Journal: PLoS ONE

Article Title: Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction

doi: 10.1371/journal.pone.0158639

Figure Lengend Snippet: The hTERT gene was transduced into 682TL and 779TL ahMNCs using a lentiviral vector. (A) Morphology of the ahMNCs was compared with that of hTERT-ahMNCs. (B) ahMNCs and hTERT-ahMNCs were cultured in vitro for a long time (>18 in vitro passages), and the numbers of cells were traced. (C) hTERT expression was confirmed by RT-PCR. (D) Telomere length was analyzed by qPCR and compared. (E) Expression of the neural stem cell marker Nestin and ahMNCs differentiation potential in vitro was compared with those of hTERT-ahMNCs. ahMNCs or hTERT-ahMNCs were cultured under differentiating conditions for 12 days. MAP2 for neurons and GFAP for astrocytes. BD; Before differentiation, AD; After differentiation.

Article Snippet: The hTERT-overexpressing lentiviral vector was co-transfected with the VSVG and PAX2 lentiviral packaging vectors into HEK293FT cells (Invitrogen, Carlsbad, CA, USA).

Techniques: Plasmid Preparation, Cell Culture, In Vitro, Expressing, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Oncogene

Article Title: The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

doi: 10.1038/s41388-020-01435-4

Figure Lengend Snippet: a Protein blot of PAX2-HA and beta actin (as loading control) of stable MCF-7-breast cancer cell inducible for PAX2-HA expression. The expression of PAX2-HA is induced by doxycycline and different concentrations. b Cell growth assay of stable MCF-7-breast cancer cell inducible for PAX2-HA expression. Doxycycline and non-doxycycline treated cells (25 or 50 ng/ml) were treated with vehicle (control) or tamoxifen (1 μM) for 96 h. Tamoxifen was added every 24 h.

Article Snippet: pLV.ExSi.P/Hygro-CMV-TET3G and pLV.ExSi.P/Puro-TRE3G-PAX2/HA vectors were purchased from Cyagen (Santa Clara, CA, USA). pCI-Neo mammalian overexpression vector was purchased from Promega (E1841).

Techniques: Expressing, Growth Assay

Journal: Oncogene

Article Title: The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

doi: 10.1038/s41388-020-01435-4

Figure Lengend Snippet: a Volcano plots of genes regulated in different treatments (tamoxifen, PAX2, and PAX2–tamoxifen) detected in GRO-seq experiment. b , c upper panel: Venn diagrams showing the overlap of up or downregulated genes detected in GRO-seq between different treatments respectively. b , c lower panel: genome browser examples illustrating different patterns of regulation of PAX2 up or downregulated genes. d Box plots of sequencing data for PAX2-HA ChIP-sequencing (left) and gene expression (right) of PAX2-regulated genes with PAX2-HA binding sites at their TSS. Genes enriched at TNF (top) and p53 (bottom) signaling are indicated. The ChIP-sequencing data are indicated as fold change of PAX2 binding of tamoxifen (DoxTam) vs non-tamoxifen (Dox). The expression data are indicated as log2 fold change of cells overexpressing PAX2 (dox) or overexpressing PAX2 and tamoxifen treatment (DoxTam) relative to non-PAX2 overexpressing cells (Veh).

Article Snippet: pLV.ExSi.P/Hygro-CMV-TET3G and pLV.ExSi.P/Puro-TRE3G-PAX2/HA vectors were purchased from Cyagen (Santa Clara, CA, USA). pCI-Neo mammalian overexpression vector was purchased from Promega (E1841).

Techniques: Sequencing, ChIP-sequencing, Expressing, Binding Assay

Journal: Oncogene

Article Title: The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

doi: 10.1038/s41388-020-01435-4

Figure Lengend Snippet: a Boxplot indicating the expression of PAX2 upregulated genes with ( n = 16) and without ( n = 79) PAX2 eRNAs. b List of enriched pathways in PAX2 upregulated genes with PAX2 eRNAs. c Kaplan–Meier curves of genes with significant overall survival (IRF1 and MAL) and relapse-free survival (BDH1). The analysis was performed by means of using public clinical databases available at UCSC Xena (browser: https://xenabrowser.net/heatmap/ ). d Paired samples (primary and metastatic tumors) of patients initially diagnosticated with ER+/HER2− tumors, which were treated with tamoxifen for 2 years and stopped responding to treatment. The expression by NanoString of BDH1 was investigated in both samples and in each of the five patients.

Article Snippet: pLV.ExSi.P/Hygro-CMV-TET3G and pLV.ExSi.P/Puro-TRE3G-PAX2/HA vectors were purchased from Cyagen (Santa Clara, CA, USA). pCI-Neo mammalian overexpression vector was purchased from Promega (E1841).

Techniques: Expressing

Journal: Oncogene

Article Title: The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

doi: 10.1038/s41388-020-01435-4

Figure Lengend Snippet: a Western blotting showing IRF1 induction by PAX2 overexpression. MCF-7-PAX2 cells were treated with Dox for 16 h, IRF1, PAX2, and β-actin levels were determined with western blotting. b PAX2 (HA antibody) ChIP experiment with MCF-7-PAX2 cells treated with vehicle (Veh) and 50 ng/ml doxycycline (Dox). Two sites located at IRF1 promoter (IRF1-P) or IRF1 enhancer (IRF1-E) were tested with real-time PCR ( n = 3, * p < 0.05, ** p < 0.01). Pull-down DNA amount was normalized to the input DNA of each sample, and data are presented as the percentage of input with ±s.d. as the error bar. c Western blotting of MCF-7-PAX2 cells transfected with nontargeting siRNA (siNT), and two siRNAs targeting IRF1 (siIRF1-1 and siIRF1-2). MCF-7-PAX2 cells were transfected with siNT, or siIRF1. IRF1, PAX2, and β-actin level were detected by Western blotting. IRF1 protein level was quantified by normalization against beta actin level. d Real-time PCR analysis of genes upregulated by PAX2. MCF-7-PAX2 cells were transfected with nontargeting siRNA (siNT) or IRF1 siRNA followed by doxycycline treatment. mRNA levels of genes were measured with real-time PCR, and result was normalized first to UBC and then to the average of Veh sample. The data are represented as the mean of independent replicates ± s.d. ( n = 3, * p < 0.05, ** p < 0.01). e MCF-7 cells were transfected with pCI-neo empty vector or with increasing amount of pCI-neo-IRF1 vector. Forty-eight hours after transfection, IRF1 and β-actin level was determined with western blotting. f MCF-7 cells were transfected with pCI-neo empty vector or with increasing amount of pCI-neo-IRF1 vector, then mRNA levels of different genes were detected with real-time PCR, result was normalized first to UBC and then to the average of Veh, and data are presented as the percentage of input with ±s.d. as the error bar ( n = 3, * p < 0.05, ** p < 0.01).

Article Snippet: pLV.ExSi.P/Hygro-CMV-TET3G and pLV.ExSi.P/Puro-TRE3G-PAX2/HA vectors were purchased from Cyagen (Santa Clara, CA, USA). pCI-Neo mammalian overexpression vector was purchased from Promega (E1841).

Techniques: Western Blot, Over Expression, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

Journal: Oncogene

Article Title: The proapoptotic gene interferon regulatory factor-1 mediates the antiproliferative outcome of paired box 2 gene and tamoxifen

doi: 10.1038/s41388-020-01435-4

Figure Lengend Snippet: a Western blotting showing induction of caspase 7 and 8 level by PAX2. MCF-7-PAX2 cells were treated with vehicle (Veh), 50 ng/ml doxycycline (Dox), 1 μM tamoxifen (Tam) or the combination of both for 48 h, then whole cell lysate was prepared and analyzed by using western blotting with caspase 7, caspase 8, and RPL13a antibodies, RPL13a is used as loading control. Cleaved caspase 7 was quantified normalization against RPL13a. b MCF-7-PAX2 cells were transfected with siNT or siIRF1 for 24 h, then treated with or without 50 ng/ml doxycycline (Dox) for 48 h. Whole cell lysate was prepared and analyzed with western blotting with caspase 7, caspase 8, and RPL13a antibodies. c , d Real-time Annexin V assay showing PS externalization. MCF-7-PAX2 cells were transfected with siNT, or siIRF1 followed by treatments with vehicle (Veh), 50 ng/ml doxycycline (Dox), 1 μM tamoxifen (Tam), or the combination of both. PS externalization was measured according to the methods session. The data are represented as the mean of independent replicates ± s.d. ( n = 5). e The model of actions of PAX2 in ER-positive breast cancer cells.

Article Snippet: pLV.ExSi.P/Hygro-CMV-TET3G and pLV.ExSi.P/Puro-TRE3G-PAX2/HA vectors were purchased from Cyagen (Santa Clara, CA, USA). pCI-Neo mammalian overexpression vector was purchased from Promega (E1841).

Techniques: Western Blot, Transfection, Annexin V Assay