence of adriamycin by immunoblotting in PITAR OE/VC U87 cells. GAPDH served as the control. L. The Cell cycle analysis was performed in the presence and absence of Adriamycin in PITAR OE/VC U87 cells. M. The relative expression of PITAR, TRIM28, and CDKN1A was measured in the presence and absence of adriamycin by qPCR in U87 cells. N. The relative expression of PITAR and TRIM28 was measured in the presence and absence of Adriamycin/CGK733 using qPCR in U87 cells. O. TRIM28 protein expression was measured by immunoblotting in the presence of Adriamycin/CGK733 in U87 cells. P. The relative expression of PITAR and TRIM28 was measured by qRT-PCR in PITAR-silenced U87 cells after Adriamycin treatment. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

Glioblastoma stem-like cell growth induced by PITAR.

A. The bright field image represents RG5 sphere growth on siNT/siPITAR condition. B&C. RG5 sphere growth was quantified by the measurement of sphere count and sphere size using ImageJ (https://imagej.nih.gov/ij/download.html). D. Limiting dilution assay was performed in RG5 cells. The graph represents the percentage of wells without spheres as a function of the number of PITAR Knockdown cells compared to control cells. E. Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR-silenced RG5 cells compared to siNT. F. The bright field image represents RG5 sphere growth on PITAR OE compared to the control vector. G&H. RG5 sphere growth was quantified by measuring sphere count and size in the PITAR OE condition compared to the control vector using ImageJ (). I. Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR OE condition of RG5 cells compared to the control vector. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

PITAR promotes glioma tumor growth and resistance to Temozolomide chemotherapy.

A. Mice (NIH nu/nu mice) were injected U87-Luc cells (0.3×106 cells/mice) transfected with siNT/siPITAR, and tumors were allowed to grow for 50 days, and the luminescence imaging was performed in the Ivis instrument. B. H&E staining was performed in formalin-fixed tumor-bearing (siNT and siPITAR) mouse brain sections. C. The tumor growth was monitored by detecting luminescence from tumor cells of siNT and siPITAR tumor-bearing mice. D. Kaplan–Meier graph showing the survival of mice bearing tumors formed by siNT and siPITAR cells. E. Immunohistochemistry analysis of TRIM28 expression in U87/siNT or U87/siPITAR cells derived intracranial tumor xenograft. The Green color represents TRIM28, and the blue depicts the nucleus. Scale bar = 100 μm. F. Mice (NIH nu/nu mice) were injected U87-Luc/PITAR OE and U87-Luc/VC cells (0.3×106 cells/mice), and tumors were allowed to grow for 11 days. The tumor-bearing mice were treated with 100 mg/kg TMZ in 25% DMSO saline solution by intraperitoneal injection for one week, and the luminescence imaging was performed in the Ivis instrument. G. The tumor growth was monitored by detecting luminescence from the tumor of VC, PITAR OE, VC+TMZ, and PITAR OE+TMZ tumor-bearing mice. H. Proposed working model in this study. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.