pcdna3 1 matf4  (Addgene inc)

Journal: Frontiers in Oncology

Article Title: Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells

doi: 10.3389/fonc.2022.767479

Figure Lengend Snippet: Primers used for qRT-PCR.

Article Snippet: Mouse ATF4 (CHOP11/cATF)-WT was a gift from David Ron (Addgene plasmid # 21845).

Techniques:

Journal: Frontiers in Oncology

Article Title: Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells

doi: 10.3389/fonc.2022.767479

Figure Lengend Snippet: Effect of hypoxia on ATF4 expression. mRNA (A) and protein (B) expression of ATF4 was determined using qRT-PCR and Western blotting in M-Wnt and metM-Wnt lung cells incubated in hypoxia (H) or normoxia (N) for 48 h. Results are expressed as mean + SEM. Asterisk (*) indicates P < 0.05 relative to normoxia, n=3. Overall survival (C) and relapse-free survival (D) of breast cancer patients with high (red) or low (black) expression of ATF4, where patient data are split by upper quartile (compared to lowest three quartiles) of ATF4 expression.

Article Snippet: Mouse ATF4 (CHOP11/cATF)-WT was a gift from David Ron (Addgene plasmid # 21845).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation

Journal: Frontiers in Oncology

Article Title: Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells

doi: 10.3389/fonc.2022.767479

Figure Lengend Snippet: Effect of ATF4 manipulation in normoxic and hypoxic cells. Viability after 48 h in hypoxia or normoxia was assessed by MTT (A) . Cells were pre-incubated in normoxia or hypoxia for 48 h and cell death was determined by flow cytometry with Zombie NIR (B) , re-plated into transwells to assess migration (C) or low attachment plates to assess viability in detached conditions (D) . metM-Wnt lung cells were transfected with siCtrl or siATF4 and incubated in hypoxia or normoxia for 48 h. ATF4 depletion was confirmed by qRT-PCR (E) . Cell viability (F) and cell death (G) were assessed at the end of incubation. Cells pre-incubated in hypoxia or normoxia were seeded into transwells to assess migration (H) or low attachment plates to assess viability (I) and cell death (J) in detached conditions. M-Wnt cells were transfected with pcDNA3.1-EV or pcDNA3.1- Atf4 and incubated in normoxia for 48 h. ATF4 overexpression was confirmed by qRT-PCR (K) . Cells were seeded into low attachment plates to assess viability in detached conditions (L) . Results are expressed as mean + SEM. Asterisk (*) indicates P < 0.05 relative to normoxia (A–D) or P < 0.05 relative to siCtrl or empty vector (E–K) , n=3-6.

Article Snippet: Mouse ATF4 (CHOP11/cATF)-WT was a gift from David Ron (Addgene plasmid # 21845).

Techniques: Incubation, Flow Cytometry, Migration, Transfection, Quantitative RT-PCR, Over Expression, 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: 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: 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: eLife

Article Title: The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals

doi: 10.7554/elife.63326

Figure Lengend Snippet: Figure 4. Use of activating transcription factor 4 (ATF4) knockout cells and a rapamycin-resistant ATF4 to validate ATF4 targets regulated by mechanistic target of rapamycin complex 1 signaling. (A) Schematic of ATF4 transcript, including upstream open reading frames (uORFs), coding sequence (CDS), and DNA-binding domain (DNABD), highlighting location of CRISPRn guides biallelic location of ATF4 mutations generated in Tsc2-/-

Article Snippet: DOI: https://doi.org/10.7554/eLife.63326 19 of 33 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct (mouse) siAtf4 GE Life Sciences/ Dharmacon L-042737-01-0020 Transfected construct (mouse) siRheb GE Life Sciences/ Dharmacon L-057044-00-0020 Transfected construct (mouse) siRhebL1 GE Life Sciences/ Dharmacon L-056074-01-0020 Transfected construct (mouse) siTsc2 GE Life Sciences/ Dharmacon L-047050-00-0020 Transfected construct (mouse) siC/ebpa GE Life Sciences/ Dharmacon L-040561-00-0005 Transfected construct (mouse) siC/ebpb GE Life Sciences/ Dharmacon L-043110-00-0005 Transfected construct (mouse) siC/ebpd GE Life Sciences/ Dharmacon L-060294-01-0005 Transfected construct (mouse) siC/ebpg GE Life Sciences/ Dharmacon L-065627-00-0005 Transfected construct (human) siATF4 GE Life Sciences/ Dharmacon L-005125-00-0020 Sequenced-based reagent qPCR primers IDT See table in Materials and methods Recombinant DNA reagent ATF4 (cDNA amplified from plasmid) Addgene RRID:Addgene_21845 Recombinant DNA reagent Pspax2 (plasmid) Addgene RRID:Addgene_12260 Recombinant DNA reagent Pmd2.G (plasmid) Addgene RRID:Addgene_12259 Recombinant DNA reagent pSpCas9n(BB) 2A-GFP (PX461) (plasmid) Addgene RRID:Addgene_48140 Recombinant DNA reagent pTRIPZ (plasmid) PMID:27088857 Alex Toker (Beth Israel Deaconess Medical Center) Recombinant DNA reagent GFP (cDNA amplified from plasmid) Addgene RRID:Addgene_19319 Recombinant DNA reagent SLC7A11 (cDNA amplified from plasmid) PMID:29259101 Alex Toker (Beth Israel Deaconess Medical Center) Recombinant DNA reagent pBabe hygro IRES-TSC2 PMID:15150095 David Kwiatkowski (Brigham and Women’s Hospital) Sequenced-based reagent CRISPR-Cas9n guides for KO of ATF4 IDT CACCGGAGG TGGAGGGGCTATGCT; AAACAGCATAGCCCC TCCACCTCC; CACCGACAATCTGCCTTC TCCAGG; AAACCC TGGAGAAGGCAGATTG TC Sequenced-based reagent Sequencing primers for Atf4-/- cell lines IDT TCGATGCTCTGTTTCGAA TG; CTTCTTCCCCC TTGCCTTAC Continued on next page Torrence et al. eLife 2021;10:e63326.

Techniques: Knock-Out, Sequencing, Binding Assay, Generated

Journal: eLife

Article Title: The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals

doi: 10.7554/elife.63326

Figure Lengend Snippet: Figure 5. Activation of activating transcription factor 4 (ATF4) contributes to the induction of protein synthesis downstream of mechanistic target of rapamycin complex 1. (A, B) Representative autoradiogram and immunoblot of wild-type (WT) and Tsc2-/- mouse embryo fibroblasts (MEFs) transfected with siRNAs targeting Atf4 or Rheb1 and Rhebl1 or non-targeting controls (siCT) and serum-deprived for 16 hr with a pulse label of [35S]-methionine for the final 20 min (A) and quantified in (B). Biological triplicates from a representative experiment are shown in (A). (B) is graphed as mean ± SEM from Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.63326 19 of 33 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Transfected construct (mouse) siAtf4 GE Life Sciences/ Dharmacon L-042737-01-0020 Transfected construct (mouse) siRheb GE Life Sciences/ Dharmacon L-057044-00-0020 Transfected construct (mouse) siRhebL1 GE Life Sciences/ Dharmacon L-056074-01-0020 Transfected construct (mouse) siTsc2 GE Life Sciences/ Dharmacon L-047050-00-0020 Transfected construct (mouse) siC/ebpa GE Life Sciences/ Dharmacon L-040561-00-0005 Transfected construct (mouse) siC/ebpb GE Life Sciences/ Dharmacon L-043110-00-0005 Transfected construct (mouse) siC/ebpd GE Life Sciences/ Dharmacon L-060294-01-0005 Transfected construct (mouse) siC/ebpg GE Life Sciences/ Dharmacon L-065627-00-0005 Transfected construct (human) siATF4 GE Life Sciences/ Dharmacon L-005125-00-0020 Sequenced-based reagent qPCR primers IDT See table in Materials and methods Recombinant DNA reagent ATF4 (cDNA amplified from plasmid) Addgene RRID:Addgene_21845 Recombinant DNA reagent Pspax2 (plasmid) Addgene RRID:Addgene_12260 Recombinant DNA reagent Pmd2.G (plasmid) Addgene RRID:Addgene_12259 Recombinant DNA reagent pSpCas9n(BB) 2A-GFP (PX461) (plasmid) Addgene RRID:Addgene_48140 Recombinant DNA reagent pTRIPZ (plasmid) PMID:27088857 Alex Toker (Beth Israel Deaconess Medical Center) Recombinant DNA reagent GFP (cDNA amplified from plasmid) Addgene RRID:Addgene_19319 Recombinant DNA reagent SLC7A11 (cDNA amplified from plasmid) PMID:29259101 Alex Toker (Beth Israel Deaconess Medical Center) Recombinant DNA reagent pBabe hygro IRES-TSC2 PMID:15150095 David Kwiatkowski (Brigham and Women’s Hospital) Sequenced-based reagent CRISPR-Cas9n guides for KO of ATF4 IDT CACCGGAGG TGGAGGGGCTATGCT; AAACAGCATAGCCCC TCCACCTCC; CACCGACAATCTGCCTTC TCCAGG; AAACCC TGGAGAAGGCAGATTG TC Sequenced-based reagent Sequencing primers for Atf4-/- cell lines IDT TCGATGCTCTGTTTCGAA TG; CTTCTTCCCCC TTGCCTTAC Continued on next page Torrence et al. eLife 2021;10:e63326.

Techniques: Activation Assay, Western Blot, Transfection