Journal: Nature communications

Article Title: Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells.

doi: 10.1038/s41467-018-04602-0

Figure Lengend Snippet: Fig. 7 MTHFD2 is deregulated in cardiovascular disease. a Glycine to serine ratio in plasma of human subjects with no atherosclerotic plaque (NP) (n = 26), stable atherosclerotic plaque (SP) (n = 26), and unstable atherosclerotic plaque (UP) (n = 26) as assessed by mass spectrometry. b Scatter plots showing expression correlation in 126 human carotid plaque samples between MTHFD2 and genes of the MTHFD2 network (colored according to Fig. 3a) as well as Nrf2 (NFE2L2) and ATF4 as calculated by Pearson correlation. c, d Relative mRNA expression of MTHFD2 and SHMT2 in plaque material of human subjects with unstable atherosclerotic plaque (UP) (n = 20), stable atherosclerotic plaque (SP) (n = 20), or non-atherosclerotic artery (NP) (n = 8) (normalized to 18SrRNA) (n = 8). e, f Western blot analysis of MTHFD2 expression (e) and quantification (f) of plaque material from human subjects with unstable atherosclerotic plaque (SP) (n = 20), stable atherosclerotic plaque (SP) (n = 20), or non-atherosclerotic artery (NP) (n = 8). g–j Relative mRNA expression of Mthfd2, Phgdh, Shmt2, and Slc3a2 in mouse thoracic aortic rings kept in organ culture and exposed to medium (1% FCS) with or without oxPAPC and rapamycin as indicated for 8 h (normalized to 18SrRNA) (n ≥4). k, l Relative mRNA expression of Mthfd2 and Shmt2 in the endothelium of partially ligated left carotid artery (LCA) compared to healthy right carotid artery (RCA) 48 h post ligation (normalized to 18S rRNA) (n = 3). (*p ≤0.05 Student’s t test). m Relative mRNA expression of Mthfd2 in the endothelium of the left carotid artery of ApoE−/−mice which were fed with high fat diet (HFD) for 0, 1, or 4 days (normalized to 18S rRNA) (n = 5). n–q Relative mRNA expression of MTHFD2, PHGDH, CEBPB and PCK2 in HAEC exposed to HDL from healthy human subjects (n = 10) or human subjects with CAD (n = 10) for 4 h. (*p ≤0.05 Mann Whitney test). r, s Western blot analysis of MTHFD2 (r) and quantification (s) of HAEC treated as in f for 24 h (n = 10) (*p ≤0.05 Mann Whitney test). Data are represented as mean ± SEM, *p ≤0.05 (ANOVA with Newman−Keuls post-hoc test if not otherwise indicated)

Article Snippet: Plasmid overexpression in endothelial cells was performed with the Neon electroporation system (Invitrogen) with FLAG-tagged ATF4 (gift from Yihong Ye, Addgene plasmid pRK-ATF4 #26114)64.

Techniques: Clinical Proteomics, Mass Spectrometry, Expressing, Western Blot, Organ Culture, Ligation, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression

doi: 10.1038/s41416-020-0872-0

Figure Lengend Snippet: a Ire1α and XBP1s expression in mock- and KSHV-infected macrophages was evaluated by western blot analysis; b ATF4, CHOP and BIP expression in mock- and KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of each protein/β-ACT. * p -value < 0.05. c PD-L1 expression on mock- and KSHV-infected macrophages was evaluated by FACS analysis. A representative experiment is shown, and the mean of fluorescence intensity is indicated. Grey peaks represent the isotype controls. d Histograms representing the mean plus SD of PD-L1 MFI (Mean fluorescence Intensity) are also reported. * p -value < 0.05.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against Kb-ZIP (Santa Cruz Biotechnology, sc-69797), pSTAT6 (1:100; Santa Cruz Biotechnology Inc., sc-136019), STAT6 (1:100; Santa Cruz Biotechnology Inc., sc-1689), mouse monoclonal anti-STAT3 (1:1000; BD Transduction Laboratories, 610189), mouse monoclonal anti-phospho-STAT3 (p-Tyr705, 1:100; Santa Cruz Biotechnology Inc., sc-8059), pSTAT1 (1:100; Santa Cruz Biotechnology Inc., sc-136229), STAT1 (1:100; Santa Cruz Biotechnology Inc., sc-464), mouse monoclonal anti-Ire1α (1:100; Santa Cruz Biotechnology, sc-390960), XBP1s (NovusBio NBP1-77681SS), ATF4 (R&D system, MAB7218), rabbit polyclonal anti-BIP (1:100; Cell Signaling, 3177), mouse monoclonal anti-CHOP (1:100; Santa Cruz Biotechnology, sc-7351), and anti-β-actin (1:10000; Sigma Aldrich, A5441).

Techniques: Expressing, Infection, Western Blot, Control, Fluorescence

Journal: British Journal of Cancer

Article Title: KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression

doi: 10.1038/s41416-020-0872-0

Figure Lengend Snippet: a IL-10, VEGF, IL-8, IL-6 and IFN-γ released by mock-, KSHV-infected macrophages and 4μ8c- (Ire1α inhibitor) or GSK 2606414 (GSK)- (PERK inhibitor) pre-treated KSHV-infected macrophages were measured by Luminex assay. Mean plus SD of three different experiments is reported. * p -value < 0.05; b and c PD-L1 expression on mock-, KSHV-infected macrophages and 4μ8c or GSK 2606414 (GSK)- pre-treated KSHV-infected macrophages was evaluated by FACS analysis. Grey peaks represent the isotype controls. Histograms representing the mean plus SD of PD-L1 MFI (Mean fluorescence Intensity) are reported. * p -value < 0.05 and a representative experiment is shown, and the mean of fluorescence intensity is indicated; d expression of K-bZIP in untreated or 4μ8c or GSK 2606414 (GSK)- pre-treated KSHV-infected macrophages; e ATF4 expression in mock-, KSHV-infected and GSK 2606414 (GSK)-pre-treated KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of each protein/ β-ACT. * p -value < 0.05.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against Kb-ZIP (Santa Cruz Biotechnology, sc-69797), pSTAT6 (1:100; Santa Cruz Biotechnology Inc., sc-136019), STAT6 (1:100; Santa Cruz Biotechnology Inc., sc-1689), mouse monoclonal anti-STAT3 (1:1000; BD Transduction Laboratories, 610189), mouse monoclonal anti-phospho-STAT3 (p-Tyr705, 1:100; Santa Cruz Biotechnology Inc., sc-8059), pSTAT1 (1:100; Santa Cruz Biotechnology Inc., sc-136229), STAT1 (1:100; Santa Cruz Biotechnology Inc., sc-464), mouse monoclonal anti-Ire1α (1:100; Santa Cruz Biotechnology, sc-390960), XBP1s (NovusBio NBP1-77681SS), ATF4 (R&D system, MAB7218), rabbit polyclonal anti-BIP (1:100; Cell Signaling, 3177), mouse monoclonal anti-CHOP (1:100; Santa Cruz Biotechnology, sc-7351), and anti-β-actin (1:10000; Sigma Aldrich, A5441).

Techniques: Infection, Luminex, Expressing, Fluorescence, Western Blot, Control

Journal: Frontiers in Endocrinology

Article Title: Metformin increases glycolysis and the stress-induced cytokine GDF15 but not FGF21 in humans

doi: 10.3389/fendo.2026.1797525

Figure Lengend Snippet: Metformin increases mRNA levels of GDF15 in human intestinal Caco-2 cells with no related increase in FGF21. Relative mRNA expression of GDF15, FGF21, SLC2A1 , and the ISR genes ATF4 and DDIT3 in differentiated Caco-2 cells after (a) 6 h or (b) 22 h without treatment (0 mM) or 0.3 mM, 1 mM, or 3 mM metformin treatment at media glucose concentrations of 5.5 mM (c) Raw ct values of GDF15 and FGF21 at 5.5 mM glucose. n = 6-8, 2 wells from 4 independent experiments evaluated in parallel. The data is presented as mean +/- SEM. **p<0.01, ***p<0.001, and ****p<0.0001 as indicated.

Article Snippet: The specific primer-probe pairs used were GDF15 (Hs00171132_m1, Life-Technologies, CA USA), FGF21 (Hs00173927_m1, Life-Technologies, CA USA) SLC2A1 (Hs00892681_m1, Life-Technologies, CA, USA), ATF4 (Hs00909569_g1, Life-Technologies, CA, USA), DDIT3 (Hs00358796_g1, Life-Technologies, CA USA), and GAPDH (Hs02786624_g1, Life-technologies, CA, USA).

Techniques: Expressing

Journal: Journal of Cancer Prevention

Article Title: Carnosic Acid Induces Apoptosis Through Reactive Oxygen Species-mediated Endoplasmic Reticulum Stress Induction in Human Renal Carcinoma Caki Cells

doi: 10.15430/JCP.2014.19.3.170

Figure Lengend Snippet: Carnosic acid-induced endoplasmic reticulum stress is involved in apoptosis. (A) Caki cells were treated with the indicated concentrations of carnosic acid for 12 hours. The protein expression levels of activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP), and actin were determined by Western blotting. The level of actin was used as a loading control. (B) Caki cells were treated with 40 μM carnosic acid for the indicated time periods. The protein expression levels of ATF4, CHOP, and actin were determined by Western blotting. The level of actin was used as a loading control. (C and D) Caki cells were transiently transfected with the ATF4 (C) or CHOP (D) small interfering RNA (siRNA), and then treated with 40 μM carnosic acid for 24 hours. The level of apoptosis was measured by the sub-diploid DNA content, sub-G1 fraction using flow cytometry (upper panel). The protein expression levels of ATF4, CHOP, cleaved poly (ADP-ribose) polymerase (PARP) and actin were determined by Western blotting. The level of actin was used as a loading control. (E) Caki cells were pretreated with 5 mM N-acetyl-L-cysteine (NAC) and 2 mM glutathione ethyl ester (GEE) for 30 minutes, and then treated with 40 μM carnosic acid for 24 hours. The protein expression levels of ATF4, CHOP and actin were determined by Western blotting. The actin was used as a loading control. The values in (C and D) represent the mean ± SD from three independent samples. * P < 0.01 compared to the carnosic acid-treated control siRNA (Cont siRNA).

Article Snippet: The ATF4 small interfering RNA (siRNA) duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Binding Assay, Western Blot, Control, Transfection, Small Interfering RNA, Flow Cytometry

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Left, image of immunostaining showing broad ATF4 expression in mouse DRG neurons. Middle, ATF4 expression was decreased in Atf4 +/− mice. Right, absence of ATF4 immunostaining upon treatment with a blocking peptide. Scale bar, 200 μm. b Size frequency distribution of ATF4-positive and total neurons in the mouse DRG. A total of 1489 neurons from n = 4 mice were analysed. c , d Colocalization of ATF4 and cell markers (IB4, CGRP and NF200) in the DRG ( c ) and sciatic nerve ( d ). Scale bar, 200 μm.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Immunostaining, Expressing, Blocking Assay

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – j The behaviours of ATF4 siRNA- and scrambled siRNA-injected mice were evaluated by the von Frey ( a ), dynamic ( b ), tape ( c ), tail clip ( d ), pinprick ( e ), Hargreaves ( f ), tail-flick ( g ), hot plate ( h ), evaporative cooling ( i ) and rotarod ( j ) tests. n = 12 mice per group in a – g , i . n = 12 mice in scrambled and n = 11 mice in ATF4 siRNA group in h . n = 10 mice per group in j . t 22 = 4.783, P < 0.0001 in f . t 22 = 5.163 , P < 0.0001 in 48 °C; t 22 = 5.661, P < 0.0001 in 50 °C; t 22 = 10.30, P < 0.0001 in 52 °C in g . t 21 = 2.357, P = 0.0282 in 52 °C; t 21 = 2.804, P = 0.0106 in 55 °C in h . k , l ATF4 siRNA abated SNL-induced thermal hyperalgesia ( k ) but not mechanical allodynia ( l ). n = 12 mice per group. F (1,22) = 56.9, P < 0.0001 in day 0, P = 0.0095 in day 4, P = 0.0013 in day 7, P = 0.0009 in day 10, P = 0.0089 in day 14 in k . m , n ATF4 siRNA abated CFA-induced heat hyperalgesia ( m ) but not mechanical allodynia ( n ). n = 8 mice per group. F (1,14) = 121.6, P < 0.0001 in day 0, P = 0.0007 in day 1, P < 0.0001 in day 3, P = 0.0098 in day 5 in m . o , p Spontaneous pain: total duration ( o ) or number ( p ) of nocifensive behaviour (paw licking or flinching within 2 min) in response to intraplantar injection of vehicle, PS (2.5 nmol/paw) or CIM0216 (2.5 nmol/paw) into ATF4 siRNA and scrambled mice. n = 10 mice per group. t 18 = 3.231, P = 0.0046 in PS, t 18 = 3.286, P = 0.0041 in CIM0216 in o . t 18 = 2.429, P = 0.0258 in PS, t 18 = 3.327, P = 0.0038 in CIM0216 in p . q Spontaneous pain: total duration of nocifensive behaviour (within 5 min) in response to intrathecal injection of vehicle, PS (1.25 nmol/paw) or CIM0216 (1.25 nmol/paw) into ATF4 siRNA and scrambled mice. n = 10 mice per group. t 18 = 3.281, P = 0.0042 in PS, t 18 = 3.024, P = 0.0073 in CIM0216. a – j , o – q Two - tailed Independent Student’s t test; k – n Two-way ANOVA followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, n.s. means not significant. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Injection, Tail Flick Test, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – j The behaviours of WT, Atf4 +/− and rescued mice were evaluated by the von Frey ( a ), dynamic ( b ), tape ( c ), tail clip ( d ), pinprick ( e ), Hargreaves ( f ), tail-flick ( g ), hot plate ( h ), evaporative cooling ( i ) and rotarod ( j ) tests. n = 10 mice in WT, n = 7 mice in Atf4 +/− and n = 6 mice in rescue group. F (2,20) = 12.11, P = 0.0004 in WT vs. Atf4 +/− , P = 0.0050 in Atf4 +/− vs. rescue in f . F (2,20) = 12.59, P = 0.0008 in WT vs. Atf4 +/− , P = 0.0009 in Atf4 +/− vs. rescue in 48 °C; F (2,20) = 20.67, P < 0.0001 in WT vs. Atf4 +/− , P = 0.0002 in Atf4 +/− vs. rescue in 50 °C; F (2,20) = 8.924, P = 0.0024 in WT vs. Atf4 +/− , P = 0.0076 in Atf4 +/− vs. rescue in 52 °C in g . F (2,20) = 7.097, P = 0.0159 in WT vs. Atf4 +/− , P = 0.0066 in Atf4 +/− vs. rescue in 50 °C; F (2,20) = 5.678, P = 0.0135 in WT vs. Atf4 +/− , P = 0.0375 in Atf4 +/− vs. rescue in 52 °C; F (2,20) = 21.31, P < 0.0001 in WT vs. Atf4 +/− , P < 0.0001 in Atf4 +/− vs. rescue in 55 °C in h . k , l Knockout and rescue of ATF4 impaired and rescued SNL-induced heat hyperalgesia ( k ) but not mechanical allodynia ( l ), respectively. n = 10 mice in WT, n = 9 mice in Atf4 +/− and n = 9 mice in rescue group. F (2,25) = 36; P = 0.0038 in WT vs. Atf4 +/− , P = 0.0098 in Atf4 +/− vs. rescue in day 0; P = 0.0002 in WT vs. Atf4 +/− , P < 0.0001 in Atf4 +/− vs. rescue in day 4; P = 0.0145 in WT vs. Atf4 +/− , P = 0.0170 in Atf4 +/− vs. rescue in day 7; P = 0.0105 in WT vs. Atf4 +/− , P = 0.0034 in Atf4 +/− vs. rescue in day 10; P = 0.0174 in WT vs. Atf4 +/− , P = 0.0087 in Atf4 +/− vs. rescue in day 14 in k . F (2,25) = 1.112; P = 0.0171 in WT vs. Atf4 +/− , P = 0.0074 in Atf4 +/− vs. rescue in day 14 in l . m , n Knockout and rescue of ATF4 impaired and rescued CFA-induced heat hyperalgesia ( m ) but not mechanical allodynia ( n ), respectively. n = 10 mice in WT, n = 9 mice in Atf4 +/− and n = 9 mice in rescue group. F (2,25) = 108.5; P = 0.0002 in WT vs. Atf4 +/− , P = 0.0005 in Atf4 +/− vs. rescue in day 0; P = 0.0045 in WT vs. Atf4 +/− , P = 0.0086 in Atf4 +/− vs. rescue in day 1; P = 0.0001 in WT vs. Atf4 +/− , P = 0.0017 in Atf4 +/− vs. rescue in day 3; P = 0.0006 in WT vs. Atf4 +/− , P < 0.0001 in Atf4 +/− vs. rescue in day 5. o , p Spontaneous pain: total duration ( o ) or number ( p ) of nocifensive behaviour (paw licking or flinching within 2 min) in response to intraplantar injection of vehicle, PS (2.5 nmol/paw) or CIM0216 (2.5 nmol/paw) into WT, Atf4 +/− and rescued mice. n = 10 mice per group. F (2,27) = 6.375, P = 0.0368 in WT vs. Atf4 +/− , P = 0.0056 in Atf4 +/− vs. rescue in PS; F (2,27) = 9.174 , P = 0.0027 in WT vs. Atf4 +/− , P = 0.0026 in Atf4 +/− vs. rescue in CIM0216 in o . F (2,27 ) = 5.976, P = 0.0201 in WT vs. Atf4 +/− , P = 0.0123 in Atf4 +/− vs. rescue in PS; F (2,27) = 7.569 , P = 0.0033 in WT vs. Atf4 +/− , P = 0.0138 in Atf4 +/− vs. rescue in CIM0216 in p . q Spontaneous pain: total duration of nocifensive behaviour (in 5 min) in response to intrathecal injection of vehicle, PS (1.25 nmol/paw) or CIM0216 (1.25 nmol/paw) into WT, Atf4 +/− and rescued mice. n = 10 mice per group. F (2,27) = 5.849, P = 0.0194 in WT vs. Atf4 +/− , P = 0.0146 in Atf4 +/− vs. rescue in PS; F (2,27) = 5.986 , P = 0.0127 in WT vs. Atf4 +/− , P = 0.0192 in Atf4 +/− vs. rescue in CIM0216. a – j , o – q , One-way ANOVA followed by Tukey’s multiple comparisons test. k – n Two-way ANOVA followed by Bonferroni’s multiple comparisons test. * P < 0.05, ** P < 0.01, n.s. means not significant. The errors bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Tail Flick Test, Knock-Out, Injection

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – j The behaviours of ATF4-overexpressing and control mice were evaluated by the von Frey ( a ), dynamic ( b ), tape ( c ), tail clip ( d ), pinprick ( e ), Hargreaves ( f ), tail-flick ( g ), hot plate ( h ), evaporative cooling ( i ) and rotarod ( j ) tests. n = 12 mice per group in a – e , g , h , j . n = 10 mice per group in f , i . t 18 = 6.308, P < 0.0001 in f . t 22 = 6.893 , P < 0.0001 in 48 °C; t 22 = 8.009, P < 0.0001 in 50 °C; t 22 = 6.831, P < 0.0001 in 52 °C in g . t 22 = 2.373, P = 0.0268 in 50 °C; t 22 = 2.245, P = 0.0352 in 52 °C; t 22 = 2.396, P = 0.0255 in 55 °C in h . k , l Spontaneous pain: total duration ( k ) or number ( l ) of nocifensive behaviour (paw licking or f l inching within 2 min) in response to intraplantar injection of vehicle, PS (2.5 nmol/paw) or CIM0216 (2.5 nmol/paw) into control and ATF4-overexpressing mice. n = 10 mice per group. t 18 = 2.411, P = 0.0268 in PS; t 18 = 2.795, P = 0.0120 in CIM0216 in k . t 18 = 2.775, P = 0.0125 in PS; t 18 = 2.334, P = 0.0314 in CIM0216 in l . m Spontaneous pain: total duration of nocifensive behaviour (in 5 min) in response to intrathecal injection of vehicle, PS (1.25 nmol/paw) or CIM0216 (1.25 nmol/paw) into control and ATF4-overexpressing mice. n = 10 mice per group. t 18 = 2.506, P = 0.0220 in PS; t 18 = 2.46, P = 0.0242 in CIM0216. a – m , Two -t ailed Independent Student’s t test, * P < 0.05, ** P < 0.01, n.s. means not significant. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Control, Tail Flick Test, Injection

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – c TRPM3 expression in the DRG membrane fraction ( a ), cytoplasmic fraction ( b ) and total lysate ( c ) from WT and Atf4 +/− mice. n = 3 mice per group. t 4 = 10.3, P = 0.0005 in a . t 4 = 6.043, P = 0.0038 in b . d TRPM3 surface levels were measured in cultured DRG neurons prepared from WT and Atf4 +/− mice using a surface biotinylation assay. n = 3 cultures per group. t 4 = 6.28, P = 0.0033. e Co-IP showing the ATF4/TRPM3 interaction in DRGs. DRG lysates were immunoprecipitated with an ATF4 (top) or TRPM3 (bottom) antibody and immunoblotted with a TRPM3 or ATF4 antibody as indicated. This experiment was repeated three times. f The SIM images show that the colocalization between ATF4 and TRPM3 in DRG neurons. Scale bar, 5 μm. g The GST pull-down assay with two purified proteins, TRPM3-GST-Flag and ATF4, showed a direct interaction between ATF4 and TRPM3. This experiment was repeated three times. h Interaction between TRPM3 and ATF4 mutants. The ATF4 mutants was transiently co-expressed with TRPM3, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. i – k TRPM3 expression in the DRG membrane fraction ( i ), cytoplasmic fraction ( j ) and total lysate ( k ) after ATF4 overexpression. n = 6 mice per group. F (2,15) = 14.31, P = 0.0007 in i . F (2,15) = 30.15, P < 0.0001 in j . l , n , p Time course of a whole-cell patch-clamp recording showing the effect of CIM0216 (3 μM) and isosakuranetin (Iso; 10 μM) on the TRPM3 current in DRG neurons from WT ( l ), Atf4 +/− ( n ) and rescued ( p ) mice. The open circle indicates the outward current recorded at + 75 mV, and the closed circle indicates the inward current recorded at −75 mV. m , o , q I–V relationship of the TRPM3 current at the time points indicated in I , n and p . r Histogram of the TRPM3 current density (outward and inward, current before CIM0216 was subtracted) of DRG neurons from WT, Atf4 +/− and ATF4 rescued mice after CIM0216 treatment. n = 6 neurons per group. F (2,15) = 8.735, P = 0.0049 in WT vs. Atf4 +/− , P = 0.0097 in Atf4 +/− vs. rescue in +75 mV; F (2,15) = 9.158, P = 0.0038 in WT vs. Atf4 +/− , P = 0.0089 in Atf4 +/− vs. rescue in −75 mV. a – d Two-tailed Independent Student’s t test. i , j , k , r , One-way ANOVA followed by Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, n.s. means not significant. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Expressing, Membrane, Cell Culture, Surface Biotinylation Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Over Expression, Patch Clamp, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a The diagram shows the experimental procedure. The hindpaws of mice were soaked in a 43 °C water bath for 30 s, and the mice were placed at RT for 2, 5, 10, 20 or 40 min. Then, behavioural, co-IP and membrane protein immunoblotting experiments were performed. b , c ATF4/TRPM3 interactions ( b ) and TRPM3 membrane expression ( c ) in mice were evaluated at different time points after heat stimulation. The experiment was repeated four times in b . n = 6 mice per group in c . F (5,18) = 20.17, P = 0.0007 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0044 in 20 min in b . F (5,30) = 10.59, P = 0.0277 in 2 min, P = 0.0022 in 5 min, P < 0.0001 in 10 min, P = 0.0027 in 20 min in c . * P < 0.05, ** P < 0.01 versus the RT group. d The diagram shows the experimental procedure. Isolate d DRG neurons were placed in a 43 °C water bath for 30 s and placed at RT for 2, 5, 10, 20 or 40 min. The neurons were then lysed to detect the membrane abundance of TRPM3. e TRPM3 membrane expression in isolated DRG neurons was evaluated at different time points after direct heat stimulation. n = 3. F (5,12) = 12.3, P = 0.0358 in 2 min, P = 0.0100 in 5 min, P < 0.0001 in 10 min, P = 0.0085 in 20 min. * P < 0.05, ** P < 0.01 versus the RT group. f – h Naïve ( f ), ATF4 siRNA-injected ( g ) and Atf4 +/− ( h ) mice were subjected to the Hargreaves test at different time points after heat stimulation. n = 12 mice per group in f , g . n = 6 mice per group in h . F (5,66) = 14.36, P = 0.0004 in 2 min, P < 0.0001 in 5 min, P < 0.0001 in 10 min, P = 0.0057 in 20 min. ** P < 0.01 versus RT group. One-way ANOVA followed by Tukey’s multiple comparisons test. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Co-Immunoprecipitation Assay, Membrane, Western Blot, Expressing, Isolation, Injection

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a – f Co-IP showing the ATF4/KIF interaction in the DRG. DRG lysates were immunoprecipitated with an ATF4 antibody and immunoblotted with a KIF17, KIF3A, KIF3B, KIF5A, KIF5B, KIFC2 or ATF4 antibody as indicated. This experiment was repeated three times. g SIM images show that the colocalization between ATF4 and KIF17 in DRG neurons. Scale bar, 5 μm. h The GST pull-down assay with two purified proteins, KIF17-GST-Flag and ATF4, showed a direct interaction between ATF4 and KIF17. This experiment was repeated three times. i Interaction between KIF17 and ATF4 mutants. The ATF4 mutants were transiently co-expressed with KIF17, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. j The interaction level between TRPM3 and KIF17 was examined by co-IP in scrambled and ATF4 siRNA-treated mice DRG lysates. DRG lysates were immunoprecipitated with a TRPM3 antibody and immunoblotted with a KIF17 or TRPM3 antibody as indicated. This experiment was repeated three times. t 4 = 12.88, P = 0.0002. k SIM images showed the colocalization between TRPM3 and KIF17 in DRG neurons from naïve, ATF4 siRNA and ATF4-overexpresing mice. Quantification data showed the colocalization rates of KIF17 with TRPM3 (colocalized yellow spots/total KIF17 positive spots) and those of TRPM3 with KIF17 (colocalized yellow spots/total TRPM3 positive spots) in DRG neurons. n = 3 mice per group. F (2,6) = 27.17, P = 0.0224 in naïve vs. ATF4-siRNA, P = 0.0255 in naïve vs. ATF4-over in KIF17 with TRPM3. F (2,6) = 33.5, P = 0.0069 in naïve vs. ATF4-siRNA, P = 0.0375 in naïve vs. ATF4-over in TRPM3 with KIF17. Scale bar, 10 μm. j Two-tailed Independent Student’s t test. k One-way ANOVA followed by Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Colocalization of the KIF17, ATF4 and TRPM3 proteins in mouse DRG sections. Scale bar, 200 μm. b Colocalization of KIF17 mRNA , ATF4 mRNA and TRPM3 mRNA in mouse DRG sections. Scale bar, 20 μm. c Colocalization of the KIF17, ATF4 and TRPM3 proteins in DRG neurons was detected by SIM. Scale bar, 5 μm. d , e Changes in the membrane expression of TRPM3 in the DRG after KIF17 knockdown ( d ) or KIF17 overexpression ( e ). n = 6 mice per group. F (2,15) = 34.36, P = 0.0002 in d . F (2,15) = 20.48, P = 0.0005 in e . f The behaviours of KIF17 siRNA- and scrambled siRNA-injected mice were evaluated by the Hargreaves test. n = 6 mice per group. t 10 = 3.485, P = 0.0059. g The behaviours of KIF17-overexpressing and control mice were evaluated by the Hargreaves test. n = 6 mice per group. t 10 = 6.776, P < 0.0001. h ATF4 knockdown suppressed the increased expression of TRPM3 in the membrane induced by KIF17 overexpression. n = 6 mice per group. F (2,15) = 16.19, P = 0.0002 in naïve vs. KIF17-over, P = 0.0014 in KIF17-over vs. KIF17-over + ATF4-siRNA. i KIF17 knockdown inhibited the increase in TRPM3 expression on the cell surface induced by ATF4 overexpression. n = 6 mice per group. F (2,15) = 12.76, P = 0.0025 in naïve vs. ATF4-over, P = 0.0010 in ATF4-over vs. ATF4-over + KIF17-siRNA. ** P < 0.01. d , e , h , i One-way ANOVA followed by Tukey’s multiple comparisons test. f , g Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Membrane, Expressing, Knockdown, Over Expression, Injection, Control, Two Tailed Test

Journal: Nature Communications

Article Title: ATF4 selectively regulates heat nociception and contributes to kinesin-mediated TRPM3 trafficking

doi: 10.1038/s41467-021-21731-1

Figure Lengend Snippet: a Cultured DRG neurons were treated with CXCL12 (1 μg/mL) for 120, 240 or 360 min, and then ATF4 expression was measured. n = 6. F (3,20) = 4.642, P = 0.0462 in 120 min, P = 0.0154 in 240 min, P = 0.0128 in 360 min. * P < 0.05 versus 0 min. b CXCL12 (1 μg in 10 μL PBS + 0.5% BSA) was intrathecally injected three times every 3 h, and ATF4 expression was measured in the DRG 2 h after the last injection. n = 6 mice per group. t 10 = 5.151 , P = 0.0004. ** P < 0.01. c ATF4 siRNA signifi c antly relieved CXCL12-induced thermal hyperalgesia. n = 12 mice per group. F (2,33) = 15.68, P < 0.0001 in vehicle vs. CXCL12, P = 0.0101 in CXCL12 vs. CXCL12 + ATF4-siRNA. * P < 0.05, ** P < 0.01. d CXCR4 siRNA but not CXCR7 siRNA abolished the increase in ATF4 expression in the DRG induced by CXCL12 in vivo. n = 6 mice per group. F (3,20) = 54.47, P < 0.0001 in CXCL12, CXCL12 + CXCR4-siRNA and CXCL12 + CXCR7-siRNA. ** P < 0.01 versus the vehicle group, ## P < 0.01 versus the CXCL12 group. e Hypothetical model illustrating that ATF4 interacts with TRPM3 and KIF17 to form a complex to regulate the membrane trafficking of TRPM3 in sensory neurons and thus contributes to thermal sensitivity. a , c , d One-way ANOVA followed by Tukey’s multiple comparisons test. b Two-tailed Independent Student’s t test. The error bars indicate the SEMs.

Article Snippet: Atf4 +/− and Trpm3 −/− mice on the C57BL/6 background were purchased from Cyagen Biosciences Inc. All animals were housed in separate cages in a temperature-controlled (24 ± 1 °C) and humidity-controlled (50-60%) room under a 12/12-h light/dark cycle.

Techniques: Cell Culture, Expressing, Injection, In Vivo, Membrane, Two Tailed Test

Journal: Aging Cell

Article Title: The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration

doi: 10.1111/acel.14165

Figure Lengend Snippet: Single‐cell RNA‐Seq analysis identifies Atf4 stress response pathway downstream of mitochondrial dysfunction. (a) Experimental setup. (b) UMAP scatterplot showing the distribution of CT and Opa1 cKO‐derived cells clusters. (c) Heatmap of cluster‐specific and genotype‐specific differential gene expression. (d) UMAP of individual genes that are differentially regulated in all the clusters highlighting Atf4 pathway. (e) Violin plots representing mitochondrial gene expression, stress response genes, and differentiation genes in each cluster split by sample. (f) RNA velocity analysis shows the differentiation direction shown by the vectors separated by sample. (g) Panels of magnified views of the transition between cluster 1 (activated NSCs) and cluster 2 (Differentiating NSCs). (h) The proportion of spliced and unspliced RNA in all the clusters split by sample.

Article Snippet: ATF4 overexpression from Origene (MR205957) was cloned into the expression vector to generate pLVX‐E2F1a‐Atf4‐Myc‐DDK‐V5‐IRES‐mCherry.

Techniques: RNA Sequencing, Derivative Assay, Gene Expression

Journal: Aging Cell

Article Title: The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration

doi: 10.1111/acel.14165

Figure Lengend Snippet: ATF4 pathway is activated by mitochondrial dysfunction and reductive metabolism under hypoxia resolves ATF4 activation. (a) Western blot of the ISR pathway‐related proteins in E12.5 embryonic cortex of CT and Opa1 KO post Opa1 deletion as mentioned in the plot. (b) Cellular oxygen consumption rate was measured using XF24 extracellular flux analyzer. Bar graphs represent the cellular respiration of basal, maximal, reserved, and ATP‐linked respiration between the listed conditions. n = 3 animals. (c) Normalized mean intensity of MitoSOX Red was calculated from live cells and plotted as bar graph. (d) Phase contrast images of neurospheres from Adult NSCs of CT and Opa1cKO animals in listed Oxygen exposure conditions. (e) Bar graph representing the average number of primary neurospheres formed in CT and Opa1 cKO neurospheres growing under normoxic and hypoxic conditions. n = 3 biological replicates; data are presented as mean ± SD (** p < 0.01, and *** p < 0.001, one‐way ANOVA). (f) Diameter size (in μm) of CT and Opa1 cKO neurospheres grown in hypoxic and normoxic conditions. 120–130 neurospheres measured with n = 3 biological replicates; data are presented as mean ± SD (**** p < 0.0001, One‐way ANOVA). (g) RT‐qPCR results of stress response genes under hypoxic and normoxic conditions. n = 3 animals; data are presented as mean ± SD (** p < 0.01, One‐way ANOVA) (h) Representative western blot image from total protein lysates of embryonic neurospheres (E12.5) treated with LV‐shCtrl or shOpa1 and grown in normoxic and hypoxic conditions. (i) Western blot quantification of ATF4, cl‐Cas3, cyclin A, and Ascl1 in CT and Opa1 KO. Mean intensity was normalized to wild type in the bar graph. n = 5 animals; data are presented as mean ± SD (* p < 0.05, ** < 0.01 and *** < 0.001 One‐way ANOVA). CT, control transgenic; OPA1 cKO, OPA1 conditional knockout; shCtrl, Lentivirus vectors shRNA scramble control; shOPA1, Lentivirus vectors shRNA to OPA1.

Article Snippet: ATF4 overexpression from Origene (MR205957) was cloned into the expression vector to generate pLVX‐E2F1a‐Atf4‐Myc‐DDK‐V5‐IRES‐mCherry.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Control, Transgenic Assay, Knock-Out, shRNA

Journal: Aging Cell

Article Title: The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration

doi: 10.1111/acel.14165

Figure Lengend Snippet: ATF4 function is required for cell proliferation and survival in normal and stressed state. (a) Representative images of EdU and DAPI staining in above mentioned conditions. Cell proliferation is measured using EdU+ cells normalized to total DAPI. (b) Quantification of the percent EdU+ over total DAPI+ cells represented in a bar graph. (c) Representative images of cleaved Caspase 3 (cl‐Cas‐3) and DAPI staining in abovementioned conditions. Cell death is measured using cl‐Cas3+ cells normalized to total DAPI. (d) Quantification of percent cleaved caspase 3+ over total DAPI+ cells represented in bar graph. n = 5–6 biological replicates; Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001, One‐way ANOVA). (e) RT‐qPCR analysis in mentioned conditions for ATF4 targets involved in amino acid transport, tRNA aminoacylation, export, and one‐carbon metabolism. n = 3–6 animals; Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, One‐way ANOVA). (f) Schematics indicating the binding of ATF4 in mouse embryonic fibroblasts as identified through ChIP on Chac1, Slc3a2, and Slc7a11 genes (Han et al., ). (g) ATF4 ChIP from shCtrl and shOpa1 KD neurosphere. (h) H3K4me3 ChIP from shCtrl and shOpa1 KD neurosphere. n = 3–6 animals; data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, and *** p < 0.001, 2‐tailed Student's t test). ATF4 OE, ATF4 overexpression vector; scrmch, Lentivirus vectors shRNA scramble mCherry; shATF4, Lentivirus vectors shRNA to ATF4; shCtrl, Lentivirus vectors shRNA scramble control; shOPA1, Lentivirus vectors shRNA to OPA1.

Article Snippet: ATF4 overexpression from Origene (MR205957) was cloned into the expression vector to generate pLVX‐E2F1a‐Atf4‐Myc‐DDK‐V5‐IRES‐mCherry.

Techniques: Staining, Quantitative RT-PCR, Binding Assay, Over Expression, Plasmid Preparation, shRNA, Control

Journal: Aging Cell

Article Title: The integrated stress response promotes neural stem cell survival under conditions of mitochondrial dysfunction in neurodegeneration

doi: 10.1111/acel.14165

Figure Lengend Snippet: Slc7a11, a key target of ATF4, and glutathione redox are required for NSC function and survival. (a–d) Histological analysis of phospho‐Histone 3(Proliferation), Ascl1(Activation), Tbr2(TAP), and Dcx(Newborn neurons) in 3 months and 6 months old wild‐type and sut/sut adult mice. n = 4–5 animals; data are presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, Student's t test). (e) In vitro monolayer culture of WT and Sut mice infected with scramble and shOpa1. Bar graph for percent EdU+ over total DAPI+ cells and (f) Cleaved caspase 3+ over total DAPI+ cells. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, Student's t test). (g, h) Glutathione measurement using HPLC of GSH:GSSG ratio and total GSH levels in embryonic neurospheres in mentioned conditions, n = 3 animals; (i) Quantification of percent EdU+ over total DAPI+ cells represented in bar graph for the mentioned conditions. (j) Quantification of percent cleaved caspase 3+ over total DAPI+ cells represented in bar graph. n = 3–4 animals; data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, one‐way ANOVA).

Article Snippet: ATF4 overexpression from Origene (MR205957) was cloned into the expression vector to generate pLVX‐E2F1a‐Atf4‐Myc‐DDK‐V5‐IRES‐mCherry.

Techniques: Activation Assay, In Vitro, Infection

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