Journal: BMC Biology

Article Title: Transcriptomic signatures of oxytosis/ferroptosis are enriched in Alzheimer’s disease

doi: 10.1186/s12915-025-02235-6

Figure Lengend Snippet: Mechanistic validation of the TrioSig gene set in cell culture. A Diagram of the ISR and NRF2 pathways. B Levels of peIF2α, total eIF2α, NRF2, ATF4, and actin after treatment of HT22 cells with 5 mM glutamate, 500 nM erastin, and 250 nM RSL3 at various timepoints were assessed by western blotting and quantified ( n = 3–4/condition). C Levels of NRF2, ATF4, and actin after knockdown with control (Ct), Nrf2, or Atf4 siRNAs ( n = 7/condition). D Survival of HT22 cells exposed to varying concentrations of glutamate, erastin, or RSL3 after knockdown with Ct, Nrf2, or Atf4 siRNAs ( n = 4/condition). E Levels of NRF2, ATF4, and actin in MC65 cells without (− Aβ) and with (+ Aβ) intracellular Aβ aggregation after 1 day (d1) and 2 days (d2), assessed by western blotting and quantified ( n = 3/condition). F Levels of NRF2, ATF4, and actin after knockdown with control (Ct), Nrf2, or Atf4 siRNAs ( n = 3/condition). G Survival of MC65 cells exposed to Aβ toxicity after knockdown with Ct, Nrf2, or Atf4 siRNAs 3 days later ( n = 4–5/condition). Two-way repeated measures ANOVA and Tukey’s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All data are mean ± SD

Article Snippet: Twenty-four hours after seeding, cells were transfected with siRNA against Atf4 (Santa Cruz, sc-351113, 17 pmol) or Nrf2 (Santa Cruz, sc-37049, 17 pmol), using lipofectamine (RNAiMAX, ThermoFisher) as the transfection reagent and Opti-MEM (GibcoTM) as the transfection medium.

Techniques: Biomarker Discovery, Cell Culture, Western Blot, Knockdown, Control

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: Uninfected NOKs (NOKs (-)), WT EBV-infected NOKs (WT-EBV) or ΔLMP1 EBV-infected NOKs (ΔLMP1) were grown in the absence of growth factors at a sub-confluent density for two days and immunoblot analyses were performed. (A) Expression levels of LMP1, p-GCN2, GCN2, and Actin are shown. (B) Expression levels of LMP1, p-PERK, PERK, and Actin are shown. (C) Expression levels of LMP1, p-PKR, PKR, and Actin are shown. The levels of phosphorylated protein in each condition were quantitated using image J and normalized to the level in uninfected NOKs (set as 1). The same extracts were used in the immunoblots in ( B ) and ( C ) and probed with different antibodies. The LMP1 and Actin blots used in (B) and (C) are also same.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Infection, Western Blot, Expressing

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: WT EBV-infected NOKs (WT-EBV) or ΔLMP1-infected NOKs (ΔLMP1), grown in the absence of growth factors at sub-confluent density for two days, were transfected with control siRNA or siRNAs targeted against PERK or GCN2, alone or in combination, as indicated and immunoblots performed. Expression levels of LMP1, PERK, GCN2, ATF4, and CHOP are shown. Tubulin served as a loading control.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

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

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: WT EBV-infected NOKs (WT-EBV) or ΔLMP1-infected NOKs (ΔLMP1), grown in the absence of growth factors at sub-confluent density for two days, were transfected with control siRNA or siRNAs targeted against PERK or GCN2, alone or in combination, as indicated and immunoblots performed. Expression levels of LMP1, PERK, GCN2, and of proteins induced by differentiation of keratinocytes (IRF6, KLF4, BLIMP1, Keratin-10, Involucrin, TGM1, and SPRR1A) are shown. Tubulin served as a loading control. The extracts used in Figure 6 immunoblots are the same as those used in . The LMP1, PERK, and GCN2 blots are the same as those used in .

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

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

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: WT EBV-infected NOKs cells were transfected with control siRNA or siRNAs against GCN2 as indicated, and then treated with TPA for 24 hours starting one day after siRNA transfection. (A) Immunoblot analysis was performed to examine the effect of GCN2 knock-down on proteins involved in the ISR pathway (including p-GCN2, GCN2, p-eIF2α, eiF2α, ATF4, and CHOP) and EBV lytic proteins (BZLF1, BRLF1, BMRF1, and p18 VCA). (B) Immunoblot analyses were performed to examine the effect of GCN2 knock-down on differentiation-induced cellular proteins Involucrin, BLIMP1, and TGM1. Tubulin served as a loading control for both panels. The same extracts were used for each panel, and the same GCN2, p-GCN2, and Tubulin blots were used for both panels.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Infection, Transfection, Control, Western Blot, Knockdown

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: ATF4 target genes activated in ΔLMP1 EBV-infected NOKs. Selected genes of interest that are upregulated in ΔLMP1 EBV-infected NOKs (ΔLMP1) compared to WT EBV-infected NOKs (WT-EBV) in the RNA-seq results are shown, along with the fold-increase in gene expression and the adjusted p value.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Gene Expression, Significance Assay

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: (A) Uninfected NOKs (NOKs (-)), NOKs infected with EBV (WT-EBV), or NOKs infected with ΔLMP1 EBV (ΔLMP1) were plated in the absence of growth factors (EGF and BPE) in KSFM for two days at a sub-confluent density, and then harvested to perform immunoblot analysis. Expression levels of LMP1, p-eIF2α, eIF2α, ATF4, and CHOP are shown. Actin served as a loading control. (B) ΔLMP1 EBV-infected NOKs infected with a LMP1-expressing lentivirus or vector control were grown in the absence of growth factors for two days at a sub-confluent density and then immunoblot analysis was performed to examine expression levels of LMP1, p-eIF2α, eIF2α, ATF4, and CHOP as shown. Tubulin served as a loading control. (C) Comparison of expression levels of LMP1 between WT-EBV and LMP1-overexpressing ΔLMP1 EBV-infected NOKs. Tubulin served as a loading control.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Infection, Western Blot, Expressing, Control, Plasmid Preparation, Comparison

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: Uninfected NOKs (NOKs (-)), NOKs infected with EBV (WT-EBV), or NOKs infected with ΔLMP1 EBV (ΔLMP1) were plated in the presence or absence of growth factors (EGF and BPE) in KSFM for two days at a sub-confluent or confluent density, as indicated, and then harvested to perform immunoblot analysis. Expression levels of LMP1, ATF4, CHOP, Keratin-10, and the EBV immediate-early lytic protein, BZLF1, are shown. Tubulin served as a loading control.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Infection, Western Blot, Expressing, Control

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: WT EBV-infected NOKs (WT-EBV) or ΔLMP1-infected NOKs (ΔLMP1), grown in the absence of growth factors at sub-confluent density for two days, were transfected with control siRNA or siRNAs targeted against PERK or GCN2, alone or in combination, as indicated and immunoblots performed. Expression levels of LMP1, PERK, GCN2, ATF4, and CHOP are shown. Tubulin served as a loading control.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

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

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: WT EBV-infected NOKs cells were transfected with control siRNA or siRNAs against GCN2 as indicated, and then treated with TPA for 24 hours starting one day after siRNA transfection. (A) Immunoblot analysis was performed to examine the effect of GCN2 knock-down on proteins involved in the ISR pathway (including p-GCN2, GCN2, p-eIF2α, eiF2α, ATF4, and CHOP) and EBV lytic proteins (BZLF1, BRLF1, BMRF1, and p18 VCA). (B) Immunoblot analyses were performed to examine the effect of GCN2 knock-down on differentiation-induced cellular proteins Involucrin, BLIMP1, and TGM1. Tubulin served as a loading control for both panels. The same extracts were used for each panel, and the same GCN2, p-GCN2, and Tubulin blots were used for both panels.

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Infection, Transfection, Control, Western Blot, Knockdown

Journal: PLOS Pathogens

Article Title: Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation

doi: 10.1371/journal.ppat.1012934

Figure Lengend Snippet: WT EBV-infected NOKs cells were transfected with control siRNA or siRNAs against ATF4 (A and C) or CHOP (B and D) as indicated, and then treated with TPA for 24 hours starting one day after siRNA transfection. (A and B) Immunoblot analyses were performed to examine the effect of ATF4 and CHOP knock-down on lytic EBV proteins BZLF1, BRLF1, BMRF1 and p18 VCA as indicated. ( C and D) Immunoblot analyses were performed to examine the effect of ATF4 and CHOP knock-down on TPA-induced differentiation markers BLIMP1, TGM1, and SPRR1A as indicated. Tubulin served as a loading control. The cellular extracts used in (A) and (C) or (B) and (D) were the same. The same ATF4 and Tubulin blots were used in (A) and (C), and the same CHOP and Tubulin blots were used for (B) and (D) .

Article Snippet: siRNAs against KLF4 (sc-35437), BLIMP1 (sc-37714), PERK (sc-36213), GCN2 (sc-45644), ATF4 (sc-35112), and control siRNAs A and C (sc-37007, sc-44230) were purchased from Santa Cruz.

Techniques: Infection, Transfection, Control, Western Blot, Knockdown