Journal: Cancer Research

Article Title: Fatty Acid Oxidation Mediated by Malonyl-CoA Decarboxylase Represses Renal Cell Carcinoma Progression

doi: 10.1158/0008-5472.CAN-23-0969

Figure Lengend Snippet: HIF regulated the expression of MLYCD through eIF4G3 microexons. A and B, Representative imaging of Western blot for detecting the MLYCD expression in OS-RC-2 or 786-O cells with/without knockdown HIF1α/HIF2α. Densitometry and statistical analysis. C, The levels of MLYCD mRNA in OS-RC-2 and 786-O cells with/without knockdown HIF2α. D, Representative imaging of Western blot for detecting the MLYCD expression in OS-RC-2 and 786-O cells with/without HIF2α overexpressing and/or treatment with DMSO or MG132 (20 nmol/L) or chloroquine (50 nmol/L). Densitometry and statistical analysis. E, Venn diagram of the translation-related DEGs according to the RNA sequencing in RCC tissues with paired normal kidney tissues ( n = 3) and 786-O or OS-RC-2 cells with knockdown HIF2α ( n = 3). DEGs were identified on the basis of P < 0.05 and |LogFC| > 2. F, The correlation of HIF2α and eIF4G3 expression in TCGA_KIRC database. G, eIF4G3 microexon splicing was detected by RT-PCR assay in OS-RC-2 and 786-O cells with/without HIF2α knockdown. Representative imaging of gel map amplifying the spliced products. The GAPDH expression acted as the loading and control. Red rectangle, microexon; orange rectangle, adjacent alternative exon. H, The predicted positions of putative HIF2α-binding motif in −2,000-bp human eIF4G3 promoter. I, ChIP-PCR assays monitoring direct binding of HIF2α to eIF4G3 promoter regions in OS-RC-2 and 786-O cells. J, Luciferase reporter assays were carried out by cotransfecting the WT eIF4G3 promoter or fragment E2-mutant eIF4G3 promoter with HIF2α overexpression vector or the negative vector in OS-RC-2 and 786-O cells. K, Representative imaging of Western blot analysis for detecting the MLYCD and eIF4G3 expression in OS-RC-2 and 786-O cells with/without HIF2α overexpressing and/or eIF4G3 knockout. Densitometry and statistical analysis. Student t test; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: After blocking the membrane in 5% nonfat milk diluted by TBST, we overnight incubated the membrane at 4°C with the primary antibodies: MLYCD (Abcam, ab234879, 1:1,000); HIF1α (ProteinTech, 66730–1-Ig, 1:1,000); HIF2α (Abcam, ab109616, 1:1,000); VHL (ProteinTech, 24756–1-AP,1:1,000); eIF4G3 (ProteinTech, 11281–1-AP, 1:2,000); p-PERK(Cell Signaling Technology, #3179, 1:1,000); ATF6 (Abcam, ab227830, 1:1,000); p-IER1α (Abcam, ab124945, 1:1,000); Bip (Cell Signaling Technology, #3177S, 1:1,000); p-eIF2α (Cell Signaling Technology, #3398S, 1:1,000); ATF4 (Cell Signaling Technology, # 11815S, 1:1,000); CHOP (Cell Signaling Technology, #2895S, 1:1,000); caspase-3 (Cell Signaling Technology, #9664S, 1:1,000); SCD (Abcam, ab236868, 1:1,000); ALB(Abcam, ab207327, 1:2,000); AQP3 (Abcam catalog no. ab125219, RRID:AB_11000698); PTGS2 (Abcam catalog no. ab179800, RRID:AB_2894871); PLIN2 (Abcam, ab52356, 1:2,000); β-actin (ProteinTech, 81115–1-RR, 1:5,000); GAPDH ((ProteinTech, catalog no. 60004–1-Ig, RRID:AB_2107436).

Techniques: Expressing, Imaging, Western Blot, Knockdown, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Control, Binding Assay, Luciferase, Mutagenesis, Over Expression, Plasmid Preparation, Knock-Out

Journal: bioRxiv

Article Title: Translational control of AMPK activity in melanoma

doi: 10.64898/2025.12.30.697000

Figure Lengend Snippet: (A) Western blot analysis of A375 and MelJuso (NRAS-mutant) melanoma cells treated with increasing concentrations of the eIF4A inhibitor Rocaglamide A (RocA) for 20 h. Both cell lines exhibited dose-dependent increases in phosphorylated AMPK, accompanied by decreased levels of LKB1 and its cofactor MO25. (B) Western blot analysis of G361 (BRAF V600E -mutant, LKB1 -null) melanoma cells treated with increasing concentrations of RocA for 20 h. AMPK activation occurred in a dose-dependent manner despite the absence of functional LKB1, confirming LKB1-independent AMPK activation. (C) Western blot analysis of A375, MelJuso, and G361 cells treated with increasing concentrations of the eIF4E-eIF4G disruptor 4E1RCat for 20 h. All three cell lines exhibited increased AMPK activity regardless of LKB1 status or driver mutation. (D) Western blot analysis of A375 and MelJuso cells transfected with two different LKB1-targeting siRNAs for 48 h, combined with 100 nM RocA treatment for the last 20 h. LKB1 knockdown did not prevent RocA-induced AMPK activation, confirming the LKB1-independent mechanism. Non-targeting control siRNA (si-NT) was used as a control. (D) Western blot analysis of A375, G361, and MelJuso cells transfected with CaMKKβ-targeting siRNAs for 48 h, combined with 100 nM RocA treatment for the last 20 h. CaMKKβ depletion did not impair RocA-induced AMPK activation, indicating that CaMKKβ is not required for AMPK activation by eIF4F inhibition. Non-targeting control siRNAs (si-NT) were used as a control. (E) Western blot analysis of A375 and MelJuso cells transfected with MLK3-targeting siRNAs for 48 h, combined with 100 nM RocA treatment for the last 20 h. MLK3 protein levels decreased following both RocA treatment and siRNA-mediated knockdown, but MLK3 depletion did not prevent RocA-induced AMPK activation. Non-targeting siRNAs (si-NT) were used as a control. Vinculin, GAPDH, or total AMPK levels served as loading controls. The control samples (CTRL) were treated with an equivalent volume of the vehicle (DMSO). The upper index letters refer to the corresponding loading control detected on the same membrane.

Article Snippet: The gene-specific siRNAs used for knockdown: LKB1 siRNA (sc-35816), eIF4E siRNA (sc-35284), eIF4G siRNA (sc-35286), eIF4AI siRNA (sc-40554), LAMTOR1 siRNA (sc-96597), PP2A Aα siRNA (sc-44033), CaMKKβ siRNA (sc-38956), MLK3 siRNA (sc-35946) from Santa Cruz Biotechnology, STK11 (4392420, ID: s13579) from ThermoFisher Scientific, and MISSION® esiRNA PPP2R1A (EHU071351) from Merck.

Techniques: Western Blot, Mutagenesis, Activation Assay, Functional Assay, Activity Assay, Transfection, Knockdown, Control, Inhibition, Membrane

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 eIF4G 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, Control, 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, Control, 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, Control

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, Control

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, Control