Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Characterizing H3K4me2 enriched lncRNAs. ( A ) ChRIP experimental workflow to identify lncRNAs bound to chromatin enriched with H3K4me2 and WDR5 in BT-549 cell line. ( B ) Computational approach used for finding chromatin-associated RNAs and enriched patterns of genomic organization with respect to nearest protein coding genes. ( C ) Scatter plot showing the enrichment of H3K4me2 lncRNAs over input. X-axis denotes log transformed expression values of H3K4me2 lncRNAs and Y-axis denotes log-fold changes of lncRNAs in H3K4me2 ChRIP sample over nuclear input sample. ( D ) Histogram shows distribution of H3K4me2 lncRNAs with antisense (X) and sense (S) pattern with respect to their protein coding partner (pPCGs). ‘n’ denotes number of lncRNA-pPCG pairs in each pattern. ( E ) Genomic organization of H3K4me2 lncRNAs (green bars) with respect to nearest protein coding genes (grey bars). XH: where lncRNA shows Head-to-Head arrangement with nearby protein coding gene. XT: lncRNA in Tail-to-Tail arrangement with protein coding gene, XI: lncRNA located inside a protein coding gene, XO: lncRNA located outside and covers the entire protein coding gene. Sense pairs means lncRNAs in the same orientation as protein coding gene. The notion of greater or less than 2 kb means that the partner genes (pPCGs) are located within 2 kb (<2 kb) or away from 2 kb (> 2 kb) but within 50 kb window with respect to H3K4me2 lncRNAs. ( F ) Distribution of different patterns of genomic arrangements as described in (E) for H3K4me2 lncRNAs and non-CAR lncRNAs.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: H3K4me2 XH lncRNAs regulate the transcriptional activity of their protein coding partners. ( A ) Nuclear expression levels of H3K4me2 XH lncRNAs and non-CAR XH lncRNAs. ( B ) Expression of H3K4me2 XH lncRNA-protein coding pairs (pPCGs) in nuclear input and H3K4me2 ChRIP samples. The P -value denotes the significance of difference between nuclear input and H3K4me2. ( C ) Gene ontology based functional enrichment analysis of protein coding genes that are partners (pPCGs) to H3K4me2 lncRNAs: divergent (XH), other antisense (XT, XI, and XO) and sense lncRNAs. The bar graph shows number of genes in corresponding ontology term and the heatmap represents the significance of individual terms ( P -values obtained using GeneSCF). The highlighted box, by the heatmap, is listed with different transcription factors from the enriched transcription related terms. ( D ) Gene expression analysis of three transcription factors ( FOXD3, GATA6 and HOXC13 ) using RT–qPCR analysis following downregulation of their neighboring H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) with siRNA in BT-549 cells. LncRNA expression is in green bars while transcription factors expression is in grey. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( E ) RT–qPCR analysis of FOXD3, GATA6 and HOXC13 gene expression after strand-specific downregulation of FOXD3-AS1, GATA6-AS1 and HOXC13-AS respectively, using LNA. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( F ) Gene expression analysis of three H3K4me2 XH lncRNAs ( FOXD3-AS1, GATA6-AS1 and HOXC13-AS ) using RT–qPCR analysis following downregulation of their respective transcription factor partners with siRNA or esiRNA in BT-549 cells. LncRNAs expression is depicted as green bars while transcription factors expression is in grey bars. Data represent the mean ± SD. of two independent biological experiments. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ns denotes non-significant.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Activity Assay, Expressing, Functional Assay, Gene Expression, Quantitative RT-PCR, esiRNA

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: XH lncRNAs are overrepresented in active lncCARs. ( A ) Scatter plot showing enrichment of WDR5 lncRNAs over nuclear input. The X-axis denotes log transformed expression and Y-axis denotes log-fold change of WDR5 lncRNAs over nuclear input. Venn diagram, in the Scatter plot, shows 209 lncRNAs that are commonly enriched in both H3K4me2 and WDR5 ChRIP pulldowns. ( B ) Distribution of different patterns of genomic arrangements as described in Figure for WDR5 lncRNAs and non-CAR lncRNAs. ( C ) Distribution of different patterns of genomic arrangements as described in Figure for active lncRNA (ChRIP pulldown using H3K4me2 and WDR5 antibodies) and inactive lncRNA (ChRIP pulldown using EZH2 and H3K27me3 antibodies). ( D ) Nuclear expression levels of WDR5 XH lncRNAs, active XH lncCAR (WDR5 and H3K4me2) and non-CAR XH lncRNAs. ( E ) Enrichment of H3K4me2, H3K4me3, WDR5 ChIP-seq signals over active XH lncCAR (in green, n = 98) and non-CAR XH lncRNA (in black, n = 335) promoters (±2 kb) in BT-549 cells. The signals presented in the plots represent log 2 ratio between ChIP and input sample. TSS denotes the transcription start site of lncRNAs. ( F ) RT-qPCR analysis of WDR5 UV-RIP. The Y-axis shows fold enrichment relative to IgG. The significance of FOXD3-AS1 and HOXC13-AS enrichment in WDR5 RIP is calculated compared to the enrichment of FOXD3 and HOXC13 mRNAs, respectively. *** P ≤ 0.001. ( G ) Left panel: f-RIP of WDR5 WT and WDR5 F266A using FLAG antibody. The Y-axis shows percentage of Input. The significance of decrease in FOXD3-AS1, HOXC13-AS and positive control HOTTIP enrichment in WDR5 F266A RIP is calculated compared to their respective enrichments in WDR5-WT. GAPDH was used as negative control. Right panel: Expression of FLAG tagged WDR5 WT and WDR5 F266A mutant in HeLa cells by western blot. Data are shown as mean ± SD. ** P ≤ 0.01.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Transformation Assay, Expressing, ChIP-sequencing, Quantitative RT-PCR, Positive Control, Negative Control, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: H3K4me2 and WDR5 enriched chromatin interacting long non-coding RNAs maintain transcriptionally competent chromatin at divergent transcriptional units

doi: 10.1093/nar/gky635

Figure Lengend Snippet: Active XH lncCARs promote transcription of their protein coding partners. ( A and B ) Promoter targeting of active XH lncCARs detected by ChOP. A) qPCR analysis of ChOP pull-downs, performed using FOXD3-AS1 and HOXC13-AS antisense probes, with primers over TSS (transcription start sites), regions spanning upstream and downstream of TSS and over transcription termination sites (TTS) of FOXD3 and HOXC13 genes in BT-549 cells. The ChOP pull-down with LacZ antisense oligo, used as a negative control (grey bars). Specific enrichment pattern for each of the XH lncCARs is depicted in green bars. The schematic in the right of the bar graph represents the genomic locations of the respective primers (grey: not enriched and rosetta: showing enrichment). Data are shown as mean ± SD (n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( B ) Similar qPCR analysis of ChOP pull-downs using FOXD3-AS1 and HOXC13-AS antisense probes upon ActD treatment in BT-549 cells. Data are shown as mean ± SD ( n = 2 biological replicates). * P < 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. ( C ) RT-qPCR analysis to detect relative levels of nascent FOXD3 and HOXC13 transcripts by Click-iT at 48 hours after the knockdown of FOXD3-AS1 and HOXC13-AS . Bar graph depicts the level of nascent protein coding transcripts upon removal of their respective active XH lncCARs. Data represent the mean ± SD of two independent biological experiments. * if P ≤ 0.05 and ** if P ≤ 0.01. ( D and E ) ChIP-qPCR analysis of the enrichment of H3K4me2 and H3K4me3 at the promoter region of FOXD3 (D) and HOXC13 (E) upon down regulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of H3K4me2 and H3K4me3 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA.The locations of ChIP primers used are depicted in schematic in the bottm of each bar graph. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( F and G ) ChIP-qPCR analysis of enrichment of WDR5 at the promoter region of FOXD3(F) and HOXC13 (G) upon downregulation of active XH lncCAR FOXD3-AS1 and HOXC13-AS respectively. Fold enrichment of WDR5 was normalized to histone H3 and IgG. After normalization the ChIP data was represented as relative enrichment compare to control siRNA. The ChIP primers used in the experiment are same as described in panel D–E. Data are shown as mean ± SD (n = 3 biological replicates). ** P ≤ 0.01 and *** P ≤ 0.001. ( H and I ) FOXD3-AS1 and HOXC13-AS overexpression: RT–qPCR analysis of FOXD3 expression, upon ectopic overexpression of FOXD3-AS1 (H) and HOXC13-AS transcript (I) respectively, at Day 2 and Day 5 post transfection in BT-549 cell line. pcDNA empty vector transfection used as a control. Data represent the mean ± SD. of two independent biological experiments. P -values has been denoted as * if P ≤ 0.05, ** if P ≤ 0.01. ns denotes non-significant. ( J ) Cell fractionation in BT-549 cells show distribution of the overexpressed FOXD3-AS1 and HOXC13-AS transcripts. Data represent the mean ± SD of two independent biological experiments. GAPDH serves as positive control for cytoplasmic fraction, U6 and KCNQ1OT1 serves as positive control for nuclear fraction.

Article Snippet: BT-549 (CLS-300312; CLS cell line service, Germany), MDA-MB-231 (ACC-732; American Type Culture Collection, USA) and Hela (ATCC CCL-2) cell lines were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic cocktail (Pen-Strep, Invitrogen) at 37°C with 5% CO 2 supply.

Techniques: Negative Control, Quantitative RT-PCR, Knockdown, IF-P, ChIP-qPCR, Control, Over Expression, Expressing, Transfection, Plasmid Preparation, Cell Fractionation, Positive Control

Journal: Life sciences

Article Title: Fluid shear stress facilitates prostate cancer metastasis through Piezo1-Src-YAP axis.

doi: 10.1016/j.lfs.2022.120936

Figure Lengend Snippet: Fig. 1. Piezo1 expression is enhanced by wall shear stress (A) The prostate tumor microenvironment was immunohistochemically stained to evaluate Piezo1 expression. Scale bars represent 250 μm and 25 μm, respectively. (B) PC3 human prostate cancer cells were plated in the microfluidics channel and applied shear stress at 0.05 dyne cm−2 for 5 h. After wall shear stress (WSS), gene expression of Piezo1 was confirmed by qRT-PCR (n = 3, independent experiments, two-tailed t- test, *** p < 0.001). (C) Immunofluorescence staining analysis was performed to evaluate Piezo1 expression (red). Cell nuclei were stained with DAPI (blue). Scale bars represent 10 μm. (D) Protein expression of Piezo1 was detected using immunoblotting at 0, 1, 5, and 12 h after WSS.

Article Snippet: PC3, PC3-Luciferase (PC3-Luc2), and DU145 human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing, Shear, Staining, Gene Expression, Quantitative RT-PCR, Two Tailed Test, Immunofluorescence, Western Blot

Journal: Life sciences

Article Title: Fluid shear stress facilitates prostate cancer metastasis through Piezo1-Src-YAP axis.

doi: 10.1016/j.lfs.2022.120936

Figure Lengend Snippet: Fig. 5. Piezo1 induces YAP activation through tyrosine 357 YAP and Src phosphorylation (A) and (B) Immunoblot analysis was measured after treatment with mock, a Piezo1 agonist (Yoda1, 1 μM), and an Src inhibitor (dasatinib, 100 nM) and exposure to static or WSS for 5 h. (C) and (D) Immunoblot analysis was measured after exposure to WSS for 5 h in PC3 cells transfected with siControl or siPiezo1. (B) and (D) Graphs showing quantification of the expression level of each phosphorylated protein compared with the expression of the non-phosphorylated isoform. (n = 3, Two Way ANOVA with Holm-Sidak multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: PC3, PC3-Luciferase (PC3-Luc2), and DU145 human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Transfection, Expressing

Journal: Life sciences

Article Title: Fluid shear stress facilitates prostate cancer metastasis through Piezo1-Src-YAP axis.

doi: 10.1016/j.lfs.2022.120936

Figure Lengend Snippet: Fig. 6. In vivo monitoring of orthotopic PC3 pros tate tumor growth and metastasis with or without shPiezo1. (A) Representative whole-body images of bioluminescence measurements for xenografted PC3 tumor growth and metastasis. Luciferase tag ged PC3-Luc/shCon and PC3-Luc/shPiezo1 cells were surgically implanted into the dorsal prostate of male nude mice. Ventral images of anesthetized representative sham, control (PC3-Luc/shCon), and shPiezo1 (PC3-Luc/shPiezo1) were taken weekly for 6 weeks to monitor spreading to other organs. All images were pseudo color set using the same intensity (sham = 4, shCon = 6, shPiezo1 = 6). (B) Histological slides include orthotopic tumors of PC3-luc/shCon and PC3-luc/shPiezo1. Representa tive images were derived from mouse prostate stained with hematoxylin and eosin (H&E). High magnified images of the top panel were presented as below. Scale bars = 25 μm and 100 μm, respectively.

Article Snippet: PC3, PC3-Luciferase (PC3-Luc2), and DU145 human prostate cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: In Vivo, Luciferase, Control, Derivative Assay, Staining

Journal: Translational Oncology

Article Title: MiR-138–5p inhibits prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B

doi: 10.1016/j.tranon.2023.101723

Figure Lengend Snippet: Differential expression and prognostic analysis of APOBEC3B. A. The mRNA expression of APOBEC3B in different tumour tissues and adjacent normal tissues based on the TCGA database. B. APOBEC3B expression in 52 pairs of PCa tissues and adjacent normal tissues in the TCGA database. C. APOBEC3B expression with Gleason Score in PCa patients based on TCGA database. D. PCa patients’ DFS for individuals in the TCGA database was assessed in terms of quartile via the Kaplan-Meier method (Log-rank test) using GEPIA2. E. APOBEC3B expression in docetaxel-resistant PCa cells (DU145/DR and PC-3/DR) and corresponding parental sensitive cells (DU145 and PC-3) measured by qRT-PCR and Western blot. U6 was used as a normalization reference at the mRNA level, and GAPDH was used as a loading control at the protein level. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.

Article Snippet: Through a series of steps, chemoresistance to docetaxel was developed in DU145 (DU145/DR) and PC-3 (PC-3/DR) cells and then maintained by growing in complete media containing 10 nM docetaxel (MCE, USA) .

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Translational Oncology

Article Title: MiR-138–5p inhibits prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B

doi: 10.1016/j.tranon.2023.101723

Figure Lengend Snippet: Silencing of APOBEC3B inhibited PCa cell proliferation and chemoresistance. A. The APOBEC3B expression in docetaxel-resistant cells DU145/DR and PC-3/DR transfected with siNC or siAPOBEC3B for 48 h was determined by qRT-PCR and Western blot. Expression of APOBEC3B at the mRNA level was normalized by U6. GAPDH was used as a loading control at the protein level. B. Cell viability of DU145/DR and PC-3/DR cells transfected with siNC or siAPOBEC3B determined by CCK-8 assay. C. Colony formation assay of DU145/DR and PC-3/DR cells transfected with siNC or siAPOBEC3B. D. Chemosensitivity analysis of DU145/DR and PC-3/DR cells to docetaxel after APOBEC3B knockdown. Docetaxel-resistant cells (DU145/DR and PC-3/DR) transfected with siRNA for 24 h in 6-well plates were digested and then seeded into 96-well plates, respectively. Adherent cells were treated with docetaxel diluted in a gradient for another 48 h before performing CCK-8 analysis. Then, calculate the IC50 of siAPOBEC group and siNC group. The IC50s of DU145/DR: siNC, 62.17; siAPOBEC3B#2, 32.87; siAPOBEC3B#3, 28.60; The IC50s of PC-3/DR: siNC, 37.14; siAPOBEC3B#2, 18.50; siAPOBEC3B#3: 17.43. *, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001.

Article Snippet: Through a series of steps, chemoresistance to docetaxel was developed in DU145 (DU145/DR) and PC-3 (PC-3/DR) cells and then maintained by growing in complete media containing 10 nM docetaxel (MCE, USA) .

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control, CCK-8 Assay, Colony Assay, Knockdown

Journal: Translational Oncology

Article Title: MiR-138–5p inhibits prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B

doi: 10.1016/j.tranon.2023.101723

Figure Lengend Snippet: miR-138–5p directly targeted APOBEC3B. A. The upstream target genes of APOBEC3B were predicted and screened by the database. B. The expression level of miR-138–5p in docetaxel-sensitive cells (DU145 and PC-3) and docetaxel-resistant cells (DU145/DR And PC-3/DR) was evaluated by qRT-PCR. C. The expression efficiency of miR-138–5p in docetaxel-resistant PCa cells transfected with mimics or inhibitors detected by qRT-PCR. d-E. Effect of miR-138–5p mimics and inhibitor on APOBEC3B expression in docetaxel-resistant PCa cells by qRT-PCR (D) and Western blot (E). U6 was used as the internal reference for miR-138–5p and APOBEC3B at the mRNA level. GAPDH was used as the loading control at the protein level. F. Schematic depiction of the interaction between miR-138–5p and APOBEC3B mRNA 3′-UTR. G. The wild-type and mutant-type plasmids of APOBEC3B mRNA 3′-UTR were constructed on the putative binding sites of miR-138–5p and APOBEC3B mRNA. H. Dual luciferase activity assay confirmed that miR-138–5p mimics negatively regulates APOBEC3B expression by binding to wild-type APOBEC3B mRNA 3′-UTR instead of mutant-type. I. Dual luciferase activity assay confirmed that miR-138–5p inhibitor upregulated APOBEC3B expression by binding wild-type APOBEC3B mRNA 3′-UTR instead of mutant-type. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: Through a series of steps, chemoresistance to docetaxel was developed in DU145 (DU145/DR) and PC-3 (PC-3/DR) cells and then maintained by growing in complete media containing 10 nM docetaxel (MCE, USA) .

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Control, Mutagenesis, Construct, Binding Assay, Luciferase, Activity Assay

Journal: Translational Oncology

Article Title: MiR-138–5p inhibits prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B

doi: 10.1016/j.tranon.2023.101723

Figure Lengend Snippet: miR-138–5p inhibited cell proliferation and chemoresistance in docetaxel-resistant PCa cells. A-B. Effects of miR-138–5p mimics (A) or inhibitor (B) on the cell viability of docet-axel-resistant PCa cells. C. Effects of miR-138–5p mimics or inhibitor on the colony formation of docetaxel-resistant PCa cells. D. miR-138–5p mimics inhibited resistance against docetaxel of docetaxel-resistant PCa cells. E. miR-138–5p inhibitor promoted resistance against docetaxel of docetaxel-resistant PCa cells. Docetaxel-resistant cells (DU145/DR and PC-3/DR) transfected with mimics or inhibitor of miR-138–5p for 24 h in 6-well plates were seeded into 96-well plates, respectively, and then treated with docetaxel diluted in a gradient for another 48 h before performing CCK-8 analysis. The IC50s were calculated based on CCK-8 data. The IC50s of DU145/DR: mimic NC, 61.48; miR-138–5p mimics, 28.33; inhibitor NC, 60.34; miR-138–5p inhibitor: 122.80. The IC50s of PC-3/DR: mimic NC, 38.25; miR-138–5p mimics, 17.52; inhibitor NC, 37.66; miR-138–5p inhibitor: 83.74. *, P <0.05; **, P <0.01; ***, P <0.001, ****, P <0.0001.

Article Snippet: Through a series of steps, chemoresistance to docetaxel was developed in DU145 (DU145/DR) and PC-3 (PC-3/DR) cells and then maintained by growing in complete media containing 10 nM docetaxel (MCE, USA) .

Techniques: Transfection, CCK-8 Assay

Journal: Translational Oncology

Article Title: MiR-138–5p inhibits prostate cancer cell proliferation and chemoresistance by targeting APOBEC3B

doi: 10.1016/j.tranon.2023.101723

Figure Lengend Snippet: APOBEC3B overexpression blocked the function of miR-138–5p on cell proliferation and docetaxel resistance. A. The overexpression efficiency of miR-138–5p in docetaxel-resistant PCa cells transfected with mimics detected by qRT-PCR. B. APOBEC3B overexpression level of docetaxel-resistant PCa cells transfected with miR-138–5p and APOBEC3B overexpression plasmid determined by Western blot. C. Cell viability of docetaxel-resistant PCa cells transfected with miR-138–5p and APOBEC3B overexpression plasmid determined by CCK-8 assay. D. Colony formation assay of docetaxel-resistant PCa cells transfected with miR-138–5p and APOBEC3B overexpression plasmid. E. The chemosensitivity of docetaxel-resistant PCa cells transfected with miR-138–5p and APOBEC3B overexpression plasmid to docetaxel was determined by CCK-8 assay. After docetaxel-resistant PCa cells were transfected with mimic NC plus OE-NC (control vector), miR-138–5p mimics plus OE-NC, mimic NC plus APOBEC3B-OE, or miR-138–5p mimics plus APOBEC3B-OE (mimic NC plus OE-NC vs miR-138–5p mimics plus OE-NC, and miR-138–5p mimics plus OE-NC vs miR-138–5p mimics plus APOBEC3B-OE-NC, respectively), CCK-8 viability assay was performed at 0, 24, 48, 72 and 96 h (B) and chemosensitivity was measured after incubation with docetaxel diluted in a gradient for another 48 h (E). The IC50s of DU145/DR: mimic NC + OE-NC, 63.77; miR-138–5p mimics + OE-NC, 31.20; miR-138–5p mimics + APOBEC3B-OE, 50.17. The IC50s of PC-3/DR: mimic NC + OE-NC, 38.43; miR-138–5p mimics + OE-NC, 18.74; miR-138–5p mimics + APOBEC3B-OE, 29.80. ns, no significance; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001, mimic NC plus OE-NC transfection group vs miR-138–5p mimics plus OE-NC transfection group. #, P <0.05; ###, P <0.001, miR-138–5p mimics plus OE-NC transfection group vs miR-138–5p mimics plus APOBEC3B-OE transfection group.

Article Snippet: Through a series of steps, chemoresistance to docetaxel was developed in DU145 (DU145/DR) and PC-3 (PC-3/DR) cells and then maintained by growing in complete media containing 10 nM docetaxel (MCE, USA) .

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Plasmid Preparation, Western Blot, CCK-8 Assay, Colony Assay, Control, Viability Assay, Incubation

Journal: International Journal of Molecular Medicine

Article Title: ERG is specifically associated with ETS-2 and ETV-4, but not with ETS-1, in prostate cancer

doi: 10.3892/ijmm.2012.1097

Figure Lengend Snippet: Expression of ERG, ETS-1, ETS-2 and ETV-4 in PC3 cell nuclear extracts and PCa tissues. Western blot analysis showed that ERG, ETS-1, ETS-2 and ETV-4 were expressed in (A) PC3 cell nuclear extracts, as well as in protein lysates prepared from (B) the PCa tissue samples of five patients.

Article Snippet: To investigate whether ERG is associated with ETS-1, ETS-2 and ETV-4 in PC3 cell nuclear extracts (Santa Cruz Biotechnology, Inc.), IP was performed using a rabbit polyclonal anti-ERG antibody (Gentex).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: ERG is specifically associated with ETS-2 and ETV-4, but not with ETS-1, in prostate cancer

doi: 10.3892/ijmm.2012.1097

Figure Lengend Snippet: ERG is specifically associated with ETS-2 and ETV-4, but not with ETS-1, in PC3 cell nuclear extracts. Immunoprecipitation (IP) using an anti-ERG antibody showed that ERG is specifically associated with (A) ETS-2 and ETV-4, but not with (B) ETS-1, in PC3 cell nuclear extracts.

Article Snippet: To investigate whether ERG is associated with ETS-1, ETS-2 and ETV-4 in PC3 cell nuclear extracts (Santa Cruz Biotechnology, Inc.), IP was performed using a rabbit polyclonal anti-ERG antibody (Gentex).

Techniques: Immunoprecipitation

Journal: Cellular signalling

Article Title: SMC2 knockdown inhibits malignant progression of lung adenocarcinoma by upregulating BTG2 expression.

doi: 10.1016/j.cellsig.2024.111216

Figure Lengend Snippet: Fig. 5. SMC2 knockdown upregulated the BTG2 expression in LUAD cells. (A) The top 10 upregulated and 10 downregulated genes through analysis of PC9 cells transcriptome. (B) The correlation between SMC2 expression and BTG2 expression in LUAD from TCGA database. (C) The expression of BTG2 in LUAD tumor tissues compared to normal tissues based on TCGA database. (D) Survival analysis of BTG2 expression in LUAD. (E) RT-qPCR assessed the mRNA level of BTG2 after SMC2 knockdown in PC9 cells. (F-G) Western blotting assessed the protein levels of BTG2 after SMC2 knockdown in PC9 cells. (H–I) Western blotting assessed the nuclear protein levels of p53 after SMC2 knockdown in PC9 cells. (J) Immunofluorescence staining of p53 after SMC2 knockdown in PC9 cells. (K-L) Western blotting assessed the protein levels of BTG2 after TP53 silencing in PC9 cells. SMC2 knockdown was achieved via SMC2-targeted lentivirus transfection. The negative control lentivirus (shCtrl) and the SMC2-targeted lentivirus (shSMC2) were transfected into PC9 cells. The negative control siRNA (siNC) and TP53-targeted siRNA (siTP53) was transfected into PC9 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The antibodies used in this study are listed as follows: anti-β-actin (Zsbio), anti-SMC2 antibody (Abclonal), anti-CDK1 antibody (CST), anti-CDK6 antibody (CST), anti-ERK1/2 (CST), anti phospho-ERK1/2 (CST), anti-AKT (CST), anti-phospho-AKT (CST), anti-BTG2 antibody (Proteintech), anti-p53 antibody (Abclonal), anti-mouse IgG HRP-linked Antibody (CST), anti-rabbit IgG HRP-linked Antibody (CST).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Transfection, Negative Control

Journal: Cellular signalling

Article Title: SMC2 knockdown inhibits malignant progression of lung adenocarcinoma by upregulating BTG2 expression.

doi: 10.1016/j.cellsig.2024.111216

Figure Lengend Snippet: Fig. 6. BTG2 silencing reversed the proliferation and invasion of SMC2-knockdown LUAD cells and restored the activation of ERK/AKT pathways. (A-C) The mRNA level of BTG2 in PC9 cells validated by RT-qPCR. (B–C) The protein level of BTG2 in PC9 cells validated by western blotting. (D) The proliferation of PC9 cells using EdU staining. (E) The percentage of EdU positive PC9 cells. (F-G) The invasive capacities of PC9 cells assessed by the transwell assay. (H) Western blotting assessed the protein levels of CDK1, CDK6, phosphorylated ERK, ERK, phosphorylated AKT, AKT in PC9 cells. SMC2 knockdown was achieved via SMC2-targeted lentivirus transfection. The negative control siRNA (siBTG2) and BTG2-targeted siRNA (siBTG2) was transfected into shCtrl or shSMC2 PC9 cells in these experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The antibodies used in this study are listed as follows: anti-β-actin (Zsbio), anti-SMC2 antibody (Abclonal), anti-CDK1 antibody (CST), anti-CDK6 antibody (CST), anti-ERK1/2 (CST), anti phospho-ERK1/2 (CST), anti-AKT (CST), anti-phospho-AKT (CST), anti-BTG2 antibody (Proteintech), anti-p53 antibody (Abclonal), anti-mouse IgG HRP-linked Antibody (CST), anti-rabbit IgG HRP-linked Antibody (CST).

Techniques: Knockdown, Activation Assay, Quantitative RT-PCR, Western Blot, Staining, Transwell Assay, Transfection, Negative Control

Journal: Cellular signalling

Article Title: SMC2 knockdown inhibits malignant progression of lung adenocarcinoma by upregulating BTG2 expression.

doi: 10.1016/j.cellsig.2024.111216

Figure Lengend Snippet: Fig. 8. The expression of BTG2 following SMC2 knockdown in lung adenocarcinoma xenograft tumor models. (A-B) Immunohistochemistry staining and score of BTG2 protein following SMC2 knockdown in the subcutaneous lung adenocarcinoma tumor model. (C–D) Immunohistochemistry staining and score of BTG2 protein following SMC2 knockdown in the intracranial lung adenocarcinoma tumor model. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The antibodies used in this study are listed as follows: anti-β-actin (Zsbio), anti-SMC2 antibody (Abclonal), anti-CDK1 antibody (CST), anti-CDK6 antibody (CST), anti-ERK1/2 (CST), anti phospho-ERK1/2 (CST), anti-AKT (CST), anti-phospho-AKT (CST), anti-BTG2 antibody (Proteintech), anti-p53 antibody (Abclonal), anti-mouse IgG HRP-linked Antibody (CST), anti-rabbit IgG HRP-linked Antibody (CST).

Techniques: Expressing, Knockdown, Immunohistochemistry, Staining

Journal: PLoS ONE

Article Title: The Specificity and Patterns of Staining in Human Cells and Tissues of p16INK4a Antibodies Demonstrate Variant Antigen Binding

doi: 10.1371/journal.pone.0053313

Figure Lengend Snippet: P16INK4A subcellular localization by immunofluorescence in HeLa cells using anti-p16INK4a antibodies F-12 ( A ), H-156 ( B ), JC8 ( C ), and E6H4 ( D ); magnification bar = 20 µm. E . Comparison of anti-p16INK4a antibodies in Western Blot analysis using p16INK4a positive Hek293 and HeLa whole cell lysates and p16INK4a negative PC3 cell extract (each loaded 20 µg whole protein per well). F . Immunoprecipitation of p16INK4a using F-12 and JC8 mouse monoclonal antibodies, the FLAG mouse monoclonal antibody was used alongside as a negative control.

Article Snippet: PC3 whole cell lysate was purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Comparison, Western Blot, Immunoprecipitation, Bioprocessing, Negative Control