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: