Journal: Oncogene

Article Title: TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.

doi: 10.1038/onc.2011.102

Figure Lengend Snippet: Figure 4 Activated ATF4 stimulates Redd1 expression in response to 2-DG. H1299, H460 and HeLa cells were transfected with siCTL or siATF4 for 20 h, followed by 30 mM 2-DG for 6 h. Protein and mRNA levels were measured via western blot and RT–PCR analyses.

Article Snippet: TXNIP (#1, SC-44943; #2, J-010814-05-0005), ATF4 (#1, sc-35112; #2, J-005125-10-0005), LKB1 (#1, sc-35816; #2, J-005035-07-0005), AMPKa1/2 (#1, sc-45312; #2, J-005027-06-0005 and J-005361-06-0005) and control siRNAs (#1, sc-37007; #2, D-001810-01-05) were purchased from Santa Cruz Biotechnology and Thermo Scientific Dharmacon (Chicago, IL, USA).

Techniques: Expressing, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.

doi: 10.1038/onc.2011.102

Figure Lengend Snippet: Figure 3 2-DG induces ATF4 transcriptional activity. (a, b) H1299, H460 and HeLa cells were treated with the indicated concentrations of 2-DG for 6 h. (c) H1299 cells were treated with 30 mM 2-DG for the indicated times. Protein levels were measured using western blot analysis (a, c) and mRNA levels with RT–PCR (b). Experiments were performed in duplicate.

Article Snippet: TXNIP (#1, SC-44943; #2, J-010814-05-0005), ATF4 (#1, sc-35112; #2, J-005125-10-0005), LKB1 (#1, sc-35816; #2, J-005035-07-0005), AMPKa1/2 (#1, sc-45312; #2, J-005027-06-0005 and J-005361-06-0005) and control siRNAs (#1, sc-37007; #2, D-001810-01-05) were purchased from Santa Cruz Biotechnology and Thermo Scientific Dharmacon (Chicago, IL, USA).

Techniques: Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Pharmaceuticals

Article Title: Mazdutide Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease by Modulating Endoplasmic Reticulum Stress, Improving Lipid Metabolism and Alleviating Inflammation

doi: 10.3390/ph19030371

Figure Lengend Snippet: Mazdutide suppresses ER stress via the PERK pathway in MASLD mouse liver tissue. ( A ) Representative immunohistochemical staining of GRP78 and CHOP in liver sections (scale bar = 50 μm, 200× magnification). ( B ) Representative Western blot images of key proteins in the PERK pathway from each experimental group. ( C ) Quantitative densitometric analysis of PERK and eIF2α phosphorylation (presented as the p-PERK/PERK and p-eIF2α/eIF2α ratios) and the protein levels of GRP78, ATF4, and CHOP. All values were normalized to β-actin. Data are presented as the mean ± SD ( n = 5). a p < 0.05 vs. the NCD group; b p < 0.05 vs. the HFD group. Animal groups are defined in .

Article Snippet: Primary antibodies for GRP78, ATF4, CHOP, and β-actin were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Western Blot, Phospho-proteomics

Journal: Pharmaceuticals

Article Title: Mazdutide Ameliorates Metabolic Dysfunction-Associated Steatotic Liver Disease by Modulating Endoplasmic Reticulum Stress, Improving Lipid Metabolism and Alleviating Inflammation

doi: 10.3390/ph19030371

Figure Lengend Snippet: Mazdutide inhibits the PERK-eIF2α-ATF4-CHOP pathway in MASLD cells. ( A ) Representative Western blot images showing the protein levels of GRP78, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, CHOP, and β-actin. ( B ) Quantitative densitometric analysis of the p-PERK/PERK and p-eIF2α/eIF2α ratios and the protein levels of GRP78, ATF4, and CHOP. The protein band intensities were normalized to β-actin. Data are presented as the mean ± SD ( n = 3). a p < 0.05 vs. the NCD group; b p < 0.05 vs. the HFD group. Cell groups are as defined in .

Article Snippet: Primary antibodies for GRP78, ATF4, CHOP, and β-actin were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Western Blot

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: E7386 induces ISR and ATF4 upregulation. A, Heatmap representation of gene signatures of pathways involving ATF4 and CBP partners in the in vivo model. Statistics: Welch t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. B, Boxplot depicting the quantification of ATF4-positive IHC staining in the murine model ( n = 4–5/arm; top), with representative images (bottom). Scale bar, 250 μm. Statistics: Kruskal–Wallis test followed by the Dunn test adjusted by the Benjamini–Hochberg test; *, P < 0.05. Error bars indicate SD. C, Heatmap representation of gene signatures associated with the ATF4 pathway in HCC cell lines treated with E7386 0.1 μmol/L or DMSO for 24 hours: sensitive to E7386 (top in green) and resistant to E7386 (bottom in red). Statistics: Student t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. D, Representative Western blot analysis and quantification of ATF4 in E7386-treated sensitive (green) and resistant (red) cell lines treated with E7386 at 0.1 μmol/L or DMSO for 24 hours. Vinculin was used for normalization. FC was calculated based on protein levels in DMSO-treated cells. Values represent the average of two independent experiments ( n = 2). Molecular weight ladder markers are displayed beside the protein of interest. Error bars indicate SD. Statistics: One-way Student t test; *, P < 0.05; **, P < 0.01. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: In Vivo, Immunohistochemistry, Western Blot, Molecular Weight

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: E7386 induces ISR via GCN2/eIF2α activation. A, Heatmap representation of gene pathways associated with ISR in the in vivo model. Statistics: Welch t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. B, Heatmap representation of gene pathways associated with ISR in HCC cell lines: sensitive to E7386 (top in green) and resistant to E7386 (bottom in red) treated with E7386 0.1 μmol/L or DMSO for 24 hours. Statistics: Student t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. C, Representative Western blot analysis of ATF4 levels following siRNA-mediated knockdown of EIF2AK1 , EIF2AK2 , EIF2AK3 , EIF2AK4 , and ATF4 with or without E7386 treatment (0.3 μmol/L for 3 hours). β-Actin was used as a loading control. D, Representative Western blot analysis of the ISR pathway–related proteins in Hep3B cells treated with DMSO or increasing concentrations of E7386 (0.03–1 μmol/L). β-Actin was used as a loading control. Molecular weight ladder markers are displayed beside the protein of interest. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: Activation Assay, In Vivo, Western Blot, Knockdown, Control, Molecular Weight

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: ATF4 targets altered by E7386 treatment in sensitive cell lines. A, Heatmap representation of genes associated with ISR, endoplasmic reticulum stress, amino acid synthesis, tRNA synthetases, cell cycle, and angiogenesis in HCC cell lines: sensitive to E7386 (in green) and resistant to E7386 (in red) treated with E7386 and DMSO. FC was corrected based on DMSO values. Only FC ≥ 1.5 (for overexpression) or FC ≤ 0.8 (for downregulation) was included. Statistics: Student t test for two group comparisons. B, Representative Western blot analysis of cyclin D1, cyclin D2, and geminin in Hep3B and SNU398 cell lines treated with E7386 and DMSO. Vinculin was used as a loading control. Quantification of the Western blot is provided in Supplementary Fig. S3. Molecular weight ladder markers are displayed beside the protein of interest. C, Quantification of VEGF secretion from Hep3B and SNU398 cells treated with DMSO, E7386, lenvatinib, or a combination of E7386 with lenvatinib. Statistics: Kruskal–Wallis test followed by the Dunn test adjusted by the Benjamini–Hochberg test; *, P < 0.05; **, P < 0.01. Error bars indicate the SD of the average from at least two independent experiments and technical duplicates. D, Heatmap representation of gene signatures associated with angiogenesis in the in vivo model. Statistics: Welch t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. E, Box plot depicting the quantification of CD31-positive IHC staining in the murine model ( n = 4–5/arm; left), with representative images (right). Scale bar, 250 μm. Statistics: Kruskal–Wallis test followed by the Dunn test adjusted by the Benjamini–Hochberg; *, P < 0.05. Error bars indicate SD. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: Over Expression, Western Blot, Control, Molecular Weight, In Vivo, Immunohistochemistry

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: E7386 and lenvatinib induce ATF4 upregulation and antiangiogenic effects in patients with uHCC treated in the context of a phase Ib/II clinical trial. A, Clinical trial schematic overview and time points for obtaining samples for RNA sequencing. Overall, seven matched cases pre- and on-treatment were included in the transcriptomic analysis. B, Heatmap representation of gene signatures associated with the ATF4 pathway and angiogenesis in matched patient samples pre- and on-treatment with the combination of E7386 and lenvatinib. Patients V and VII, highlighted in a red box, carry gain-of-function missense mutations in CTNNB1 , whereas the rest are WT. Patient VII also harbors an AXIN1 mutation. For on-treatment samples, the percentage of MTS is indicated on the red–green scale. Statistics: Student t test for two group comparisons. Signatures are listed in the order in which they appear in Supplementary Table S1. QD, once a day; BID, twice a day. Statistically significant P values ( P < 0.05) are shown in bold and trends ( P < 0.1) are shown in italics.

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: RNA Sequencing, Mutagenesis

Journal: Clinical Cancer Research

Article Title: E7386 Enhances Lenvatinib’s Antitumor Activity in Preclinical Models and Human Hepatocellular Carcinoma

doi: 10.1158/1078-0432.CCR-25-0725

Figure Lengend Snippet: Proposed novel mechanism of action for E7386. E7386 treatment triggers ATF4 overexpression and ATF4-dependent ISR, along with downregulation of cell-cycle proteins, VEGFA upregulation, and secretion. Concomitant treatment of E7386 with lenvatinib reverses ATF4/VEGFA-related angiogenesis and potentiates antitumor responses. (Created with BioRender.com. Llovet, J. [2025] https://BioRender.com/io86bef .)

Article Snippet: Samples were incubated for 2 hours at room temperature with the anti-CD31 antibody from Abcam (ab28364; RRID: AB_726362; 1:50) or overnight with the anti-ATF4 antibody from Novus Biologicals (SD20-32; RRID: AB_2877169; 1:200) and the anti-CHOP antibody (15204-1-AP; RRID: AB_2292610; 1:200), the anti-CHAC1 antibody (15207-1-AP; RRID: AB_2878118; 1:500), the anti-REDD1 antibody (10638-1-AP; RRID: AB_2245711; 1:500), and the anti-geminin antibody (10802-1-AP; RRID: AB_2110945; 1:200) from Proteintech.

Techniques: Over Expression