Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibition of JAK2/STAT3 Signaling Pathway Suppresses Proliferation of Burkitt’s Lymphoma Raji Cells via Cell Cycle Progression, Apoptosis, and Oxidative Stress by Modulating HSP70

doi: 10.12659/MSM.910170

Figure Lengend Snippet: Expression of HSP70 as detected by qRT-PCR ( A ) and Western blotting ( B, C ) in Raji cells. * P <0.05 compared with the Control group and NC siRNA group; # P <0.05 compared with the HSP70 siRNA group.

Article Snippet: AG490 was purchased from Abmole Bioscience (M1646, Shanghai, China), recombinant human JAK2 was purchased from Thermo Fisher Scientific (PV4393, Rockford, USA), and HSP70 siRNA and NC siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibition of JAK2/STAT3 Signaling Pathway Suppresses Proliferation of Burkitt’s Lymphoma Raji Cells via Cell Cycle Progression, Apoptosis, and Oxidative Stress by Modulating HSP70

doi: 10.12659/MSM.910170

Figure Lengend Snippet: Expression of JAK2/STAT3 signaling pathway-related proteins in Raji cells as determined by Western blotting. * P <0.05 compared with the Control group and NC siRNA group; # P <0.05 compared with the HSP70 siRNA group; & P <0.05 compared with the AG490 group.

Article Snippet: AG490 was purchased from Abmole Bioscience (M1646, Shanghai, China), recombinant human JAK2 was purchased from Thermo Fisher Scientific (PV4393, Rockford, USA), and HSP70 siRNA and NC siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibition of JAK2/STAT3 Signaling Pathway Suppresses Proliferation of Burkitt’s Lymphoma Raji Cells via Cell Cycle Progression, Apoptosis, and Oxidative Stress by Modulating HSP70

doi: 10.12659/MSM.910170

Figure Lengend Snippet: Effects of the JAK2/STAT3 pathway on the proliferation and cycle distribution of Raji cells via regulating HSP70 . ( A ) Proliferation of Raji cells (12 h, 24 h, 36 h, 48 h, and 72 h) as assessed by MTT assay; ( B, C ) Detection of Raji cell cycle distribution in each group by flow cytometry.

Article Snippet: AG490 was purchased from Abmole Bioscience (M1646, Shanghai, China), recombinant human JAK2 was purchased from Thermo Fisher Scientific (PV4393, Rockford, USA), and HSP70 siRNA and NC siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: MTT Assay, Flow Cytometry

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibition of JAK2/STAT3 Signaling Pathway Suppresses Proliferation of Burkitt’s Lymphoma Raji Cells via Cell Cycle Progression, Apoptosis, and Oxidative Stress by Modulating HSP70

doi: 10.12659/MSM.910170

Figure Lengend Snippet: Effect ofJAK2/STAT3 pathway-regulated HSP70 on BL Raji cell apoptosis as detected by Annexin V-FITC/PI staining ( A ) and Hoechst 33342/PI staining ( B ).

Article Snippet: AG490 was purchased from Abmole Bioscience (M1646, Shanghai, China), recombinant human JAK2 was purchased from Thermo Fisher Scientific (PV4393, Rockford, USA), and HSP70 siRNA and NC siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Staining

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Inhibition of JAK2/STAT3 Signaling Pathway Suppresses Proliferation of Burkitt’s Lymphoma Raji Cells via Cell Cycle Progression, Apoptosis, and Oxidative Stress by Modulating HSP70

doi: 10.12659/MSM.910170

Figure Lengend Snippet: Blocking the JAK2/STAT3 pathway can inhibit HSP70 to promote oxidative stress in Raji cells. ( A ) Mean fluorescence intensity (MIF) of ROS in each group as measured by the DCFH-DA assay; ( B, C ) MDA content ( B ), SOD activity ( C ) and GSH-Px activity ( D ) of cells in each group; * P <0.05 compared with the Control group and NC siRNA group; # P <0.05 compared with the HSP70 siRNA group; & P <0.05 compared with the AG490 group.

Article Snippet: AG490 was purchased from Abmole Bioscience (M1646, Shanghai, China), recombinant human JAK2 was purchased from Thermo Fisher Scientific (PV4393, Rockford, USA), and HSP70 siRNA and NC siRNA were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Blocking Assay, Fluorescence, DCFH-DA Assay, Activity Assay

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70 and eIF4G expression are significantly higher in hepatocellular carcinoma (HCC) tumor specimens. ( A ) Representative pictures of H&E and IF staining. Magnification, 20×; Scale bar, 50μm. ( B ) The protein levels of HSP70 and eIF4G in HCC tumor specimens were significantly higher than those of adjacent non-tumor specimens. ( C ) Heatmap showing the relative protein expression of eIF4A, eIF4E, eIF4G, 4EBP1, and HSP70 in all HCC patients tissue samples based on IF staining results. ( D ) Heatmap showing the average relative protein expression of eIF4A, eIF4E, eIF4G, 4EBP1, and HSP70 in all patients in each TNM stage. ( E , F ) Protein expression of HSP70 and eIF4G in HCC patients displayed according to the TNM stage. ( G , H ) The scatter plot of correlation between the AFP level and the protein expression of HSP70 and eIF4G. * p < 0.05, *** p < 0.001 compared with the adjacent non-tumor groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: Clinicopathological Characteristics of HCC Patients.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: Correlation between HSP70 Expression and Clinicopathological Characteristics in HCC Patients.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: The expression of HSP70 and eIF4G positively correlated in tumor specimens of patients with HCC. High and low expression of HSP70 or eIF4G are defined in Materials and Methods. ( A ) Correlation plot showing the relationships between HSP70 and eIF4A, eIF4E, eIF4G, and 4EBP1, respectively. Light blue indicates the highest correlation while light red indicates the lowest correlation. ( B ) The relative protein expression of HSP70 and eIF4G in HCC tissues was positively correlated. ( C , D ) The OS and PFS in HCC patients with high and low HSP70 expression were evaluated. ( E , F ) The OS and PFS of HCC patients were assessed based on high and low eIF4G expression. ( G , H ) HCC patients were divided into four groups of low HSP70 expression/low eIF4G expression, high HSP70 expression/low eIF4G expression, low HSP70 expression/high eIF4G expression, and high HSP70 expression/high eIF4G expression. The OS and PFS of each group were analyzed. ( I – K ) The PFS of HCC patients was compared according to clinical features of AFP level ( I ), tumor size ( J ), and TNM stage ( K ). The survival curves were plotted by the Kaplan-Meier method, and p values were calculated by the log-rank test.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Expressing

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70 interacts with eIF4G in vitro. ( A , B ) Cell lysates were analyzed with a Western blot to detect the protein level by using primary antibodies targeting eIF4A, eIF4E, eIF4G, 4EBP1, and HSP70. β-actin was used as an internal control. ( C ) Flag-HSP70 immunoprecipitated eIF4G from the cell lysates of HepG2 and Huh7 cells transfected with or without Flag-HSP70 plasmid. Immunoprecipitates were analyzed with Flag or eIF4G antibodies via Western blot. ( D ) The HSP70-eIF4G interaction was detected by in situ PLA. Flag-HSP70 transfected cells and control cells were incubated with mouse anti-HSP70 mAb and rabbit anti-eIF4G mAb. Red signal spots are shown to indicate the protein interaction between HSP70 and eIF4G. Magnification 20×; Scale bar 50 μm. Quantitative data from three experiments with similar results were analyzed. Data are presented as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: In Vitro, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, In Situ, Incubation, Standard Deviation

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: The HSP70 and eIF4G interaction increases under hypoxia. ( A ) HepG2 and Huh7 cells were treated as described in the methods section. IC50 of CoCl2 in HepG2 and Huh7 cells were calculated based on cell viability assessed by MTT assay, and the results presented as a ratio with the control group. ( B , C ) Cells were treated with 10 µM CoCl 2 for 12 h and the protein levels of the eIF4F complex and HSP70 detected by Western blot. β-actin was used as an internal control. ( D ) Cells were incubated with 10 µM CoCl2 for 12 h and the protein of eIF4G immunoprecipitated from the cell lysates of HepG2 and Huh7 cells with HSP70 antibody. The immunoprecipitates were analyzed via Western blot with HSP70 and eIF4G antibodies. ( E ) Cells were stimulated with 10 µM CoCl 2 for 12 h and the HSP70–eIF4G interaction in situ was detected by PLA assay. Representative images are shown. Magnification 20×; Scale bar 50 μm. Quantitative data from three experiments with similar results were analyzed. Data are presented as mean ± SD. ** p < 0.01 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: MTT Assay, Western Blot, Incubation, Immunoprecipitation, In Situ

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70–eIF4G interaction promotes cellular protein synthesis. ( A , B ) Cells were incubated with 10 µM CoCl 2 for 12 h; each group was then examined via protein synthesis assay. Representative pictures of protein synthesis are shown. Magnification 20×; Scale bar 50 μm. ( C , D ) Cells were cultivated with 10 µM CoCl 2 for 12 h and the eIF4G-eIF4E interaction examined in situ for each group. Representative images of the PLA assay are presented. Magnification 20×; Scale bar 50 μm. Quantitative data from three experiments with similar results were analyzed. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Incubation, In Situ

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70–eIF4G interaction promotes cell proliferation. ( A , B ) Cells were incubated with 10 μM CoCl 2 for 5 days; cell viability was measured daily. ( C , D ) Cells treated with 10 μM CoCl 2 ; colony units were counted after 14 days of incubation. Quantitative data from three experiments with similar results were summarized. Data are presented as mean ± SD. ns: not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001 compared with corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Incubation

Journal: Cancers

Article Title: HSP70–eIF4G Interaction Promotes Protein Synthesis and Cell Proliferation in Hepatocellular Carcinoma

doi: 10.3390/cancers12082262

Figure Lengend Snippet: HSP70–eIF4G interaction inhibits cell apoptosis. ( A , B ) Cells were incubated with 10 μM CoCl 2 for 48 h. Annexin-V and PI were used to stain the treated cells and flow cytometry analysis was performed to assess the rate of cell apoptosis. ( C , D ) Cells treated with 10 μM CoCl 2 for 48 h. The percentage of the Sub-G1 portion of the cell cycle distribution was quantified by flow cytometry analyses after PI staining. Quantitative data from three experiments with similar results were summarized. Data presented as mean ± SD. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to corresponding control groups.

Article Snippet: Cells were seeded at a density of 1.5 × 10 5 –2.5 × 10 5 in 6-well plates and incubated overnight in normal growth medium without antibiotics. eIF4A shRNA Plasmid (h) (SC-40554-SH), eIF4G shRNA Plasmid (h) (SC-40558-SH), eIF4E shRNA Plasmid (h) (SC-35284-SH), 4EBP shRNA Plasmid (h) (SC-29594-SH), HSP70 shRNA Plasmid (h) (SC-29352-SH), and eIF4G siRNA (h) (sc-35286) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pCMV3-N-FLAG-HSP70 plasmid (HG11660-NF) was obtained from Sino Biological (Eschborn, Germany).

Techniques: Incubation, Staining, Flow Cytometry