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

A. The Scatterplot depicts the differentially regulated protein-coding (Blue dots) and lncRNA (Red dots) transcripts. The X-axis shows a differentially regulated gene (log2 fold change) in GBM vs. normal (TCGA patient cohort), and the Y-axis represents differentially regulated genes (log2 fold change) in the GSC Vs. DGC dataset (GSE54791). 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). GlioVis 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=79). 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 immortalized human 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 U87 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 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 (0.25µg/ml) and Temozolomide (300µM) in U87 cells compared to control cells. J. The viable cell count of human astrocytes (IHA) 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 gene sets, results derived from PITAR-silenced 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. The qRT-PCR of PITAR and TRIM28 RNA was performed in ChIRP-RNA pull-down samples. The Probes for Odd and LacZ were used to pull down endogenous PITAR and interacting TRIM28 mRNA in 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 U87 cells. The panel shows the 3D reconstructed cell images of the merged 2D image (Imaris image analysis software). 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 (ΔTRIM28 3′ UTR), co-transfected with VC and PITAR OE. I. Relative expression of TRIM28 in PITAR-silenced U87 cells was measured by qRT-PCR. 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 qRT-PCR. 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 pcDNA3.1-PITAR (PITAR OE)/ empty vector control plasmid (pcDNA3.1) and harvested 48h post-transfection for qRT-PCR (PITAR, TRIM28, TP53, and CDKN1A) and immunoblotting with indicated antibodies (TRIM28, p53, and p21). GAPDH served as the control. E. The Half-life of the p53 protein was measured in PITAR-silencing (siPITAR) and control (siNT) U87 cells with the treatment of cycloheximide (CHX; 50 μg/mL). The relative expression of the remaining p53 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 pcDNA3.1-PITAR (PITAR OE)/empty vector plasmid (pcDNA3.1) stable U87 cells by p53 immunoprecipitation followed by immunoblotting with the indicated antibodies in the presence and absence of MG132. G, H & I. The relative expression of PITAR, TRIM28, and CDKN1A was measured by qRT-PCR in U87/siPITAR#1 and U87/siNT cells with exogenously overexpressed TRIM28 conditions. J. The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in U87/siPITAR#1 and U87/siNT cells with exogenously overexpressed TRIM28 condition. K, L & M. The relative expression of PITAR, TRIM28, and CDKN1A was measured by qRT-PCR in U87/shTRIM28 and U87/shNT cells with exogenously overexpressed PITAR conditions. N. The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in U87/shTRIM28 and U87/shNT cells with exogenously overexpressed PITAR conditions. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

DNA damage-induced PITAR diminishes the DNA damage response by p53 through TRIM28.

A. The relative expression of CDKN1A was measured in the presence and absence of Adriamycin by qRT-PCR in PITAR OE/VC U87 cells. B. 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. C. The viable cell count was performed in the presence and absence of Adriamycin in PITAR OE/VC U87 cells. D. The Cell cycle analysis was performed in the presence and absence of Adriamycin in PITAR OE/VC U87 cells. E. The relative expression of PITAR, TRIM28, TP53, and CDKN1A was measured by qRT-PCR in Ad-p53 and Ad-GFP-infected U87 cells. F. The protein expression of TRIM28 and p53 was measured by immunoblotting in Ad-p53 and Ad-GFP-infected U87 cells. G. The relative expression of PITAR, TRIM28, and CDKN1A was measured in the presence and absence of Adriamycin by qRT-PCR in U87 cells. H. The immunoblot depicting the expression of TRIM28, p53, and p21 upon treatment of Adriamycin. I & J. The qRT-PCR was performed to measure the relative expression of PITAR and TRIM28 in the p53 knockdown condition upon Adriamycin treatment. K. The relative expression of PITAR and TRIM28 was measured by qRT-PCR in Adriamycin-treated PITAR-silenced U87 cells. 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) were injected with siNT/siPITAR#1 transfected U87-Luc cells (0.3x106 cells/mice), and tumors were allowed to grow for 50 days, and the luminescence imaging was performed using the IVIS instrument (Perkin Elmer IVIS system). B. H&E staining was performed in formalin-fixed tumor-bearing (siNT and siPITAR#1) mouse brain sections. C. The tumor growth curve of siNT and siPITAR#1 tumor-bearing mice was quantified over time using IVIS. D. The Kaplan–Meier graph shows the survival of mice-bearing tumors formed by siNT and siPITAR#1 cells. E. The Immunohistochemistry assay was performed to show the TRIM28 protein expression in the tumor tissue section derived from U87-Luc/siNT and U87-Luc/siPITAR#1 tumors. The Green color represents the TRIM28 protein, and the blue depicts the nucleus stained with DAPI. Scale bar = 100 μm. F. Mice (NIH nu/nu) were injected with U87-Luc/PITAR OE and U87-Luc/VC cells (0.3x106 cells/mice), and tumors were allowed to grow for 30 days. The tumor-bearing mice were treated with 100 mg/kg TMZ in 25% DMSO saline solution after 11 days by intraperitoneal injection for one week, and the luminescence imaging was performed using the IVIS instrument. G. The tumor growth curve of VC, PITAR OE, VC+TMZ, and PITAR OE+TMZ tumor-bearing mice was quantified over time using IVIS. H. Mice (NIH nu/nu) were injected with U87-Luc/PITAR OE+shNT, U87-Luc/VC+shNT, U87-Luc/PITAR OE+shTRIM28 and U87-Luc/VC+shTRIM28 cells (0.3x106 cells/mice), and tumors were allowed to grow for 50 days. I. The tumor growth curve of VC+shNT, PITAR OE+shNT, VC+shTRIM28, and PITAR OE+shTRIM28 tumor-bearing mice was quantified over time using IVIS. Luminescence was evaluated twice per 10 days and before sacrifice. Bars indicate standard error. Data are shown as mean ± SD (n=3). ***P-value <0.001, **P-value <0.01, *P-value <0.05.

Proposed working model of this study. PITAR inhibits p53 by binding and stabilizing TRIM28 mRNA (created with BioRender.com).

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