Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a The genes involved in m 6 A modification are shown in the table as candidate targets of ZFHX3 and were analyzed by Chip-Enrich using the ZFHX3 Chip-Seq results. b ZFHX3 correlated with WTAP , FTO , and ALKBH5 when using a cluster of PCa patients containing mRNA expression of these genes. c The m 6 A level was detected by immunofluorescence in RWPE1 cells after knocking down ZFHX3, and the arrows indicated m 6 A translocating from the nucleus to the cytoplasm. n = 3. d The expression of ZFHX3, FTO, and ALKBH5 in human prostate epithelial cell lines, was determined by western blot. The efficiency of ZFHX3 knockdown was detected by western blot, and the global m 6 A levels were reduced by ZFHX3 knockdown and detected by RNA dot blot in RWPE1 ( e ) and LNCaP ( f ). n = 3. MB (methylene blue) represents the loading control of the RNA samples. *** P < 0.001.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Modification, ChIP-sequencing, Expressing, Immunofluorescence, Western Blot, Knockdown, Dot Blot, Control

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a , b Knockdown of ZFHX3 upregulated FTO expression in RWPE1 and LNCaP cells at the protein and mRNA levels, as detected separately by western blot and qRT-PCR. n = 3. c The wild type and mutant of the FTO promoter were constructed, and the critical sequences are shown in the panel. Red rectangles indicate mutant sequences, and blue rectangles indicate the regions within the FTO promoter for PCR amplification. d ZFHX3 knockdown increased the activity of the wild-type promoter of FTO , but the effect on the mutant promoter of FTO was partly impaired compared to the wild-type promoter. n = 3. e Expression plasmids for control-vector (Flag) or Flag-ZFHX3 were transfected in 293T cells and the efficiency was detected by western blot. f Detection of ZFHX3-bound FTO promoter DNA in ZFHX3-overexpressed cells or control cells was completed using ChIP and regular PCR. g Binding of ZFHX3 to FTO promoter region 1 and region 2 was found using ChIP and qRT-PCR. n = 3. ** P < 0.01; *** P < 0.001; ns not significant.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Mutagenesis, Construct, Amplification, Activity Assay, Control, Plasmid Preparation, Transfection, Binding Assay

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a , d The knockdown efficiency of FTO shRNA with lentivirus constructs in RWPE1 or siRNAs against FTO in LNCaP was confirmed by western blot. b Acini formation was decreased in the FTO knockdown cell line. The arrows indicated the normal acini structure. n = 3. c , f Western blot showed that MYC expression was repressed when FTO was knocked down by shRNA or siRNAs, separately in RWPE1 and LNCaP. e Sphere formation was decreased after knocking down FTO (siFTO-1) in LNCaP. The average number of spheres with a diameter >75 µm/well was counted. n = 3. g We determined the m 6 A abundance in mRNA in RWPE1 cells upon FTO inhibitor FB23-2 treatment under various concentrations as indicated for 72 h via dot blot assay. h FB23-2 suppressed cell proliferation of RWPE1 detected by SRB assay. The cells treated with different concentrations of FB23-2 were collected at indicated time. n = 3. i – k Knockdown of ZFHX3 by siRNA was accompanied by three concentrations of siRNAs against FTO as indicated. The efficiency of ZFXH3 and FTO knockdown was tested by western blot and FTO knockdown eliminated the promoting effect of ZFHX3’s inhibition on acini formation in RWPE1 cells. n = 3. * P < 0.05; ** P < 0.01; *** P < 0.001, ns not significant.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Knockdown, shRNA, Construct, Western Blot, Expressing, Dot Blot, Sulforhodamine B Assay, Inhibition

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a Immunoblotting of FTO in FTO knockdown RWPE1 and control RWPE1 cells is presented. b The distribution of peaks is shown with a significant change in m 6 A level in FTO knockdown RWPE1 cells compared to control RWPE1 cells. c A Venn diagram shows the shared genes between increased m 6 A peaks upon FTO deletion and FTO-relation genes analyzed by RNA-seq. A total of 236 genes were observed. d , e Pathway analysis and KEGG analysis of the above 236 shared genes showed that the cell cycle was altered in FTO knockdown cells. f The mapped reads represent enriched RNA fragments by MeRIP-seq. RNA methylation profiles were loaded in IGV software and m 6 A modification peak alterations in CDKN2C and E2F2 mRNA full length were visualized. g MeRIP-qRT-PCR was used to detect the m 6 A levels alterations of CDKN2C and E2F2 after knocking down FTO in RWPE1 cells. h Cell cycle distribution of FTO silencing cells and control cells was analyzed by flow cytometry. i E2F2 and CDKN2C were detected by western blot in ZFHX3 knockdown cells or control cells where FTO was silenced with siRNA targeting FTO or siCtrl. ** P < 0.01; *** P < 0.001, ns not significant.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Western Blot, Knockdown, Control, RNA Sequencing, Methylation, Software, Modification, Quantitative RT-PCR, Flow Cytometry

Journal: Cell Death Discovery

Article Title: ZFHX3 acts as a tumor suppressor in prostate cancer by targeting FTO-mediated m 6 A demethylation

doi: 10.1038/s41420-024-02060-w

Figure Lengend Snippet: a , b The potential m 6 A sites of ZFHX3 and the consensus motifs modified by m 6 A predicted by SRAMP are shown. c The m 6 A peaks in the black rectangles (R1, R2, and R3) visualized by IGV are those that co-localized with predicted sites of ZFHX3 . d The m 6 A levels of fragments in ZFHX3 (R1, R2, and R3) were elevated by MeRIP-qRT-PCR after knocking down FTO in RWPE1 cells. n = 3. e Western blot and RT-qPCR assays showed that ZFHX3 expression at the protein level or mRNA level was elevated after depletion of FTO (shFTO) in RWPE1 cells. n = 3. f The mRNA half-life ( t 1/2 ) was increased in RWPE1 cells with depleted expression of FTO (shFTO). g ZFHX3 protein expression or mRNA expression was upregulated after knockdown of FTO (siFTO-1 and siFTO-2) in LNCaP cells. n = 3. h The mRNA half-life ( t 1/2 ) was increased in LNCaP cells with depleted expression of FTO (siFTO-1). *** P < 0.001.

Article Snippet: Human immortalized prostate epithelial cell line RWPE1 and other human PCa cell lines were purchased from KeyGEN Biotech (Jiangsu, China).

Techniques: Modification, Quantitative RT-PCR, Western Blot, Expressing, Knockdown

Journal: Cells

Article Title: Role of Mitochondrial Glycerol-3-Phosphate Dehydrogenase in Metabolic Adaptations of Prostate Cancer

doi: 10.3390/cells9081764

Figure Lengend Snippet: Energy metabolism and mGPDH content in prostate cancer cells. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) ( A ), as well as OCR/ECAR ratio ( B ) in prostate cancer cells (LNCaP) compared to control epithelial cell line (PNT1A). Values were calculated from the rates in basal conditions, i.e. at the presence of 10 mM glucose, and determined by a Seahorse XFe analyzer ( n = 5). ( C ) Enzyme activity of mGPDH measured spectrophotometrically using 10 mM glycerol-3-phosphate as a substrate ( n = 6). ( D ) ROS generation in intact LNCaP cells compared to control PNT1A measured by the CM-H 2 DCFDA probe. To determine the FCCP-sensitive portion of ROS production, 1 μM uncoupler was used. ( E ) Cell lysates (15 μg protein) were separated on SDS-PAGE and mGPDH content was analyzed by Western blotting using a specific antibody against mGPDH, actin was used as a loading control. Representative blot of 5 independent experiments is depicted. Antibody signals were quantified densitometrically as the total mGPDH levels normalized to actin levels and the results are expressed as % of control values. ( F ) Processing of mGPDH was determined densitometrically as a ratio of the lower band and total mGPDH content ( n = 5). Data represent the means ± S.D., * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The normal human immortalized prostate epithelial cell line PNT1A (95012614, Merck/Sigma, Darmstadt, Germany) and androgen-sensitive human prostate cancer cell line LNCaP (kindly provided by Dr. Hodny, IMG CAS, Prague, Czech Republic) and human embryonic kidney cells (HEK293) were purchased from ATCC (CRL-1573, ATCC, Manassas, VA, USA).

Techniques: Activity Assay, SDS Page, Western Blot

Journal: Cells

Article Title: Role of Mitochondrial Glycerol-3-Phosphate Dehydrogenase in Metabolic Adaptations of Prostate Cancer

doi: 10.3390/cells9081764

Figure Lengend Snippet: Analysis of mGPDH forms. ( A ) mGPDH topology by the Protter visualization tool. Predicted phosphorylation (green) and targeting sequences by Uniprot (yellow) and Phobius (orange) are depicted. ( B ) Table of experimental and calculated differences of two mGPDH forms. Experimental difference ( n = 5) was determined by the Molecular Weight Analysis tool (Image Lab software, Bio-Rad). ( C ) Cell lysates (50 μg protein) of HEK293 cells overexpressed with FLAG in different places of the GPD2 sequence were separated on SDS-PAGE (Hoefer System) and mGPDH forms were analyzed by Western blot using a specific antibody against FLAG ( n = 3). C-term—GPD2 with FLAG tag at the C-terminal, N-term—GPD2 with FLAG following initial start codon (N-term), 27AA—GPD2 with FLAG tag following 27th amino acid and 42AA—GPD2 with FLAG tag following 42nd amino acid. ( D ) Cell lysates (15 μg protein) of control PNT1A and cancer (LNCaP) cells were separated on SDS-PAGE and IMMP2L peptidase content was analyzed by Western blotting using a specific antibody ( n = 5). Actin was used as a loading control ( C–D ). Data represent the means ± S.D., ** p < 0.01.

Article Snippet: The normal human immortalized prostate epithelial cell line PNT1A (95012614, Merck/Sigma, Darmstadt, Germany) and androgen-sensitive human prostate cancer cell line LNCaP (kindly provided by Dr. Hodny, IMG CAS, Prague, Czech Republic) and human embryonic kidney cells (HEK293) were purchased from ATCC (CRL-1573, ATCC, Manassas, VA, USA).

Techniques: Molecular Weight, Software, Sequencing, SDS Page, Western Blot, FLAG-tag