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: Nature Communications

Article Title: Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy

doi: 10.1038/s41467-021-24215-4

Figure Lengend Snippet: a Representative western blots for ATF6α-N (50 kDa, nuclear), phospho-IRE1α (Ser 724; arrowhead), total IRE1α, phospho- and total PERK, phospho-eIF2α (Ser 51), total-eIF2α, ATF4, and DNAJC3. Phospho-PERK was analyzed using Phos-tag gels. The heart protein extracts are from the indicated mice at 4 weeks of age. Gapdh serves as a processing and loading control. b , c Western blot for Thbs1 and PERK following immunoprecipitation (IP) of PERK ( b ) or Thbs1 ( c ) from protein extracts of tTA cont. and Thbs1 DTG hearts at 6 weeks of age. IPs with corresponding IgG served as negative controls. Vinculin is an input loading control. d Schematic diagram of Thbs1 with the GST-Thbs1 fusion proteins regions shown (red bars). Below the schematic a representative western blot for PERK following GST pull-down with the different Thbs1 domains from primary neonatal rat ventricular cardiomyocyte extracts. GST protein serves as a negative control. e - i , Quantitative RT-PCR for Eif2ak3 (PERK protein; * P = 0.0317 vs tTA control), Atf4 (* P = 0.0006 vs tTA control), Atf3 (* P = 0.0058 vs tTA control), Ddit3 (Chop protein; * P < 0.0001 vs tTA control)) and Fgf21 (* P = 0.0023 vs tTA control) from mRNA isolated from hearts of tTA cont. (n = 6 biologically independent animals) and Thbs1 DTG mice (n = 5 biologically independent animals) at 6 weeks of age. Statistical analysis was performed using two-tailed Student’s t test. Error bars are ±standard error of the mean. Source data are provided as a Source Data File.

Article Snippet: Full-length mouse Atf4 cDNA was obtained from Addgene (plasmid #21845, deposited by Dr. David Ron), amplified by PCR using CloneAMP HiFi PCR premix (Takara Bio, #639298) and cloned into the Cla1 and XhoI sites of pAAV-MCS vector (Cell biolabs, #VPK-410) using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, #E2621).

Techniques: Western Blot, Control, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy

doi: 10.1038/s41467-021-24215-4

Figure Lengend Snippet: a Low magnification images of wild type and Thbs1 −/− whole mount cardiac histological sections stained with Hematoxylin & Eosin, 2 weeks after TAC surgery at 8 weeks of age. Scale bar is 1 mm. b HW/BW ratio and c FS percentage 2 weeks after TAC or sham surgery at 8 weeks of age. The number of biologically independent animals analyzed is indicated on the graphs for panels “ b – d ”. d – p Quantitative RT-PCR results for Eif2ak3 (PERK protein) , Atf6, Ern1 (IRE1α protein) , Manf (Armet protein) , Hspa5 (BiP protein) , Calr (Calreticulin protein), Atf4, Atf3, Fgf21, Map1lc3b (LC3b protein) , Trim63 (MuRF1 protein) , Fbxo32 (Atrogin-1 protein), and Trib3 mRNA isolated from hearts of wild type and Thbs1 −/− mice, 2 weeks after TAC or sham surgery at 8 weeks of age. For panels “ d – i ”, n = 6 biologically independent samples per group; for panels “ j and p ”, n = 6 biologically independent samples for sham wild type and Thbs1 −/− 2 weeks after TAC surgery, n = 5 biologically independent samples for sham Thbs1 −/− and n = 9 biologically independent samples for wild-type 2 weeks after TAC surgery; for panels “ k – o ”, n = 6 biologically independent samples for sham wild-type, sham Thbs1 −/− and Thbs1 −/− 2 weeks after TAC surgery and n = 9 biologically independent samples for wild-type 2 weeks after TAC surgery. Data are represented as fold expression over sham wild type. All statistical analysis was performed using one-way ANOVA and Tukey multiple comparisons test. P -values are shown in each graph. Error bars are ±standard error of the mean. Source data are provided as a Source Data File.

Article Snippet: Full-length mouse Atf4 cDNA was obtained from Addgene (plasmid #21845, deposited by Dr. David Ron), amplified by PCR using CloneAMP HiFi PCR premix (Takara Bio, #639298) and cloned into the Cla1 and XhoI sites of pAAV-MCS vector (Cell biolabs, #VPK-410) using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, #E2621).

Techniques: Staining, Quantitative RT-PCR, Isolation, Expressing

Journal: Nature Communications

Article Title: Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy

doi: 10.1038/s41467-021-24215-4

Figure Lengend Snippet: a Low magnification cardiac histological images from tTA cont. Eif2ak3 fl/fl (PERK), tTA Eif2ak3 fl/fl βMHC-Cre ( Eif2ak3 CKO ), Thbs1 DTG Eif2ak3 fl/fl , and Thbs1 DTG Eif2ak3 CKO mice stained with Masson’s trichrome at 8 weeks of age. Scale bar is 2 mm. b VW/BW ratio, c FS percentage and d LW/BW ratio at 8 weeks of age in the indicated groups of mice. The number of biologically independent animals analyzed and P -values are indicated on the graphs for panels “ b – d ”. Statistical analysis was performed using one-way ANOVA and Tukey multiple comparisons test for panels “ b – d ”. Error bars are ±standard error of the mean. e Kaplan–Meier survival plot from tTA cont. Eif2ak3 fl/fl , Thbs1 DTG Eif2ak3 fl/fl , tTA cont. Eif2ak3 CKO and Thbs1 DTG Eif2ak3 CKO . The number of biologically independent animals analyzed are indicated on the graph. Statistical analysis was performed using a two-tailed log-rank test. *P < 0.0001 vs tTA cont. Eif2ak3 fl/fl , Eif2ak3 CKO and Thbs1 DTG Eif2ak3 CKO , # P = 0.0678 vs tTA cont. Eif2ak3 fl/fl , and # P = 0.0404 vs Eif2ak3 CKO . f Representative western blots for Thbs1, PERK, ATF4, LC3b, and p62 from cardiac protein extracts isolated from the groups shown at 8 weeks of age. Protein extracts were from dissociated adult mouse heart cardiomyocytes. Gapdh serves as a loading control. Source data are provided as a Source Data File.

Article Snippet: Full-length mouse Atf4 cDNA was obtained from Addgene (plasmid #21845, deposited by Dr. David Ron), amplified by PCR using CloneAMP HiFi PCR premix (Takara Bio, #639298) and cloned into the Cla1 and XhoI sites of pAAV-MCS vector (Cell biolabs, #VPK-410) using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, #E2621).

Techniques: Staining, Two Tailed Test, Western Blot, Isolation, Control

Journal: Nature Communications

Article Title: Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy

doi: 10.1038/s41467-021-24215-4

Figure Lengend Snippet: a Schematic diagram depicting the experimental protocol. Either 1E11 genomic copies of adeno-associated virus 9 (AAV9)-PERK or AAV9-luciferase (Lucif.) control were injected into the mediastinum of 7-day-old WT mouse pups. Hearts were harvested at 8 weeks of age for further analysis. b Low magnification of whole mount cardiac histological images from mice injected with AAV9-PERK or AAV9-Lucif. control stained with Masson’s trichrome at 8 weeks of age. Scale bar is 2 mm. c Representative western blots for PERK, ATF4, LC3b, and p62 from cardiac protein extracts of 8-week-old mice injected with either AAV9-Lucif. or AAV9-PERK. Gapdh serves as a loading control. d Representative western blot for ubiquitin-conjugated proteins (Ubiq.) and Gapdh as loading control on cardiac protein extracts of 8-week-old mice injected with either AAV9-Lucif. or AAV9-PERK. e HW/BW ratio (* P = 0.0051 vs AAV9-Lucif.) and f FS% at 8 weeks of age in the 2 indicated groups of mice. * P = 0.0060 vs AAV9-Lucif. g Representative immunohistochemistry for PERK (green), nuclei with DAPI (blue) and WGA (purple)-stained membranes from heart sections of AAV9-PERK or AAV9-Lucif. injected mice killed at 8 weeks of age. Scale bars are 100 μm. h Quantitative analysis of AAV9-Lucif. versus or AAV9-PERK positive (Pos.) and negative (Neg.) cross sectional area (CSA) determined by WGA staining of cardiac histological sections. * P = 0.0087 vs AAV9-Lucif. and * P = 0.0050 vs AAV9-PERK Neg. The number of biologically independent animals analyzed are indicated on the graphs. Statistical analysis was performed using a two-tailed Student’s t -test in panels “ e , f ”, and one-way ANOVA and Tukey multiple comparisons test in panel “ h ”. All error bars are ±standard error of the mean. Source data are provided as a Source Data File.

Article Snippet: Full-length mouse Atf4 cDNA was obtained from Addgene (plasmid #21845, deposited by Dr. David Ron), amplified by PCR using CloneAMP HiFi PCR premix (Takara Bio, #639298) and cloned into the Cla1 and XhoI sites of pAAV-MCS vector (Cell biolabs, #VPK-410) using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, #E2621).

Techniques: Virus, Luciferase, Control, Injection, Staining, Western Blot, Ubiquitin Proteomics, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy

doi: 10.1038/s41467-021-24215-4

Figure Lengend Snippet: a Schematic diagram depicting the experimental protocol. Either 0.5E11 or 1E10 genomic copies (gc) of AAV9-ATF4 or AAV9-Lucif. control were injected into the mediastinum of 7-day-old wild-type mouse pups. Hearts were harvested at 4 weeks of age for further analysis. b Representative western blots for PERK and ATF4 from cardiac protein extracts of 4-week-old mice treated with the indicated AAV9. Gapdh serves as a loading control. c , d Representative heart sections with Masson’s Trichrome staining ( c ) and immunohistochemistry for ATF4 (green) and WGA (purple)-stained membranes and nuclei with DAPI (blue) ( d ) of AAV9-ATF4 or AAV9-Lucif. injected mice (both 0.5E11 gc) at 4 weeks of age. Scale bars are 2 mm and 100 μm, respectively. e CSA of AAV9-Lucif. versus AAV9-ATF4 positive cardiomyocytes determined by ATF4 and WGA staining of histological sections as shown in panel “ d ”. * P = 0.0548 vs AAV9-Lucif. f Representative Masson’s trichrome stained images of hearts from mice injected with 1E10 gc AAV9-Lucif or -ATF4 and harvested at 4 weeks of age. Scale bar is 2 mm. g HW/BW ratio at 4 weeks of age in the indicated groups of mice. * P = 0.0235 vs AAV9-Lucif. h Representative immunohistochemistry for ATF4 (green), nuclei with DAPI (blue) and WGA (purple)-stained membranes from heart sections of AAV9-ATF4 or AAV9-Lucif. injected mice (both 1E10 gc) at 4 weeks of age. Scale bars are 100 μm. i CSA of ATF4 positive cardiomyocytes determined by ATF4 and WGA staining as shown in panel “ h ”. * P = 0.0116 vs AAV9-Lucif. j Representative western blots for LC3b, p62, and ubiquitin-conjugated proteins (Ubiq.) from cardiac protein extracts of 4-week-old mice treated with 1E10 gc AAV9-Lucif. or -ATF4. Gapdh serves as a loading control. k , l Representative micrographs of fluorescent LC3 puncta ( k ) and quantification thereof ( l ) in cultured primary neonatal rat ventricular myocytes 48 h after infection with adenoviruses to overexpress tandem mRFP-GFP-LC3 (Ad-tf-LC3) and ATF4 or βgal expressing control. Yellow dots represent autophagosomes, whereas red dots indicate autolysosomes. Scale bars are 50 μm. * P < 0.0001 vs Adβgal. Number of biologically independent animals or cells analyzed is indicated in each panel. All statistical analysis were performed using two-tailed Student’s t test. Error bars are ±standard error of the mean. Source data are provided as a Source Data File.

Article Snippet: Full-length mouse Atf4 cDNA was obtained from Addgene (plasmid #21845, deposited by Dr. David Ron), amplified by PCR using CloneAMP HiFi PCR premix (Takara Bio, #639298) and cloned into the Cla1 and XhoI sites of pAAV-MCS vector (Cell biolabs, #VPK-410) using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, #E2621).

Techniques: Control, Injection, Western Blot, Staining, Immunohistochemistry, Ubiquitin Proteomics, Cell Culture, Infection, Expressing, Two Tailed Test

Journal: Cell death and differentiation

Article Title: A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

doi: 10.1038/cdd.2015.24

Figure Lengend Snippet: Figure 1 L-Pro rapidly remodels the ESC transcriptome and modulates the AAR pathway. (a) Genes deregulated (≥1.5-fold; FDRo0.0001) by L-Pro (0.5 mM) in TBV2 ESCs at 24 and 48 h, as inferred from microarray analysis (Supplementary Table 1). (b) Time course of qPCR analysis of selected genes in L-Pro-treated versus untreated ESCs. Data are presented as fold change compared with control after normalisation to Gapdh. (c) Gene ontology (GO) analysis of L-Pro-treated ESC transcriptome showing gene enrichment in KEGG pathway. (d) Schematic representation of the AAR (Gcn2-Eif2α-ATF4) pathway. (e) Heat-map diagram showing AAR-related genes downregulated (green) in L-Pro- treated ESCs or upregulated (red) by histidinol (HisOH) and halofuginone (HF). (f) Western blot analysis of phospho-(51Ser)-Eif2α and Atf4 in ESCs treated (8 h) with L-Pro (0.5 mM) or HF (8 nM) either alone or in combination. Gapdh was used as a loading control. (g) Effects of different NEAAs on the expression of AAR-related and AAR-unrelated genes. qPCR analysis of Lefty1 and Slc1a4 in ESCs treated with individual NEAA (0.5 mM) or left untreated as a control (24 h). (h) Effects of different NEAAs on ESC proliferation (36 h). Proliferation was measured by the CyQuantR assay and expressed as relative fluorescence units (RFU). (i) Effect of different NEAAs on the ESC to PiC transition. Colony- type frequency (domed versus flat) of ESCs +/ −individual NEAA as assessed (~300 colonies scored/condition) at day 5 after plating. See Supplementary Figure 1. Data represent the mean ± S.D. from (b, f, g and i) three or (h) five independent experiments, *Po0.001

Article Snippet: Atf4 Tet-Off ESCs (Atf4 GOF) were generated as previously described.46 Briefly, mouse Atf4 cDNA (OriGene; cat. n. MR29597) was cloned in the exchange vector pPTHC/MCS47 and then targeted to the Rosa26 locus of the EB3 ESCs, as described.19 A PCR-based assay on genomic DNA was used to identify the positive clones.19 Two independent clones were tested for Atf4 mRNA and protein induction upon tetracycline (Sigma-Aldrich, 1 μg/ml) removal.

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

Journal: Cell death and differentiation

Article Title: A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

doi: 10.1038/cdd.2015.24

Figure Lengend Snippet: Figure 3 Biological effects of L-Pro-dependent modulation of the AAR pathway in ESCs. (a and b) Effect of Atf4 KD on ESC proliferation. FACS-based analysis (EdU incorporation) of Atf4 KD (shATF4) and control (shNT) ESCs proliferation (a) performed at 36 h after plating. The results are expressed as the fold change compared with control. Representative FACS plots of EdU incorporation (b) in Atf4 KD ESC +/ −individual NEAAs (0.5 mM) at 36 h after plating. (c) Western blot analysis of Atf4 in untreated or HF- treated Atf4 KD and control ESCs (36 h). The densitometric analysis is expressed in ADU as the Atf4/Gadph ratio. (d) Proliferation of control and Atf4 KD ESCs after 48 h treatment with HF, either alone or with L-Pro, was measured by CyQuantR and expressed as RFU. (e and f) Effect of Atf4 KD on cell colony formation. Representative photomicrographs (e) of colonies generated from Atf4 KD and control ESCs plated +/ −L-Pro (0.2 mM) and stained with crystal violet at day 5. Scale bar, 200 μm. Quantification of crystal violet staining of cell colonies generated in the different culture conditions (f). (g–j) Effect of Atf4 overexpression on ESCs behaviour. Immunoblotting analysis of Atf4 protein (g) in Atf4 Tet-OFF ESCs +/ −Tet at 48 h. The densitometric analysis is expressed in ADU as the Atf4/Gadph ratio. Representative FACS plots of EdU incorporation (h) in Atf4 Tet-OFF ESCs +/ −Tet. Representative photomicrographs of colonies (i) generated from Atf4 Tet-OFF ESCs treated with +/ −Tet and +/ −L-Pro (0.2 mM) and stained with crystal violet at day 5 after plating. Scale bar, 50 μm. Effect of Atf4 overexpression on L-Pro-induced cell motility (j). Control (+Tet) and Atf4 GOF ( −Tet) ESCs were treated with +/ −L-Pro and migration was assessed at day 5 after plating. The results show the average numbers of cells migrating towards an FBS gradient (1–15%). (k) qPCR analysis of Aldh18a1 and Pycr1 in control (+Tet) and Atf4 GOF ( −Tet) at 24 h and 48 h. (l) The intracellular free L-Pro concentration was measured in control (+Tet) and Atf4 GOF ( −Tet) ESCs at day 5 after plating. The results are expressed as the fold change compared with control. (m) Schematic representation of the L-Pro–AAR/Atf4 regulatory feedback loop. Data represent the mean ± S.D. from (a, b, c, f, g, h, j, k, and l) three or (d) five independent experiments, *Po0.001

Article Snippet: Atf4 Tet-Off ESCs (Atf4 GOF) were generated as previously described.46 Briefly, mouse Atf4 cDNA (OriGene; cat. n. MR29597) was cloned in the exchange vector pPTHC/MCS47 and then targeted to the Rosa26 locus of the EB3 ESCs, as described.19 A PCR-based assay on genomic DNA was used to identify the positive clones.19 Two independent clones were tested for Atf4 mRNA and protein induction upon tetracycline (Sigma-Aldrich, 1 μg/ml) removal.

Techniques: Control, Western Blot, Generated, Staining, Over Expression, Migration, Concentration Assay

Journal: Cell death and differentiation

Article Title: A novel autoregulatory loop between the Gcn2-Atf4 pathway and (L)-Proline [corrected] metabolism controls stem cell identity.

doi: 10.1038/cdd.2015.24

Figure Lengend Snippet: Figure 6 Model depicting the role of L-Pro ↔AAR axis in the control of stem cell identity. The ESC identity depends on a highly specific intrinsic nutrient stress condition generated by a L-Pro-AAR/Atf4 axis. Under L-Pro shortage/AAR activation condition, ESCs grow as a compact, adherent cell mass (i.e., like the inner cell mass in the blastocyst). Alleviation of this stress (L-Pro fullness/AAR inactivation) initially improves ESC proliferation, but later on it induces the esMT, which is accompanied by a huge induction of macroautophagy and apoptosis. During the esMT, ESCs acquire mesenchymal-like features, becoming highly motile and invasive pluripotent stem cells. Targeting of the L-Pro ↔AAR/Atf4 axis genetically (Atf4 LOF and GOF approaches) or pharmacologically (HF, Z-VAD) modulates esMT

Article Snippet: Atf4 Tet-Off ESCs (Atf4 GOF) were generated as previously described.46 Briefly, mouse Atf4 cDNA (OriGene; cat. n. MR29597) was cloned in the exchange vector pPTHC/MCS47 and then targeted to the Rosa26 locus of the EB3 ESCs, as described.19 A PCR-based assay on genomic DNA was used to identify the positive clones.19 Two independent clones were tested for Atf4 mRNA and protein induction upon tetracycline (Sigma-Aldrich, 1 μg/ml) removal.

Techniques: Control, Generated, Activation Assay