Journal: Nature Communications

Article Title: Germinal center output is sustained by HELLS-dependent DNA-methylation-maintenance in B cells

doi: 10.1038/s41467-023-41317-3

Figure Lengend Snippet: a GSEA showing enrichment of signatures related to mTORC1 activation, UPR, ATF4 targets and cholesterol biosynthesis in Mb1Hells KO day 14 GC B cells. NES normalized enrichment score, FDR false discovery rate. b Venn Diagram of upregulated genes in day-14 Mb1Hells KO GC B cells compared to mTORC1 targets upregulated in activated B cells (GSE141423) and ATF4-dependent mTORC1 targets (GSE158605). Genes commonly upregulated in day-14 Mb1Hells KO GC B cells and in GSE141423 and/or GSE158605 are represented in blue on the volcano plot (gray and blue = p < 0.05, calculated by a two-tailed test). Source data are provided in Source Data File.

Article Snippet: Murine ATF4 coding sequence without upstream ORFs was PCR-amplified from Mouse ATF4 (CHOP11/cATF)-WT vector, a gift from David Ron (Addgene plasmid # 21845; http://n2t.net/addgene:21845 ; RRID:Addgene_21845), and cloned into Bgl II and Xho I sites of the pMIG retroviral vector, a gift from William Hahn (Addgene plasmid # 9044; http://n2t.net/addgene:9044 ; RRID:Addgene_9044), to generate pMIG-ATF4 expression vector.

Techniques: Activation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Germinal center output is sustained by HELLS-dependent DNA-methylation-maintenance in B cells

doi: 10.1038/s41467-023-41317-3

Figure Lengend Snippet: a Single-cell transcriptional analysis of day 7 and 14 GC B cells. UMAP displaying 429 day-14 GC B, 466 day-7 GC B Mb1Hells KO , 414 day-14 GC B, and 448 day-7 GC B control cells, colored by shared nearest neighbor clusters collected. Bar charts show the frequencies of GC B cells within the 4 clusters. n = 2 mice for each genotype and timepoint; one of the two control mice for day-7 GC B cells was Mb1 Cre/WT Hells F/WT . b Selected gene expression for the 4 different clusters shown in ( a ), presented in dot plot scaled on normalized UMI counts. c Representative CD98 staining of CD95 + GL7 + CCR6 neg CD38 neg GC B cells and quantification of CD98 geometric MFI (Mean Fluorescent Intensity). d Representative intracellular ATF4 staining within CD95 + GL7 + CCR6 neg GC B cells and quantification of ATF4 geometric MFI (Mean Fluorescent Intensity). e Representative CD98 staining of B220 + day-8 iGB cell cultures and quantification of CD98 geometric MFI (Mean Fluorescent Intensity). f Expression of Asns and Chac1 in sorted B220 + CD138 neg day-8 iGB cells measured by RT-qPCR after normalization to Ubc . g Representative CD98 staining of GFP + and GFP neg day-8 iGB cell cultures after transduction of empty or ATF4 coding retroviral vectors, and quantification of CD98 geometric MFI (Mean Fluorescent Intensity). h GSEA showing enrichment of ASC signature in Mb1Hells KO vs. control day 14 GC B cells. The ASC signature consists in the top 200 genes upregulated in GSE60927. NES normalized enrichment score, FDR false discovery rate. n = 4 for each genotype in ( c ), ( d ), ( e ), ( f ) and ( h ); experiments were done twice and one representative experiment is shown. n = 5 for ( g ); experiment was done twice and data were pooled. Bar charts and error bars represent the mean ± SD. Unpaired two-tailed t -test were performed for ( c ) to ( g ). Source data are provided in Source Data File.

Article Snippet: Murine ATF4 coding sequence without upstream ORFs was PCR-amplified from Mouse ATF4 (CHOP11/cATF)-WT vector, a gift from David Ron (Addgene plasmid # 21845; http://n2t.net/addgene:21845 ; RRID:Addgene_21845), and cloned into Bgl II and Xho I sites of the pMIG retroviral vector, a gift from William Hahn (Addgene plasmid # 9044; http://n2t.net/addgene:9044 ; RRID:Addgene_9044), to generate pMIG-ATF4 expression vector.

Techniques: Control, Gene Expression, Staining, Expressing, Quantitative RT-PCR, Transduction, Retroviral, Two Tailed Test

Journal: Antioxidants

Article Title: Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1

doi: 10.3390/antiox10101543

Figure Lengend Snippet: Oligonucleotide sequences used in the present study.

Article Snippet: An expression plasmid encoding mouse ATF4 was a gift from Dr. David Ron (Addgene plasmid #21845).

Techniques: Recombinant, Plasmid Preparation

Journal: Antioxidants

Article Title: Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1

doi: 10.3390/antiox10101543

Figure Lengend Snippet: Cystine and methionine deficiency activates the integrated stress response (ISR). HepG2 and L-O2 cells were exposed to CST/Met (−) for 6 ( a , b ), 12 ( d , f ), 24 ( c , e ), or 24–36 h ( g ). ( a ) Phosphorylation of GCN2 and eIF2α. Equal protein loading was verified by β-actin immunoblotting. ( b ) SUnSET assay. Puromycin-integrated polypeptides of 25–170 kDa were quantified by densitometry (left and right). Equal protein loading was verified by Ponceau S staining (upper middle) and β-actin immunoblotting (lower middle). ( c ) ATF4 expression in cells exposed to CST/Met (−) with or without Fer-1 (10 μM) was normalized to β-actin. ( d ) mRNA levels of ISR target genes were determined by qPCR analysis. ( e – g ) Lipid peroxidation ( e ), PTGS2 mRNA ( f ), and cell viability ( g ) were determined after HepG2 cells were exposed to ISRIB (1 μM) under CST/Met (−). ** p < 0.01, * p < 0.05, versus control; ## p < 0.01, # p < 0.05, versus CST/Met (−): Con, control; ISRIB, ISR inhibitor; N.S., not significant.

Article Snippet: An expression plasmid encoding mouse ATF4 was a gift from Dr. David Ron (Addgene plasmid #21845).

Techniques: Phospho-proteomics, Western Blot, Staining, Expressing, Control

Journal: Antioxidants

Article Title: Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1

doi: 10.3390/antiox10101543

Figure Lengend Snippet: Cystine and methionine deficiency induces BTG1 by activating ATF4. HepG2 and L-O2 cells were exposed to CST/Met (−) for 1–12 ( a , d ), or 24 h ( b , c , e , f ). ( a ) The level of BTG1 mRNA was determined by qPCR. Fer-1 (10 μM, 12 h) or ISRIB (1 μM, 12 h) was simultaneously applied to HepG2 cells under CST/Met (−) (left). ( b ) BTG1 protein. Open arrowheads and dashed lines in immunoblot images indicate nonspecific bands and cropped images of the same membrane, respectively (upper). ( c ) BTG1 transactivation. Schematic illustration of constructed reporter plasmids containing the human and murine BTG1 gene promoter (upper). An ATF4 expression plasmid was co-transfected with hpBTG1-luc, mpBTG1-luc, or mpBTG1-ΔA4RE-luc. pCDNA3.2/V5-DEST was used for mock transfection (lower). ( d – f ) Effect of siATF4 on BTG1 induction. A siRNA targeting human ATF4 ( d , e ) or murine ATF4 ( f ) was transfected into HepG2 cells, and expression of BTG1 ( d , e ) and transactivation ( f ) were determined by qPCR, immunoblot, and reporter gene assays, respectively. ** p < 0.01, * p < 0.05, versus control ( a , b , d , e ) or mock transfection ( c , f ); ## p < 0.01, # p < 0.05, versus HepG2 cells exposed to CST/Met (−) ( a , d , e ) or ATF4-transfected cells ( c , f ); †† p < 0.01, between basal level of ATF4 mRNA ( d ): A4RE, putative ATF4 response element; Con, control.

Article Snippet: An expression plasmid encoding mouse ATF4 was a gift from Dr. David Ron (Addgene plasmid #21845).

Techniques: Western Blot, Membrane, Construct, Expressing, Plasmid Preparation, Transfection, Control

Journal: Antioxidants

Article Title: Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1

doi: 10.3390/antiox10101543

Figure Lengend Snippet: Putative ATF4 response elements in BTG1 promoter.

Article Snippet: An expression plasmid encoding mouse ATF4 was a gift from Dr. David Ron (Addgene plasmid #21845).

Techniques: Binding Assay, Sequencing

Journal: Antioxidants

Article Title: Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1

doi: 10.3390/antiox10101543

Figure Lengend Snippet: BTG1 induces ferroptosis under cystine and methionine deficiency. WT and BTG1 KO HAP1 cells were exposed to CST/Met (−) for 1 ( a —lower), 12 ( c , e ), 24 ( a —upper, and b , d ), or 0–24 h ( f ). ( a ) Fluorescence intensity was measured after incubating HAP1 cells with CST/Met (−) and DCFH-DA (lower). The phenotype of BTG1 KO HAP1 cells was verified by BTG1 immunoblotting. Open arrowheads and dashed lines in immunoblot images indicate nonspecific bands and cropped images of the same membrane, respectively (upper). ( b ) Effect of BTG1 KO on CST/Met (−)-inducible expression of ATF4. ( c ) mRNA levels of ISR target genes in HAP1 cells. ( d – f ) Lipid peroxidation ( d ), PTGS2 mRNA level ( e ), and necrotic cell death ( f ) in HAP1 cells exposed to CST/Met (−). ** p < 0.01, * p < 0.05, versus control; ## p < 0.01, between HAP1 cells exposed to CST/Met (−); N.S., not significant.

Article Snippet: An expression plasmid encoding mouse ATF4 was a gift from Dr. David Ron (Addgene plasmid #21845).

Techniques: Fluorescence, Western Blot, Membrane, Expressing, 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: 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 Assay, Derivative Assay, Expressing

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