Journal: Cancers

Article Title: Exosomal Long Non-Coding Ribonucleic Acid Ribonuclease Component of Mitochondrial Ribonucleic Acid Processing Endoribonuclease Is Defined as a Potential Non-Invasive Diagnostic Biomarker for Bladder Cancer and Facilitates Tumorigenesis via the miR-206/G6PD Axis

doi: 10.3390/cancers15215305

Figure Lengend Snippet: Positive correlation between RMRP and G6PD expression in BLCA Cells. ( A – E ) In vivo experimental scheme of BIU-87 allografts. ( A ) Experimental scheme of in vivo antitumor experiment ( n = 5 mice per group). ( B ) The tumor volume of each treatment group. ( C ) Photos of mice and tumors from all groups. ( D ) The tumor weight at the end point of the animal experiment. ( E ) Representative images of H and E, PCNA, and Ki67 staining in the tumor, scale bar = 100 µm. ( F ) G6PD expression in BLCA and normal tissues in TCGA database. ( G ) G6PD expression in tumor tissues from normal and BLCA patients. ( H ) Correlation analysis of RMRP and G6PD expression in BLCA patients. Each dot represents one tumor tissue. ( I ) RT-qPCR and ( J ) WB analyses of G6PD expression in 5637 and BIU-87 cells after treatment of sh-NC or sh-RMRP. The uncropped blots are shown in . All data are presented as the mean ± SEM of triplicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST buffer and incubated with primary antibodies against CD9 (1:1000, 13174S; Cell Signaling Technology (CST), Danvers, MA, USA), TSG101 (1:1000, Ab83; Abcam, Cambridge, UK), MG130 (1:1000, 12480S; CSTUSA); G6PD (1:1000, 12263S; CST, USA) and β-Actin (1:1000, 4967S; CST, USA) overnight at 4 °C.

Techniques: Expressing, In Vivo, Staining, Quantitative RT-PCR

Journal: Cancers

Article Title: Exosomal Long Non-Coding Ribonucleic Acid Ribonuclease Component of Mitochondrial Ribonucleic Acid Processing Endoribonuclease Is Defined as a Potential Non-Invasive Diagnostic Biomarker for Bladder Cancer and Facilitates Tumorigenesis via the miR-206/G6PD Axis

doi: 10.3390/cancers15215305

Figure Lengend Snippet: RMRP serves as an miRNA sponge of miR-206 to target G6PD. ( A ) The construction of a ceRNA network including RMRP, miR-206, and G6PD. ( B ) qRT-PCR analyses of miR-206 expression in 5637 and BIU-87 cells after treatment of sh-NC or sh-RMRP. ( C , D ) Anti-AGO2 RIP was performed followed by RT-PCR to detect the expression of ( C ) miR-206 or ( D ) RMRP associated with AGO2. ( E ) Schematic representation of the 3′-UTR of RMRP with the predicted target site for miR-206. The mutant site of RMRP 3′-UTR is indicated (without line). ( F ) Luciferase reporter constructs containing either RMRP WT or RMRP MUT at the predicted miR-206 target sequences were co-transfected into HEK293T cells, along with miR-206 or miR-NC mimics. ( G ) mRNA expression of G6PD in 5637 cells treated with inhibitor NC or inhibitor 206. ( H ) mRNA expression of G6PD in BIU-87 cells treated with mimics NC or mimics 206. ( I ) WB results showed the expression of G6PD in BLCA cells treated with mimic NC or mimic 206, and inhibitor NC or inhibitor 206. Actin was served as a loading control. ( J ) Schematic representation of the 3′-UTR of G6PD with the predicted target site for miR-206. The mutant site of G6PD 3′-UTR is indicated (without line). ( K ) Luciferase reporter constructs containing either G6PD WT or G6PD MUT at the predicted miR-206 target sequences were co-transfected into HEK293T cells, along with miR-206 or miR-NC mimics. ( L ) mRNA and ( M ) protein expression of G6PD in BIU-87 and 5637 cells treated with sh-NC, sh-RMRP or sh-RMRP + inhibitor 206. The uncropped blots are shown in . All data are presented as the mean ± SEM of triplicate experiments. ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST buffer and incubated with primary antibodies against CD9 (1:1000, 13174S; Cell Signaling Technology (CST), Danvers, MA, USA), TSG101 (1:1000, Ab83; Abcam, Cambridge, UK), MG130 (1:1000, 12480S; CSTUSA); G6PD (1:1000, 12263S; CST, USA) and β-Actin (1:1000, 4967S; CST, USA) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Luciferase, Construct, Transfection

Journal: Cancers

Article Title: Exosomal Long Non-Coding Ribonucleic Acid Ribonuclease Component of Mitochondrial Ribonucleic Acid Processing Endoribonuclease Is Defined as a Potential Non-Invasive Diagnostic Biomarker for Bladder Cancer and Facilitates Tumorigenesis via the miR-206/G6PD Axis

doi: 10.3390/cancers15215305

Figure Lengend Snippet: Regulation of RMRP on tumor progression in BLCA through miR-206/G6PD axis. ( A ) Cell proliferation of 5673 cells was determined at 0, 20, 40, 60 and 80 h after inhibitor NC or inhibitor 206 treatment. ( B ) Colony formation assays of 5637 cells treated with inhibitor NC or inhibitor 206 for 14 days. ( C ) Cell migration and invasion assays using Transwell in 5637 cells treated with inhibitor NC or inhibitor 206. ( D – I ) BIU-87 and 5673 cells were treated with sh-NC or sh-RMRP. Then, BIU-87 and 5673 cells with RMRP knockdown had the appearance of inhibitor 206. ( D ) Cell proliferation of BIU-87 and 5673 cells was determined by CCK-8. ( E ) Colony formation assays of BIU-87 and 5673 cells were detected at day 14 in different treatment groups. ( F , G ) EdU assays were used to detect the proliferation rate of BIU-87 and 5673 cells in different treatment groups. Columns are the average of three independent experiments. ( H , I ) Cell migration and invasion assays using Transwell in BIU-87 and 5673 cells. ( J ) Schematic showing the functional and molecular mechanisms of RMRP/miR-206/G6PD in tumor progression of BLCA. All data are presented as the mean ± SEM of triplicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST buffer and incubated with primary antibodies against CD9 (1:1000, 13174S; Cell Signaling Technology (CST), Danvers, MA, USA), TSG101 (1:1000, Ab83; Abcam, Cambridge, UK), MG130 (1:1000, 12480S; CSTUSA); G6PD (1:1000, 12263S; CST, USA) and β-Actin (1:1000, 4967S; CST, USA) overnight at 4 °C.

Techniques: Migration, CCK-8 Assay, Functional Assay

Journal: Virology Journal

Article Title: Dysregulation of intracellular redox homeostasis by the SARS-CoV-2 ORF6 protein

doi: 10.1186/s12985-023-02208-7

Figure Lengend Snippet: Cellular antioxidant response is induced by ORF6 at the transcriptional level. The effects of SARS-CoV-2 ORF6 protein, wt or mutated forms, on cellular antioxidant modulation are evaluated at the transcriptional level in ( A ) HEK-293T and ( B ) A549 cells. Total RNA is purified from empty plasmid (Ctr-) or ORF6-transfected cells collected at 24 h post-transfection. Specific Nuclear factor erythroid 2-related factor 2 (NRF2), Glucose-6-Phosphate Dehydrogenase (G6PD) and Heme Oxygenase-1 (HO-1) mRNA levels are detected by quantitative reverse-transcription polymerase chain reaction (RT‒qPCR). 18S gene expression is used for relative quantification based on the 2 −ΔΔCt method. Data are expressed as mean values ± standard deviations (SD) of at least three ( n ≥ 3) independent experiments, each performed in duplicate. Significance is determined with respect to the negative control (Ctr-), transfected with empty plasmid, sample as ** p < 0.005; * p < 0.05

Article Snippet: After blocking with Intercept Blocking Buffer (LI-COR Biosciences, Germany), antioxidant proteins are detected following membrane incubation with anti-G6PD, anti-NRF2 (Cell Signaling Technologies, Milan, Italy), anti-SOD1, anti-HO-1 (Santa Cruz Biotechnologies, Milan, Italy), anti-p38 and phosphorylated p38 (Santa Cruz Biotechnologies, Milan, Italy), anti-phosphorylated NRF2 (Ser40) (Thermo Fisher Scientific, Milan, Italy), anti-PKC (Santa Cruz Biotechnologies, Milan, Italy) or anti GSK-3β (Santa Cruz Biotechnologies, Milan, Italy).

Techniques: Purification, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: Virology Journal

Article Title: Dysregulation of intracellular redox homeostasis by the SARS-CoV-2 ORF6 protein

doi: 10.1186/s12985-023-02208-7

Figure Lengend Snippet: ORF6 protein hinders scavenger protein content. The effect of SARS-CoV-2 ORF6 protein, wt- or mutated variants, on the cellular antioxidant response is further analysed by immunoblotting for ( A ) NRF2 and ( B ) G6PD and HO-1 on 50 μg of whole cell lysates (WCL) of transfected HEK-293T cells. ( C ) The effects of ORF6 protein mutants on endogenous NRF2 expression is further investigated on WCL of A549 cells expressing ORF6 protein variants. The loading control is represented by immuno-detection of actin protein. Densitometric analysis of reactive bands is performed by ImageJ software and the results are plotted as the mean fold change in target proteins, normalized with respect to relative actin levels, from ( n = 3) independent experiments, performed in duplicate, ± SD. Significance is determined with respect to the negative control (Ctr-), transfected with empty plasmid, sample as ** p < 0.005; * p < 0.05

Article Snippet: After blocking with Intercept Blocking Buffer (LI-COR Biosciences, Germany), antioxidant proteins are detected following membrane incubation with anti-G6PD, anti-NRF2 (Cell Signaling Technologies, Milan, Italy), anti-SOD1, anti-HO-1 (Santa Cruz Biotechnologies, Milan, Italy), anti-p38 and phosphorylated p38 (Santa Cruz Biotechnologies, Milan, Italy), anti-phosphorylated NRF2 (Ser40) (Thermo Fisher Scientific, Milan, Italy), anti-PKC (Santa Cruz Biotechnologies, Milan, Italy) or anti GSK-3β (Santa Cruz Biotechnologies, Milan, Italy).

Techniques: Western Blot, Transfection, Expressing, Software, Negative Control, Plasmid Preparation