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

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: Frontiers in Molecular Neuroscience

Article Title: Repeated Exposure to D -Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

doi: 10.3389/fnmol.2016.00165

Figure Lengend Snippet: Sequences of PCR primers.

Article Snippet: Primary antibodies against p-eIF2α (Ser51) (1:1000; Cell Signaling, #3398), eIF2α (1:1000; Cell Signaling, #5324), p-eEF2 (Thr56) (1:1000; Cell Signaling, #2331), p-p70S6K (Thr389) (1:1000; Cell Signaling, #9234), p-4EBP1 (Thr37/46) (1:500; Cell Signaling, #2855), 4EBP1 (1:500; Cell Signaling, #9644), OPHN1 (1:1000; Cell Signaling, #11939), ATF4 (1:1000; NeuroMab, #75-345), MAP2 (1:2000; Sigma, #M4403) from Sigma, CaMKIIa (1:1000; Millipore, #05-532), puromycin [1:1000; ( )], and β-actin (1:40000; Abcam, #AB6276) were used.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Repeated Exposure to D -Amphetamine Decreases Global Protein Synthesis and Regulates the Translation of a Subset of mRNAs in the Striatum

doi: 10.3389/fnmol.2016.00165

Figure Lengend Snippet: Repeated d-amphetamine administration increases the translation of a subset of uORF-containing mRNAs. (A,B,E,G) Relative mRNA expression levels of Ophn1 (A) , Atf4 (B) , Ppp1r15a (E) , and Ddit3 (G) in non-polysomal (NP) and polysomal (P) fractions analyzed by qRT-PCR in the striatum of mice chronically treated with D -amphetamine (10 mg/kg, once daily for 5 days) or saline ( n = 5 mice/group). All candidate mRNAs were normalized to β- actin or Gapdh mRNA and expressed as a percentage of saline control. (C,D) Representative western blot (top) and quantification (bottom) of OPHN1 (C) and ATF4 (D) (normalized to β-actin) expression levels in the striatum 60 or 120 min after the last injection of saline (sal) or D -amphetamine ( D -amph). Data are expressed as a percentage of saline control ( n = 5 mice/group). (F,H) Ratio of non-polysomal (NP) and polysomal (P) fractions of Ppp1r15a (F) and Ddit3 (H) mRNAs from the results represented in (E,G) , respectively. Results are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01 by unpaired Student t -test (saline versus D -amphetamine).

Article Snippet: Primary antibodies against p-eIF2α (Ser51) (1:1000; Cell Signaling, #3398), eIF2α (1:1000; Cell Signaling, #5324), p-eEF2 (Thr56) (1:1000; Cell Signaling, #2331), p-p70S6K (Thr389) (1:1000; Cell Signaling, #9234), p-4EBP1 (Thr37/46) (1:500; Cell Signaling, #2855), 4EBP1 (1:500; Cell Signaling, #9644), OPHN1 (1:1000; Cell Signaling, #11939), ATF4 (1:1000; NeuroMab, #75-345), MAP2 (1:2000; Sigma, #M4403) from Sigma, CaMKIIa (1:1000; Millipore, #05-532), puromycin [1:1000; ( )], and β-actin (1:40000; Abcam, #AB6276) were used.

Techniques: Expressing, Quantitative RT-PCR, Saline, Control, Western Blot, Injection

Journal: PLoS ONE

Article Title: Medial prefrontal area reductions, altered expressions of cholecystokinin, parvalbumin, and activating transcription factor 4 in the corticolimbic system, and altered emotional behavior in a progressive rat model of type 2 diabetes

doi: 10.1371/journal.pone.0256655

Figure Lengend Snippet: Representative immunofluorescent images of 8-week-old OLETF and LETO rats in the anterior cingulate cortex (ACC; A , B , C and D ), basolateral amygdala (BLA; E , F , G , and H ), and hippocampal cornu ammonis area 3 (CA3; I , J , K , and L . Labeling for activating transcription factor 4 (ATF4; A , E , and I ) and neuronal nuclei (NeuN; B , F , and G ) and merged images ( C , G , and K for OLETF rats; D , H , and L for LETO rats). Arrows or arrow heads indicate neurons expressing ATF4 and NeuN or only NeuN, respectively. Scale bars = 100 μm. Rad: radiatum layer; Py: pyramidal cell layer; Or: oriens layer. The percentages of neurons co-expressing ATF4 and NeuN in the ACC, prelimbic cortex (PL), infralimbic cortex (IL), lateral amygdala (LA), BLA, CA1, CA2, CA3, and dentate gyrus (DG; M ). ATF4+: ATF4-positive; NeuN+: NeuN-positive. Values represent mean ± standard error of means. * Significant difference from age-matched LETO rats, p < 0.05, two-way ANOVA.

Article Snippet: After rinsing, the sections were incubated simultaneously with mouse anti-ATF4 antibody (1:1000; Novus Biologicals) and rabbit anti-NeuN antibody (1:4000; Merck Millipore, Burlington, MA, USA), rabbit anti-CCK antibody (1:4000; Sigma), or rabbit anti-PV antibody (1:5000; Swant, Marly, Switzerland) at 4°C.

Techniques: Labeling, Expressing

Journal: PLoS ONE

Article Title: Medial prefrontal area reductions, altered expressions of cholecystokinin, parvalbumin, and activating transcription factor 4 in the corticolimbic system, and altered emotional behavior in a progressive rat model of type 2 diabetes

doi: 10.1371/journal.pone.0256655

Figure Lengend Snippet: Representative immunofluorescent images of 8-week-old OLETF and LETO rats in the anterior cingulate cortex (ACC; A , B , C , and D ), basolateral amygdala (BLA; E , F , G , and H ), and hippocampal cornu ammonis area 3 (CA3; I , J , K , and L ). Labeling for activating transcription factor 4 (ATF4; A , E , and I ) and cholecystokinin (CCK; B , F , and J ) and merged images ( C , G , and K for OLETF rats; D , H , and L for LETO rats). Arrows or arrow heads indicate neurons expressing ATF4 and CCK or only CCK, respectively. Scale bars = 100 μm. Rad: radiatum layer; Py: pyramidal cell layer; Or: oriens layer. The percentages of neurons co-expressing ATF4 and CCK in the ACC, prelimbic cortex (PL), infralimbic cortex (IL), lateral amygdala (LA), BLA, CA1, CA2, CA3, and dentate gyrus (DG; M ). ATF4+: ATF4-positive; CCK+: CCK-positive. Values represent mean ± standard error of means. * Significant difference from age-matched LETO rats, p < 0.05; † significant difference from same strain at 8 weeks of age, p < 0.01, two-way ANOVA.

Article Snippet: After rinsing, the sections were incubated simultaneously with mouse anti-ATF4 antibody (1:1000; Novus Biologicals) and rabbit anti-NeuN antibody (1:4000; Merck Millipore, Burlington, MA, USA), rabbit anti-CCK antibody (1:4000; Sigma), or rabbit anti-PV antibody (1:5000; Swant, Marly, Switzerland) at 4°C.

Techniques: Labeling, Expressing

Journal: PLoS ONE

Article Title: Medial prefrontal area reductions, altered expressions of cholecystokinin, parvalbumin, and activating transcription factor 4 in the corticolimbic system, and altered emotional behavior in a progressive rat model of type 2 diabetes

doi: 10.1371/journal.pone.0256655

Figure Lengend Snippet: Representative immunofluorescent images of 8-week-old OLETF and LETO rats in the prelimbic cortex (PL; A , B , C and D ), basolateral amygdala (BLA; E , F , G , and H ), and hippocampal cornu ammonis area 3 (CA3; I , J , K , and L ). Labeling for activating transcription factor 4 (ATF4; A , E , and I ) and parvalbumin (PV; B , F , and J ) and merged images ( C , G , and K for OLETF rats; D , H , and L for LETO rats). Arrows or arrow heads indicate neurons expressing ATF4 and PV or only PV, respectively. Scale bars = 100 μm. Rad: radiatum layer; Py: pyramidal cell layer; Or: oriens layer. The percentages of neurons co-expressing ATF4 and PV in the anterior cingulate cortex (ACC), PL, infralimbic cortex (IL), lateral amygdala (LA), BLA, CA1, CA2, CA3, and dentate gyrus (DG; M ). ATF4+: ATF4-positive; PV+: PV-positive. Values represent mean ± standard error of means. * Significant difference from age-matched LETO rats, p < 0.01; † significant difference from same strain at 8 weeks of age, p < 0.05, two-way ANOVA.

Article Snippet: After rinsing, the sections were incubated simultaneously with mouse anti-ATF4 antibody (1:1000; Novus Biologicals) and rabbit anti-NeuN antibody (1:4000; Merck Millipore, Burlington, MA, USA), rabbit anti-CCK antibody (1:4000; Sigma), or rabbit anti-PV antibody (1:5000; Swant, Marly, Switzerland) at 4°C.

Techniques: Labeling, Expressing

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