gapdh probe/primer set  (Thermo Fisher)

Journal: Scientific Reports

Article Title: PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

doi: 10.1038/s41598-024-75142-5

Figure Lengend Snippet: PKR interacts with 4.1R depending on its kinase activity . ( A ) COS7 cells were transfected with the indicated plasmids, followed by IP and WB. The top panel shows the co-precipitation of PKR with 4.1R by IP with anti-Flag antibody and WB with anti-Myc antibody. The second panel shows the expression of 4.1R by IP with anti-Flag antibody and WB with anti-Flag antibody. The lower two panels show the expression level of each protein without IP. ( B ) COS7 cells were transfected with the indicated plasmids, followed by IP and WB. The top panel shows the co-precipitation of 4.1R with PKR by IP with anti-Flag antibody and WB with anti-Myc antibody. The second panel shows the expression of PKR by IP with anti-Flag antibody and WB with anti-Flag antibody. The lower two panels show the expression level of each protein without IP. ( C ) COS7 cells were transfected with the indicated plasmids, followed by IP and WB. The top panel shows the co-precipitation of wild type PKR or PKR K296R with 4.1R by IP with anti-Flag antibody and WB with anti-Myc antibody. The second panel shows the expression of 4.1R by IP with anti-Flag antibody and WB with anti-Flag antibody. The lower two panels show the expression level of each protein without IP.

Article Snippet: The PKR, 4.1R, and GAPDH primer and probe sets were obtained from Thermo Fisher Scientific (ID nos.

Techniques: Activity Assay, Transfection, Expressing

Journal: Scientific Reports

Article Title: PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

doi: 10.1038/s41598-024-75142-5

Figure Lengend Snippet: The expression level of 4.1R protein is regulated by protein expression and activation levels of PKR in HCC cell lines. ( A ) Three HCC cell lines, HuH7, HLE, and HepG2 cells, were seeded in a 6-well, flat-bottomed plate, and cell lysates from three independent wells of each cell line were loaded, followed by WB with anti-4.1R antibody. Overexpression ( B ) and knockdown ( C ) of PKR and C16 treatment ( D ) affected the expression level of 4.1R protein. ( B ) HuH7 cells were transfected with or without Flag-PKR, followed by WB with anti-PKR antibody (top panel), anti-4.1R antibody (middle panel), and anti-actin antibody (bottom panel). The quantified PKR:actin ratio (center panel) and the 4.1R at 130 kDa:actin ratio (right panel) of the WB are shown. ( C ) HuH7 cells were transfected with control siRNA and two PKR siRNAs, followed by WB with anti-PKR antibody (top panel), anti-4.1R antibody (middle panel), and anti-actin antibody (bottom panel). The quantified PKR:actin ratio (center panel) and the 4.1R at 130 kDa:actin ratio (right panel) of WB are shown. ( D ) HuH7 cells were treated with DMSO or 1, 2, or 3 μM of C16 for 24 h, respectively, followed by WB with anti-phospho-PKR antibody (top panel), anti-PKR antibody (second panel), anti-4.1R antibody (third panel), and anti-actin antibody (bottom panel). The quantified PKR:actin ratio (center panel) and the 4.1R at 130 kDa: actin ratio (right panel) of WB are shown. The values in the figure represent fold change to those of control. Means ± SD values of three independent experiments are shown (*p < 0.05, **p < 0.01, ***p < 0.001 compared to control groups by the two-sided Student’s t -test or Tukey’s test).

Article Snippet: The PKR, 4.1R, and GAPDH primer and probe sets were obtained from Thermo Fisher Scientific (ID nos.

Techniques: Expressing, Activation Assay, Over Expression, Knockdown, Transfection, Control

Journal: Scientific Reports

Article Title: PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

doi: 10.1038/s41598-024-75142-5

Figure Lengend Snippet: High expression of 4.1R in HCC patients leads to a poor prognosis. ( A ) 4.1R levels in HCC tissue and normal adjacent tissue were analyzed by the UALCAN database (p = 1.62E-12 compared to the normal group by Student’s t -test). ( B , C ) Survival rates based on the expression level of 4.1R ( B ) and PKR ( C ) were analyzed by the Kaplan–Meier Plotter database (p = 0.016, 0.0015, respectively, compared to the low expression group by the log-rank test).

Article Snippet: The PKR, 4.1R, and GAPDH primer and probe sets were obtained from Thermo Fisher Scientific (ID nos.

Techniques: Expressing

Journal: Scientific Reports

Article Title: PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

doi: 10.1038/s41598-024-75142-5

Figure Lengend Snippet: Suppression of anchorage-independent proliferation by silencing of PKR or 4.1R in HCC cell lines. ( A ) HuH7 cells (top panel) and HepG2 cells (bottom panel) on culture dishes were transfected with control siRNA or two 4.1R siRNAs, followed by MTT assay. The black line, blue line, and yellow line show the results for control, 4.1R siRNA1, and 4.1R siRNA2, respectively. ( B ) Huh7 and HepG2 cells were transfected with control siRNA or two 4.1R siRNAs, followed by colony formation assay. Three representative images of colony formation on soft agar and their quantified colony area with control siRNA or two 4.1R siRNAs, respectively, in HuH7 (B, top panels) and HepG2 cells ( B , bottom panels) are shown. ( C ) HuH7 cells (top panel) and HepG2 cells (bottom panel) on culture dishes were transfected with control siRNA or two PKR siRNAs, followed by MTT assay. The black line, blue line, and yellow line show the results for control, PKR siRNA1, and PKR siRNA2, respectively. ( D ) Huh7 and HepG2 cells were transfected with control siRNA or two PKR siRNAs, followed by colony formation assay. Three representative images of colony formation on soft agar and their quantified colony area with control siRNA or two PKR siRNAs, respectively, in HuH7 ( D , top panels) and HepG2 cells ( D , bottom panels) are shown. ( E ) Huh7 and HepG2 cells were transfected with the indicated siRNA and plasmid, respectively, followed by colony formation assay. Three representative images of colony formation on soft agar and their quantified colony area after transfection of the indicated siRNA and plasmid, respectively, in HuH7 ( B , top panels) and HepG2 cells ( B , bottom panels) are shown. The values indicate the mean ± SD values of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group by the two-sided Student’s t -test or Tukey’s test.

Article Snippet: The PKR, 4.1R, and GAPDH primer and probe sets were obtained from Thermo Fisher Scientific (ID nos.

Techniques: Transfection, Control, MTT Assay, Colony Assay, Plasmid Preparation

Journal: Scientific Reports

Article Title: PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

doi: 10.1038/s41598-024-75142-5

Figure Lengend Snippet: Increased anchorage-independent proliferation by overexpression of PKR or 4.1R in HCC cell lines. ( A ) HuH7 cells (top panel) and HepG2 cells (bottom panel) were transfected with or without Flag-4.1R, followed by MTT assays on culture dishes. The blue line and black line show the results with and without Flag-4.1R, respectively. ( B ) Huh7 and HepG2 cells were transfected with or without Flag-4.1R, followed by colony formation assay. Two representative images of colony formation on soft agar and their quantified colony area with or without Flag-4.1R in HuH7 (B, top panels) and HepG2 cells (B, bottom panels) are shown. ( C ) HuH7 cells (top panel) and HepG2 cells (bottom panel) were transfected with or without Flag-PKR, followed by MTT assay on culture dish. The blue line and black line show the results with and without Flag-PKR, respectively. ( D ) Huh7 and HepG2 cells were transfected with or without Flag-PKR, followed by colony formation assay. Two representative images of colony formation on soft agar and their quantified colony area with or without Flag-PKR in HuH7 (D, top panels) and HepG2 cells (D, bottom panels) are shown. ( E ) Huh7 and HepG2 cells were transfected with the indicated plasmid and siRNA, respectively, followed by colony formation assay. Three representative images of colony formation on soft agar and their quantified colony area after transfection of indicated siRNA and plasmid, respectively, in HuH7 cells (top panels) and HepG2 cells (bottom panels) are shown. The values indicate the mean ± SD values of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group by the two-sided Student’s t -test or Tukey’s test.

Article Snippet: The PKR, 4.1R, and GAPDH primer and probe sets were obtained from Thermo Fisher Scientific (ID nos.

Techniques: Over Expression, Transfection, Colony Assay, MTT Assay, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: PKR associates with 4.1R to promote anchorage-independent growth of hepatocellular carcinoma and lead to poor prognosis

doi: 10.1038/s41598-024-75142-5

Figure Lengend Snippet: Schematic depicting the mechanism by which PKR enhances anchorage-independent proliferation via 4.1R and anchorage-dependent proliferation via mitogen-activated protein kinase (MAPK).

Article Snippet: The PKR, 4.1R, and GAPDH primer and probe sets were obtained from Thermo Fisher Scientific (ID nos.

Techniques:

Journal: Communications Biology

Article Title: A carboxy-terminal ubiquitylation site regulates androgen receptor activity

doi: 10.1038/s42003-023-05709-x

Figure Lengend Snippet: a LNCaP cells stably expressing wild-type or mutant ARs were treated for 2 days with an AR siRNA targeting the 5’ UTR (present only in the endogenous transcript), followed by 24 h in CSS medium with or without addition of DHT (10 nM). Lysates were then immunoblotted with an anti-AR or anti-PSA Ab. b LNCaP cells stably expressing wild-type or mutant ARs were treated for 2 days with an AR siRNA targeting the 5’ UTR, followed by 24 h in CSS medium with or without addition of DHT (1 nM or 10 nM). Levels of PSA and NKX3.1 mRNA were then determined by qRT-PCR. Data are mean of biological replicate samples. c LNCaP cells stably expressing wildtype or mutant ARs (V5 tagged) were cultured for 24 h in CSS medium and then stimulated with 10 nM DHT for 4 h. Recruitment of total AR or V5 tagged AR to the AREs in the PSA and NKX3.1 genes were then determined by ChIP-qPCR. Data are expressed as % of input DNA and mean of biological replicates are shown.

Article Snippet: Primers were annealed at 55 °C for 30 s follow by extension at 72 °C for 60 s. The TaqMan primer-probe sets for PSA/NKX3.1 (FAM labeled) and the internal control GAPDH (VIC-TAMRA labeled) transcripts were purchased as inventoried mixes from Applied Biosystems.

Techniques: Stable Transfection, Expressing, Mutagenesis, Quantitative RT-PCR, Cell Culture

Journal: PLoS ONE

Article Title: NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

doi: 10.1371/journal.pone.0024638

Figure Lengend Snippet: In silico analysis identified a cluster of potential Sp1/Osterix binding sites within approximately 70 bp of the 5′ flanking region of the human NELL-1 gene. Within the cluster, Site A contains three overlapping potential Sp1 sites from −71 to −96 bp and Site B contains a single Sp1 site from −133 to −142 bp. Four OSE2 sites to which Runx2 binds in human NELL-1 promoter are also shown (labeled A, B, C and H1). A similar pattern is seen in mouse Nell-1 promoter. Multiple putative Sp1 sites (labeled Site a∼e) are located before three OSE2 sites (labeled 1, m1 and 2). The relative location of primers for CHIP assay are indicated on the human 2.2 kb promoter.

Article Snippet: TaqMan primer-probe sets for osteocalcin (Ocn) , osteopontin ( Opn ), NELL-1 , Osterix , and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were purchased (Applied Biosystems, Foster City, CA) and analyzed by real time PCR as previously described .

Techniques: In Silico, Binding Assay, Labeling

Journal: PLoS ONE

Article Title: NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

doi: 10.1371/journal.pone.0024638

Figure Lengend Snippet: ( A ) Human NELL-1 promoter is responsive to Osterix in a dose-dependent manner shown by reporter assay. Graph detailing the luciferase activity in Saos-2 cells co-transfected with either the p2213WT-Luc or the p325WT-Luc promoter-luciferase constructs as well as 0, 0.25, 0.5, or 1 ug of Osterix expression vector (pOsx). Data are expressed as a percentage of the luciferase activity of the p325WT-Luc construct in the absence of control vector (pCtr). ( B ) Osterix overexpression decreased NELL-1 mRNA level in Saos-2 cells at 2 days and 7 days post-transfection. Western blot showed Osterix protein levels at 2 days and 7 days post-transfection. (*p<0.05) ( C ) Osterix transcriptional repression of NELL-1 promoter in osteoblastic and non-osteoblastic cells. A graph detailing the luciferase activity of p325WT-Luc 2 days post-transfection in Saos-2, U2OS, Hela and Glioma cell lines co-transfected with pCtr or pOsx. (*p<0.05)

Article Snippet: TaqMan primer-probe sets for osteocalcin (Ocn) , osteopontin ( Opn ), NELL-1 , Osterix , and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were purchased (Applied Biosystems, Foster City, CA) and analyzed by real time PCR as previously described .

Techniques: Reporter Assay, Luciferase, Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation, Over Expression, Western Blot

Journal: PLoS ONE

Article Title: NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

doi: 10.1371/journal.pone.0024638

Figure Lengend Snippet: ( A ) Schematic of p325WT-Luc, p325mut all-Luc, p325mutSiteA, and p325mutSiteB constructs. Blue represents mutated Sp1 sites. ( B ) Graph depicting promoter activity on Saos-2 cells cotransfected with pOsx as well as p325WT-Luc or mutated NELL-1 promoter-luciferase constructs (p325mut all-Luc, p325mutSiteA-Luc, and p325mutSiteB-Luc, respectively). Data are reported as percent activity of control cells transfected with pCtr. (*p<0.05)

Article Snippet: TaqMan primer-probe sets for osteocalcin (Ocn) , osteopontin ( Opn ), NELL-1 , Osterix , and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were purchased (Applied Biosystems, Foster City, CA) and analyzed by real time PCR as previously described .

Techniques: Construct, Activity Assay, Luciferase, Transfection

Journal: PLoS ONE

Article Title: NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

doi: 10.1371/journal.pone.0024638

Figure Lengend Snippet: ( A ) EMSA of Saos-2 nuclear proteins transfected with pCtr or pOsx binding to SiteA (containing three proximal Sp1 sites) and SiteB probes. Supershifts with specific Osterix antibody indicate the specific Osterix-DNA complexes. ( B ) EMSA depicting primary human osteoblast cell (hOB) nuclear proteins transfected with pOsx binding to the SiteA probes, with competition by 20x and 200x unlabeled SiteA and 200x unlabeled mutated SiteA (Mut A ) oligonucleotides. Note that the MutA probes failed to bind nuclear proteins. The same pattern is also seen by competition of SiteB element and Mut B . ( C–D ) Osterix binds to endogenous Sp1 sites of the human NELL-1 promoter in Saos-2 (C) and hOB (D) cells. The NELL-1 -1 kb primer set covers 1 kb proximal promoter region containing all Sp1/Osterix binding sites and three OSE2 sites. The NELL-1 -2 kb primer set covers NELL-1 promoter region from −1 kb to −2 kb where no Sp1/Osterix binding site but one OSE2 site exists. The qPCR products depict DNA amplified from Chromatin Immunoprecipitation with cells utilizing Control IgG and Osterix antibody. Input DNA represents positive genomic DNA control.

Article Snippet: TaqMan primer-probe sets for osteocalcin (Ocn) , osteopontin ( Opn ), NELL-1 , Osterix , and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were purchased (Applied Biosystems, Foster City, CA) and analyzed by real time PCR as previously described .

Techniques: Transfection, Binding Assay, Amplification, Chromatin Immunoprecipitation

Journal: PLoS ONE

Article Title: NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

doi: 10.1371/journal.pone.0024638

Figure Lengend Snippet: ( A ) Graph depicting promoter activity in Saos-2 cells after co-transfection with control empty pcDNA3.1 vector (pCtr), pcDNA-Runx2 (pRunx2) or pRunx2 plus pOsx expression vectors as well as p2213-Luc, p325-Luc or p325mut-Luc NELL-1 promoter-luciferase constructs. Data are reported as fold changes in comparison to control cells transfected with pCtr and p325WT-Luc constructs. (p<0.05) ( B ) Osterix affects binding of RNA polymerase II to NELL-1 's promoter, but does not compete with Runx2 binding of OSE2 sites. The NELL-1 -1 kb primer set used covers 1 kb proximal promoter region where all Sp1/Osterix binding sites and three OSE2 sites are located. The qPCR products depict DNA amplified from Chromatin Immunoprecipitation with Saos-2 cells transfected with pCtr or pOsx utilizing Control IgG, Osterix antibody, Runx2 antibody or RNA polymerase II antibody. Input DNA represents positive genomic DNA control. (*p<0.05)

Article Snippet: TaqMan primer-probe sets for osteocalcin (Ocn) , osteopontin ( Opn ), NELL-1 , Osterix , and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were purchased (Applied Biosystems, Foster City, CA) and analyzed by real time PCR as previously described .

Techniques: Activity Assay, Cotransfection, Plasmid Preparation, Expressing, Luciferase, Construct, Transfection, Binding Assay, Amplification, Chromatin Immunoprecipitation

Journal: PLoS ONE

Article Title: NELL-1, an Osteoinductive Factor, Is a Direct Transcriptional Target of Osterix

doi: 10.1371/journal.pone.0024638

Figure Lengend Snippet: Real time PCR analysis of Nell-1 , OCN and OPN transcripts in Saos-2 cells transiently transfected with Control (pCtr) or Osterix expression vector (pOsx) at 2 days and 7 days (top panel). The effects with transcient transfection of Negative control siRNA (NC-siRNA) or Osterix siRNA mixture (Osx-siRNA) were revealed by Real time PCR analysis at 2 days and 7 days (bottom panel). ( C ) Diagram of the regulatory relationship between Runx2, Osterix and NELL-1. Runx2 positively regulates Osterix and NELL-1. Osterix negatively controlled NELL-1 expression in this study. NELL-1 was also shown to positively affect Runx2 through phosphorylation and negatively feedback on Osterix during osteogenesis.

Article Snippet: TaqMan primer-probe sets for osteocalcin (Ocn) , osteopontin ( Opn ), NELL-1 , Osterix , and glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) were purchased (Applied Biosystems, Foster City, CA) and analyzed by real time PCR as previously described .

Techniques: Real-time Polymerase Chain Reaction, Transfection, Expressing, Plasmid Preparation, Negative Control