Journal: Journal of Biological Chemistry

Article Title: Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells

doi: 10.1074/jbc.m109.014092

Figure Lengend Snippet: FIGURE 1. Fenretinide and bortezomib regulate eIF2 signaling in neuroectodermal tumor cells. Shown are Western blots for eIF2, phospho-eIF2, ATF4 (indicated by the asterisk), ATF3, Noxa, and -actin in SH-SY5Y, A375, and SK-MEL-28 cells treated with fenretinide (SH-SY5Y, 5 M; and A375/SK-MEL-28, 10 M), bortezomib (SH-SY5Y, 5 nM; and A375/SK-MEL-28, 30 nM), or thapsigargin (SH-SY5Y, 1.5 M; and A375/SK-MEL- 28, 7.5 M) for 0–24 h.

Article Snippet: Blots were probedwith antibodies to ATF4 (Calbiochem); ATF3 and GADD153 (B-3, Santa Cruz Biotechnology); Noxa (Alexis Biochemicals); c-Myc, eIF2 , phospho-eIF2 , phospho-c-Jun, and IRE1 (Cell Signaling Technology); and spliced XBP-1 (Cambridge BioScience).

Techniques: Western Blot

Journal: Journal of Biological Chemistry

Article Title: Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells

doi: 10.1074/jbc.m109.014092

Figure Lengend Snippet: FIGURE 3. ATF4 mediates fenretinide- and bortezomib-induced cell death. A and B, SH-SY5Y and A375 cells were transfected with siRNAs for ATF4 or with a non-silencing control siRNA (ctrl) prior to treatment with fenretinide (FenR) (SH-SY5Y, 5 M; and A375, 10 M), bortezomib (Bort) (SH-SY5Y, 5 nM; and A375, 30 nM), or thapsigargin (Thap) (SH-SY5Y, 1.5 M; and A375, 7.5 M) for 6 h. ATF4 or Noxa mRNA was measured by real-time PCR relative to -actin as an internal control (A). ATF4 (lower band indicated by the asterisk in SH-SY5Y cells), ATF3, Noxa, and -actin expression was determined by Western blotting (B). C, SH-SY5Y and A375 cells were transfected with siRNAs for ATF4 or with a non-silencing control siRNA prior to treatment with fenretinide (SH-SY5Y, 10 M; and A375, 15 M), bortezomib (SH-SY5Y, 5 nM; and A375, 50 nM), or thapsigargin (SH-SY5Y, 3 M; and A375, 10 M) for 24 h. Apoptosis was measured by flow cytometry of propidium iodide-stained cells to determine the sub-G1 fraction. Data are expressed as the percentage total population or relative to control untreated cells; each point is the mean S.E. (n 3). D, A375 cells were treated with fenretinide (15 M), bortezomib (50 nM), or thapsigargin (10 M) for 6 h. Recruitment to the Noxa promoter was determined by promoter pulldown assays in the absence (control) or presence of the Noxa promoter DNA fragment, followed by Western blotting for ATF4. Data are shown for whole cell extracts; similar results were obtained with nuclear extracts (not shown).

Article Snippet: Blots were probedwith antibodies to ATF4 (Calbiochem); ATF3 and GADD153 (B-3, Santa Cruz Biotechnology); Noxa (Alexis Biochemicals); c-Myc, eIF2 , phospho-eIF2 , phospho-c-Jun, and IRE1 (Cell Signaling Technology); and spliced XBP-1 (Cambridge BioScience).

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry, Staining

Journal: Journal of Biological Chemistry

Article Title: Regulation of Endoplasmic Reticulum Stress-induced Cell Death by ATF4 in Neuroectodermal Tumor Cells

doi: 10.1074/jbc.m109.014092

Figure Lengend Snippet: FIGURE 5. Fenretinide- and bortezomib-induced cell death is independent of PERK. A and B, SH-SY5Y and A375 cells were transfected with siRNAs for PERK or with a non-silencing control siRNA (ctrl) prior to treatment with fenretinide (FenR) (SH-SY5Y, 5 M; and A375, 10 M), bortezomib (Bort) (SH-SY5Y, 5 nM; and A375, 30 nM), or thapsigargin (Thap) (SH-SY5Y, 1.5 M; and A375, 7.5 M) for 6 h. PERK or ATF4 mRNA was measured by real-time PCR relative to -actin as an internal control (A). eIF2, phospho-eIF2, ATF4, ATF3, and -actin expression was determined by Western blotting (B). C, SH-SY5Y and A375 cells were transfected with siRNAs for PERK or with a non-silencing control siRNA prior to treatment with fenretinide (SH-SY5Y, 10 M; and A375, 15 M), bortezomib (SH-SY5Y, 5 nM; and A375, 50 nM), or thapsigargin (SH-SY5Y, 3 M; and A375, 10 M) for 24 h. D, apoptosis was measured by flow cytometry of propidium iodide-stained cells to determine the sub-G1 fraction. Data are expressed as the percentage total population; each point is the mean S.E. (n 3).

Article Snippet: Blots were probedwith antibodies to ATF4 (Calbiochem); ATF3 and GADD153 (B-3, Santa Cruz Biotechnology); Noxa (Alexis Biochemicals); c-Myc, eIF2 , phospho-eIF2 , phospho-c-Jun, and IRE1 (Cell Signaling Technology); and spliced XBP-1 (Cambridge BioScience).

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Flow Cytometry, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids

doi: 10.1016/j.jcmgh.2024.101366

Figure Lengend Snippet: Antibodies Used

Article Snippet: ATF3 , Rabbit , Novus, NBP1-85816 , 1:500.

Techniques:

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a-c , DFFB expression in parental and persister cells. a , A375 cells treated with 250 nM dabrafenib and 25 nM trametinib. b , PC9 cells treated with 2.5 μM erlotinib. c , BT474 cells treated with 500 nM lapatinib. d-f , DFFB loss of expression in CRISPR KO clones. cl, clone. d , A375 DFFB KO clones. e , PC9 DFFB KO clones. WT cl1 is a control WT clone which failed to lose DFFB expression during CRISPR editing. f , BT474 DFFB KO clones. g , Caspase 9 expression in CRISPR-mediated caspase 9-depleted (pooled KO) A375 cells used in Fig. . h , PC9 cells treated with 2.5 μM erlotinib were assessed for DNA damage marker γH2AX. Parental cells were treated for 24 h with 500 nM staurosporine as a positive control. i , PC9 cells treated with 2.5 μM erlotinib for 5 weeks were analyzed for DTEP formation. n = 3 biological replicates, mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± s.d.; two-tailed Student’s t-test.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: Expressing, CRISPR, Clone Assay, Control, Marker, Positive Control, Two Tailed Test

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a-b , A375 DFFB KO cells form tumours and respond to drug in vivo similarly to WT cells. cl, clone. a , A375 xenograft tumour formation and b , initial drug response to dabrafenib and trametinib in mice. n = 6 biological replicates; mean ± s.d. is shown; P values calculated with two-tailed Student’s t-test; ns, not significant. c-f , Clonal variability in parental cell growth rates in cell culture is independent of DFFB WT or loss of function (LOF) status. DFFA-CR, cleavage resistant DFFA. g-k , WT and DFFB LOF cell initial 3 day drug treatment response. g , k , A375 cells treated with 250 nM dabrafenib and 25 nM trametinib. h , PC9 cells treated with 2.5 μM erlotinib. i , PC9 cells treated with 500 nM osimertinib. j , BT474 cells treated with 2 μM lapatinib. l-p , WT and DFFB LOF persister cell formation. l , p , A375 cells treated with dabrafenib and trametinib. m , PC9 cells treated with erlotinib. n , PC9 cells treated with 500 nM osimertinib. o , BT474 cells treated with 2 μM lapatinib. For c-p , n = 3 biological replicates; mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± s.d. is shown; P values calculated with two-tailed Student’s t-test. ns, not significant. q , Cleaved caspase 3 analyzed in A375 WT and DFFB KO parental and persister cells. r , DFFB essentiality in CRISPR screen data from the PICKLES database . Negative scores reflect non-essentiality.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: In Vivo, Two Tailed Test, Cell Culture, CRISPR

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a , ATF3 protein levels in A375 WT, DFFB-KO and DFFA-CR parental and persister cells. b , A375 DFFB-KO persister cells treated with or without 100 μM etoposide for 48 h and analysed for DNA damage, ATF3 and STAT1. c , A375 wild-type persister cells cotreated with 1 μM JAKi and analysed for ATF3 expression. d , Total STAT1 and ATF3 levels in A375 cells with CRISPR-mediated ATF3 depletion (pooled KO). e , Bulk RNA-seq gene set enrichment analysis of Hallmarks IFNα response gene set using differentially expressed genes between A375 ATF3-KO and wild-type persister cells. The P value was adjusted with the Benjamini–Hochberg correction. f , Total STAT1 and ATF3 protein levels in A375 DFFB-KO persister cells with ectopically expressed ATF3. g , DTEP colony formation in A375 wild-type and ATF3-depleted (pooled KO) cells. n = 3 biological replicates; mean ± s.d.; two-tailed Student’s t -test. h , i , Bulk RNA-seq gene set enrichment analysis of A375 ATF3-KO ( h ) and DFFB-KO ( i ) persister cells with versus without cotreatment with 20 μM AP1 inhibitor T-5224. The IFN-related gene sets are coloured red. n = 3 biological replicates. P values were adjusted with the Benjamini–Hochberg correction. j , Summary diagram. EMT, epithelial-to-mesenchymal transition.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: Expressing, CRISPR, RNA Sequencing, Two Tailed Test

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a , ATF3 expression levels between A375 WT and DFFB KO persister and DTEP cells measured with scRNAseq. P values calculated with the two-sided Wilcoxon Rank Sum test with Bonferroni correction. cl, clone. b , ATF3 expression in PC9 WT and DFFB KO cells treated with 500 nM osimertinib. c , A375 WT and DFFB KO cells treated with 250 nM dabrafenib and 25 nM trametinib analyzed for ATF3 at the indicated times. d , A375 WT and DFFB KO cells treated with 250 nM dabrafenib and 25 nM trametinib analyzed for γH2AX, ATF4, and ATF3 at the indicated times. e , PC9 persister cells derived from 500 nM osimertinib with or without 5 μM JAK inhibitor ruxolitinib and analyzed for ATF3 levels.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: Expressing, Derivative Assay

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a , A375 WT cells treated with ER stress inducer 1 μM thapsigargin for 4 h, BH3 mimetics 5 μM ABT-737 and 10 μM S63845 for 2.5 h followed by 24 h recovery, and persister cells analyzed for ISR genes (phosphorylated (p) eIF2α, total eIF2α, ATF4) and ATF3. b , PC9 WT cells treated with 1 μM thapsigargin, BH3 mimetics 1.5 μM ABT-737 and 3 μM S63845 for 4 h followed by 2 h recovery, and persister cells analyzed for ISR genes and ATF3. c-f , Flow cytometry analysis of cytochrome C release in A375 ( c,d) and PC9 ( e , f ) parental, persister, and BH3 mimetic-treated cells. d and f , Fractional loss of cytochrome C (geometric means) are plotted on the right graphs. n = 3 biological replicates; mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± s.d. is shown; P values calculated with two-tailed Student’s t-test. g,h , A375 ( g ) and PC9 ( h ) WT and ATF4 CRISPR-depleted (pooled KO) persister cells were analyzed for ATF3 levels.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: Flow Cytometry, Two Tailed Test, CRISPR

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a , b , A375 ATF3-depleted (pooled KO) cells assessed for parental cell proliferation ( a ) and persister cell formation ( b ). n = 3 biological replicates; mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± s.d. is shown; P values calculated with two-tailed Student’s t-test. ns, not significant. c , A375 WT DTEP cycling (MKI67 + ) and noncycling (MKI67-) cell ATF3 scRNAseq expression. P values calculated with the two-sided Wilcoxon Rank Sum test. d , STRING analysis of ATF3 interactions. e , Bulk RNAseq gene set enrichment analysis of A375 WT persister cells versus persister cells co-treated with 20 μM AP1 inhibitor T-5224. n = 3 biological replicates. EMT, epithelial-to-mesenchymal transition.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: Two Tailed Test, Expressing

Journal: Nature Cell Biology

Article Title: DNA fragmentation factor B suppresses interferon to enable cancer persister cell regrowth

doi: 10.1038/s41556-025-01810-x

Figure Lengend Snippet: a , b , ScRNAseq anastasis gene set signature scores in A375 parental, persister, and DTEP timepoint DFFB WT and KO cells ( a ) and PC9 parental and persister cells ( b ). Anastasis remains elevated at the DTEP timepoint, indicating that DTEP cells remain drug stressed. cl, clone. c , Anastasis gene set expression analyzed in on-treatment versus pretreatment patient melanoma tumours treated with BRAF + /- MEK targeted therapy. Normalized enrichment score, NES. P value calculated with a two-sided permutation test with family-wise error rate correction. d , Anastasis gene set signature scores in treatment naïve (TN), residual disease (RD), and progressive disease (PD) patient non-small cell lung cancer tumours treated with EGFR targeted therapy. e , MKI67 (Ki-67) expression in pretreatment and on-treatment melanoma patient tumours. f , Ki-67 expression in each lung cancer treatment response stage. g-h , Hallmarks IFN alpha response gene set expression analyzed in on-treatment versus pretreatment patient melanoma ( g ) and lung cancer tumour treatment response stages ( h ). g , P value adjusted with Benjamini–Hochberg correction. i , ATF3 expression in pretreatment and on-treatment patient melanoma tumours. j , ATF3 expression in each lung cancer treatment response stage. P value calculated with the two-sided Wilcoxon Rank Sum test with Bonferroni correction. k,l , A375 ( k ) and PC9 ( l ) WT and DFFB KO parental and persister cells analyzed for STING expression. a,b,d , h , P values calculated with two-sided Mann-Whitney test. d,h , n = 1,073 (TN), 572 (RD), and 2,109 (PD) cells analyzed from 15 (TN), 12 (RD), and 17 (PD) patients. e , i , n = 11 patients. P values calculated with two-sided paired ratio t-test.

Article Snippet: Caspase 9-KO cells were generated with Santa Cruz caspase 9 CRISPR plasmids (h) (sc-400257-KO-2), ATF3-KO cells with Santa Cruz ATF3 CRISPR plasmids (h) (sc-416577) and ATF4-KO cells with Santa Cruz ATF4 CRISPR plasmids (h2) (sc-400155-KO-2) following manufacturer’s protocols.

Techniques: Expressing, MANN-WHITNEY

Journal: Cell reports

Article Title: Optic nerve regeneration screen identifies multiple genes restricting adult neural repair

doi: 10.1016/j.celrep.2021.108777

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were incubated with tissue sections overnight at 4°C: anti-Iba1 (1:200, Abcam), anti-CD68 (1:250, Bio-Rad), anti-GFAP (1:500, Abcam), anti-DLK (1:100, Genetex), anti-Atf3 (1:200, Novus Biologicals), anti-GFP (1:500, Abcam), anti-Il22Ra1 (1:200, Bioss), anti-Rbpms (1:100; PhosphoSolutions), anti-βIII-tubulin antibody (1:500, Promega).

Techniques: Virus, Recombinant, SYBR Green Assay, DNA Extraction, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Gene Expression, shRNA, Expressing, Plasmid Preparation, Software