Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: (A) E6AP KO HeLa cells were transfected with HA-tagged WT or S218A E6AP, as in . Cells were treated with 0.1 μM ETO for 18 hours, and released in fresh medium for recovery. Cells were then harvested at the indicated time points (after the removal of ETO) for IF using an anti-phospho-Aurora A/B/C antibody. The activation of Aurora phosphorylation (shown in red) and chromosome condensation (in blue) indicated mitosis. The percentages of cells in mitosis were quantified and shown. The mean values and standard deviations were calculated from three experiments. An unpaired 2-tailed Student’s t test was used to determine the statistical significance (** p<0.01). (B) E6AP KO HeLa cells expressing HA-tagged WT or S218A E6AP, as in panel A, were treated with 2 mM HU for 18 hours. Cells were then released in fresh medium, and incubated as indicated, for recovery. Cell cycle progression was analyzed by FACS. (C) WT or S218A E6AP was expressed in E6AP KO HEK293 cells. Cells were treated without or with 0.1 μM ETO for 18 hours, released in fresh medium for recovery, and incubated as indicated. Cells were analyzed by immunoblotting for phospho-CDK substrates and histone H3. (D) WT or S218A E6AP was expressed in E6AP KO HEK293 cells, as in panel C. Cells were treated without or with 1 μM CPT for 90 minutes, and analyzed by immunoblotting for phospho-ATM/ATR substrates, phospho-SMC1 Ser-957, and α-tubulin.

Article Snippet: Other antibodies include α-tubulin (Santa Cruz Biotechnology, #sc-5286), E6AP (Bethyl Laboratories, A300-351), HA (Cell signaling technology #3724), γ-H2AX Ser-139 (Cell signaling technology #9718S), phospho-ATM/ATR substrate motif (Cell signaling technology, #6966S), phospho-SMC1 Ser-957 (Cell signaling technology, #58052), phospho-CHK1 Ser-345 (Cell signaling technology, #2348), phospho-CHK2 Thr-68 (Cell signaling technology, #2197), Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (Cell signaling technology, #2914), Aurora A (Cell signaling technology, #14475), Aurora B (Cell Signaling technology, #3094), CDK1 (Cell signaling technology, #9112), Cyclin B (Cell signaling technology, #4138), phosphor-CDK substrates (Cell signaling technology, #2325), RPA32 (Thermo Fisher Scientific, # PA5-22256), S5a (Boston Biochem, #SP-400), ubiquitin (Cell Signaling Technology, #3936), and GFP (Cell Signaling Technology, #2555).

Techniques: Transfection, Activation Assay, Expressing, Incubation, Western Blot

Journal: bioRxiv

Article Title: The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

doi: 10.1101/2023.02.22.529521

Figure Lengend Snippet: DNA damage induces ATM/ATR activation and checkpoint signaling. Activated ATM/ATR also phosphorylates E6AP Ser-218, leading to the dissociation of E6AP from MASTL and reduced MASTL degradation. The subsequent accumulation of MASTL promotes de-activation of the DNA damage checkpoint and initiates cell cycle resumption by inhibiting dephosphorylation of CDK substrates.

Article Snippet: Other antibodies include α-tubulin (Santa Cruz Biotechnology, #sc-5286), E6AP (Bethyl Laboratories, A300-351), HA (Cell signaling technology #3724), γ-H2AX Ser-139 (Cell signaling technology #9718S), phospho-ATM/ATR substrate motif (Cell signaling technology, #6966S), phospho-SMC1 Ser-957 (Cell signaling technology, #58052), phospho-CHK1 Ser-345 (Cell signaling technology, #2348), phospho-CHK2 Thr-68 (Cell signaling technology, #2197), Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (Cell signaling technology, #2914), Aurora A (Cell signaling technology, #14475), Aurora B (Cell Signaling technology, #3094), CDK1 (Cell signaling technology, #9112), Cyclin B (Cell signaling technology, #4138), phosphor-CDK substrates (Cell signaling technology, #2325), RPA32 (Thermo Fisher Scientific, # PA5-22256), S5a (Boston Biochem, #SP-400), ubiquitin (Cell Signaling Technology, #3936), and GFP (Cell Signaling Technology, #2555).

Techniques: Activation Assay, De-Phosphorylation Assay

Journal: International Journal of Biological Sciences

Article Title: ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

doi: 10.7150/ijbs.71327

Figure Lengend Snippet: USP11 is phosphorylated by activated ERK1/2 at serine 905. (A and B) MCF-7 cell lysates were immunoprecipitated using anti-USP11 (A) or anti-ERK1/2 antibody (B). The immunoprecipitates were then examined with indicated antibodies. (C) MCF-7 cells were treated with DMSO or U0126 (5 µM) for 6 h. Cell lysates were immunoprecipitated with anti-USP11 antibody, followed by western blotting with anti-p-Ser/Thr antibody and other indicated antibodies. (D) Sequence alignment of the ERK1/2 phosphorylation site within USP11 orthologs from different species. (E) MCF-7 cells were transfected with Flag-USP11 wt , Flag-USP11 S905A , empty vector plasmids for 24 h, and then treated with DMSO or U0126 (5 µM) for 6 h. Cell lysates were immunoprecipitated using anti-Flag antibody, followed by western blotting with anti-phospho-MAPK/CDK substrates antibody and other indicated antibodies.

Article Snippet: UltraSignal ECL (catalog no. 4AW011-100) was purchased from 4A Biotech Co., Ltd. Antibodies information: anti-USP11 (Santa Cruz, catalog no. sc-365528/ Abcam, catalog no. ab109232); anti-p21 (Santa Cruz, catalog no. sc-397/ Cell Signaling Technology, catalog no. 2947S); anti-Flag (MBL, catalog no. M185-3L); anti-Myc (MBL, catalog no. M192-3); anti-HA (MBL, catalog no. M180-3); anti-ERK1/2 (Cell Signaling Technology, catalog no. 4695S); anti-phospho-ERK1/2 (Cell Signaling Technology, catalog no. 4376S); anti-phospho-MAPK/CDK Substrates (Cell Signaling Technology, catalog no. 2325S); anti-phosphoserine/threonine (BD Transduction Laboratories, catalog no. M180-3); anti-GAPDH (COOLRUN Life Science, catalog no. AT0002); Dylight 488 (Thermo Fisher Scientific, catalog no. #35502); Dylight 594 (Thermo Fisher Scientific, catalog no. #35560).

Techniques: Immunoprecipitation, Western Blot, Sequencing, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

doi: 10.1371/journal.pone.0009951

Figure Lengend Snippet: A. As indicated, RNAs or proteins were isolated from trunk tissues (Tk), hindlimbs (HL), and forelimbs (FL) of chicken embryos (E4 = day 4 in ovo ) and fetuses (E12), and also from differentiated cultures of mouse C 2 C 12 myogenic cells (C 2 ) and primary cultures of E4 and E12 chicken hindlimb cells. RNAs were analyzed by RT-PCR with exon-spanning primers specific for chicken Prdm1 and, as a positive control, GAPDH as indicated. Proteins were analyzed by immunoblotting with antibodies specific for Prdm1 (Cell Signaling Technology) or GAPDH. Bands with the expected ∼240 bp size of the Prdm1 RT-PCR product or the expected ∼100 kDa M r of the Prdm1 protein were obtained from all chicken embryonic and fetal tissues and from both E4 and E12 chicken myogenic cell cultures. Prdm1 mRNA and protein were not obtained from cells of the mouse C 2 C 12 myogenic cell line, though GAPDH was found. Specificity of the amplified cDNAs for Prdm1 was confirmed by restriction enzyme analysis (not shown). Bands from some gels were re-arranged for presentation. B. Full-length Prdm1 immunoblot (Ab from Cell Signaling Technology) of the same tissue samples shown in panel A, demonstrating lack of non-specific bands. MW = molecular standards with number = kDa.

Article Snippet: From E4 embryos (HH stage 23–25), the forelimb buds, hindlimb buds, and trunk tissue from between the fore- and hindlimbs were dissected and homogenized on ice in a buffer (recommended by Cell Signaling Technology) consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium Pyrophosphate, 1 mM β-glycerophosphate, Sodium Vanadate, and Calbiochem III Protease Inhibitors.

Techniques: Isolation, In Ovo, Reverse Transcription Polymerase Chain Reaction, Positive Control, Western Blot, Amplification

Journal: PLoS ONE

Article Title: Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

doi: 10.1371/journal.pone.0009951

Figure Lengend Snippet: Double immunofluorescence analysis for fast MyHC(s) with mAb F59 ( A , red fluorescence) and slow MyHC(s) with mAb S58 ( B , green fluorescence) showed that fast and slow MyHCs were co-expressed by all differentiated myocytes in somite cultures. In addition, Prdm1 immunostaining (Ab from Cell Signaling Technology) was found in the nuclei of all myocytes, as well as in many MyHC-negative cells ( C , merged image, green fluorescence = S58, red fluorescence = Prdm1, blue fluorescence = nuclei). D . Prdm1 (green fluorescence, Ab from Abcam) was expressed throughout the myotome (mt, arrows) of a mature somite at the forelimb bud level at E4. Additional Prdm1 staining was found in some cells of the sclerotome (sct) and neural tube (nt). Arrows indicate Prdm1-positive nuclei within myocytes. Bar in Panel A = 40 µm for panels A and B); 15 µm for panel C; and 50 µm for panel D.

Article Snippet: From E4 embryos (HH stage 23–25), the forelimb buds, hindlimb buds, and trunk tissue from between the fore- and hindlimbs were dissected and homogenized on ice in a buffer (recommended by Cell Signaling Technology) consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium Pyrophosphate, 1 mM β-glycerophosphate, Sodium Vanadate, and Calbiochem III Protease Inhibitors.

Techniques: Immunofluorescence, Fluorescence, Immunostaining, Staining

Journal: PLoS ONE

Article Title: Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

doi: 10.1371/journal.pone.0009951

Figure Lengend Snippet: As indicated, individual cultures, in this case a proliferating E4 hindlimb culture prior to myotube formation, were analyzed by double immunofluorescence for Prdm1 ( A , Ab from Cell Signaling Technology) and the myoblast marker Pax7 ( B ). The myoblasts illustrated here all co-expressed Prdm1 and Pax7. C . Quantitative analysis of proliferating E4 cultures prior to myotube formation. Cultures were double immunostained for Prdm1 (Ab from Cell Signaling Technology) and Pax7 and staining patterns were quantified. Cells positive for both Pax7 and Prdm1 were the most abundant cell type in somitic (S), forelimb bud (F), and hindlimb bud (H) cultures (yellow bars). A small percentage of cells was positive for Pax7 but negative for Prdm1 (red bars). Some cells were also negative for Pax7 but positive for Prdm1 (green bars) or negative for both (gray bars). Bar = 10 µm.

Article Snippet: From E4 embryos (HH stage 23–25), the forelimb buds, hindlimb buds, and trunk tissue from between the fore- and hindlimbs were dissected and homogenized on ice in a buffer (recommended by Cell Signaling Technology) consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium Pyrophosphate, 1 mM β-glycerophosphate, Sodium Vanadate, and Calbiochem III Protease Inhibitors.

Techniques: Immunofluorescence, Marker, Staining

Journal: PLoS ONE

Article Title: Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

doi: 10.1371/journal.pone.0009951

Figure Lengend Snippet: Somite tissues were dissected from E4 embryos (HH stage 23–25), cultured for 2 days, and analyzed for expression of Prdm1 and MyHC isoforms. A, C, E . In the presence of a non-specific oligonucleotide, cells derived from the main explant formed a surrounding monolayer of cells (A, nuclei) and both Prdm1-positive cells (C, Ab from Cell Signaling Technology) and MyHC-expressing myocytes (E, S58-positive) were found among these cells. The main tissue explant (exp) is to the lower right of each panel. B, D, F . In the presence of Prdm1 antisense nucleotides, formation of the monolayer was not affected (B, nuclei), but expression of both Prdm1 (C, Ab from Cell Signaling Technology) and slow MyHC(s) (F, mAb S58) was inhibited. Staining with mAb F59, and thus fast MyHC expression, was also inhibited by Prdm1 knockdown (not shown). See text for quantitative analysis. Bar in A = 75 µm.

Article Snippet: From E4 embryos (HH stage 23–25), the forelimb buds, hindlimb buds, and trunk tissue from between the fore- and hindlimbs were dissected and homogenized on ice in a buffer (recommended by Cell Signaling Technology) consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium Pyrophosphate, 1 mM β-glycerophosphate, Sodium Vanadate, and Calbiochem III Protease Inhibitors.

Techniques: Cell Culture, Expressing, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: a , Western blots showing time-dependent changes of proteins in MEFs pretreated with DMSO control or ConA (200 nM) for 1 h, and then co-treated with Dox (30 μg/ml) for 0-8 h. All ConA-treated conditions were thus treated with ConA for a total time of 9 h. b , Western blots of MEFs treated with ConA (200 nM) or Torin1 (250 nM) for 0-24 h. c , Western blots of MEFs treated with cycloheximide (CHX), in the absence or presence of ConA (200 nM) for 0-150 min. d , Schematic diagram of the ATF4 translational reporter, comprising the upstream open reading frames (uORF1 and uORF2) of the ATF4 5′ untranslated region (5′UTR) followed by HA-mScarlet tag replacing the ATF4 coding sequence, built on a lentiviral expression vector. The GFP control is directly driven by the cytomegalovirus (CMV) promoter. e , Western blots of MEFs stably expressing the ATF4 translational reporter and GFP control treated with or without Dox (30 μg/ml) or Tunicamycin (TM, 1.5 μg/ml) for 3 h, in the presence of DMSO control or ConA (200 nM). f , Western blots of HEK293T cells expressing control (ctrl), ATP6V0C ( V0C ) or ATP6V0D1 ( V0D1 ) sgRNA, and treated with or without Dox (30 μg/ml) for 24 h. g , Western blots of MEFs pretreated with PBS control or 25-100 μM chloroquine (CQ) for 1 h, and then co-treated with or without Dox (30 μg/ml) for 24 h. h , ATF4 ChIP-qPCR analysis ( n = 4 biologically independent samples) of the promoters of ATF4 targeted genes in MEFs pretreated with DMSO or ConA (200 nM) for 1 h, and then co-treated with or without Dox (30 μg/ml) for 3 h. Error bars denote S.E.M. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test (* P < 0.05; ** P < 0.01; *** P < 0.001; N.S., not significant).

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: Western Blot, Sequencing, Expressing, Plasmid Preparation, Stable Transfection

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: a , ATF4 phosphorylation recognized by a context-dependent (S*P) phosphorylation-specific antibody is increased by Rheb co-expression and inhibited by Torin1. HEK293T cells transfected with the indicated plasmids were immuno-precipitated (IP) with anti-Flag antibody and analyzed by western blots. When applicable, Torin1 (250 nM) were added 2 h before harvest. TCL, total cell lysate. b , mTOR directly phosphorylates ATF4 in vitro . In vitro kinase assay was performed with recombinant GST-tagged human mTOR purified from baculovirus-infected insect cells and recombinant His-tagged human ATF4 with or without Torin1 (250 nM). Arrows indicate the mobility shifts likely separating the hyperphosphorylated and nonphosphorylated ATF4. c , The ratios of the phosphorylated and nonphosphorylated peptides containing the phosphorylation sites of ATF4 from a kinase assay performed similar to ( b ), as determined by mass spectrometry. d , The identified mTOR-targeted phosphorylation sites on ATF4 with the vertebrate orthologs aligned below, with numbering according to the amino acid sequence of human ATF4 protein. NTD, N-terminal domain; BD, Basic domain; CLZ, C-leucine zipper. The highly conserved putative TOR signaling (TOS) motif was also highlighted. e , Validation of the two commercially available antibodies that specifically recognize ATF4 phosphorylation at Ser 166 and Thr 173 , respectively. HEK293T cells transfected with the indicated plasmids were immuno-precipitated with anti-Flag antibody and analyzed by western blots. Torin1 (250 nM) were added 2 h before harvest. f , Increased ATF4 Ser 166 and Thr 173 phosphorylation upon Dox treatment, which was inhibited by ConA and Torin1. Wild-type MEFs were pretreated with DMSO, ConA (200 nM) or Torin1 (250 nM) for 1 h, and then co-treated with or without Dox (30 μg/ml) for 2 h, immuno-precipitated with anti-ATF4 antibody and analyzed by western blots. g , Increased ATF4 phosphorylation upon mitochondrial, but not ER stress inducers. Wild-type MEFs were with treated with Antimycin A (AntiA, 2 μM), Oligomycin (Olig, 2 μM), or Tunicamycin (TM, 1.5 μg/ml) for 2 h, immuno-precipitated with anti-ATF4 antibody and analyzed by western blots. Similar amount of immuno-precipitated ATF4 protein was loaded for different conditions to compare phosphorylation changes in ( f ) and ( g ).

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: Expressing, Transfection, Western Blot, In Vitro, Kinase Assay, Recombinant, Purification, Infection, Mass Spectrometry, Sequencing

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: a , mTORC1 directly phosphorylates ATF4 in vitro . In vitro kinase assay was performed with Flag-tagged mTORC1 immunoprecipitated from HEK293T cells and recombinant His-tagged ATF4, with or without Torin1 (250 nM). b , The representative spectrums for the phosphorylated peptides of human ATF4 identified by Liquid Chromatograph Triple Quadrupole Mass Spectrometer (LC-MS/MS), with numbering according to the amino acid sequence of human ATF4 protein

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: In Vitro, Kinase Assay, Immunoprecipitation, Recombinant, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: a , qRT-PCR results ( n = 4 biologically independent samples) of wild-type (WT) and Atf4 −/− MEFs treated with or without Dox (30 μg/ml) or Antimycin A (AntiA, 2 μM) for 24 h. b , The oxygen consumption rate (OCR) of WT or Atf4 −/− MEFs at basal or after sequential addition of Oligomycin (Olig), FCCP and AntiA/Rotenone. The basal and maximum OCR was statistically analyzed ( n = 6 biologically independent samples). c , Atf4 knockout leads to disrupted mitochondrial network upon mitochondrial stress. MitoTracker staining of WT or Atf4 −/− MEFs treated with or without Dox (30 μg/ml) or AntiA (2 μM) for 24 h. The average mitochondrial network perimeter and area were analyzed by ImageJ with a Mito-Morphology macro ( n = 3 independent experiments). Scale bar, 10 μm. d , Western blots of Atf4 −/− MEFs stably expressing empty vector (vector), the wild-type ATF4 (WT-ATF4), the phospho-defective mutant (5A-ATF4), and an ATF4 mutant carrying a point mutation of the bulky phenylalanine residue 94 in the TOS motif to alanine (F94A-ATF4). e , qRT-PCR results ( n = 4 biologically independent samples) of Atf4 −/− MEFs stably expressing vector, wild-type, 5A or F94A forms of ATF4, treated with or without Dox (30 μg/ml) for 24 h. Error bars denote S.E.M. Statistical analysis was performed by two-tailed unpaired Student’s t -test in ( b ), or by ANOVA followed by Tukey post-hoc test in ( a ), ( c ) and ( e ) (** P < 0.01; *** P < 0.001; N.S., not significant).

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: Quantitative RT-PCR, Knock-Out, Staining, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: a , ATF4 phosphorylation defective (5A) and TOS motif disrupted (F94A) mutants failed to bind to the promoters of UPR mt genes in Atf4 −/− MEFs upon Dox treatment. ATF4 ChIP-qPCR analysis ( n = 4 biologically independent samples) of the promoters of the UPR mt genes in Atf4 −/− MEFs stably expressing empty vector, wild-type, 5A or F94A forms of ATF4, with or without Dox (30 μg/ml) treatment for 3 h. b , qRT-PCR results ( n = 4 biologically independent samples) of Atf4 −/− MEFs stably expressing empty vector, wild-type, 5A or F94A forms of ATF4, with or without Tunicamycin (TM, 1.5 μg/ml) treatment for 24 h. Error bars denote S.E.M. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test (* P < 0.05; ** P < 0.01; *** P < 0.001; N.S., not significant).

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: a , MitoTracker staining of Atf4 −/− MEFs stably expressing wild-type (WT), 5A or F94A forms of ATF4, with or without Dox (30 μg/ml) or AntiA (2 μM) treatment for 24 h. The average mitochondrial network perimeter and area were analyzed by ImageJ with a Mito-Morphology macro ( n = 3 independent experiments). Scale bar, 10 μm. b , The oxygen consumption rate (OCR) of Atf4 −/− MEFs stably expressing WT, 5A or F94A forms of ATF4, after DMSO control (ctrl) or AntiA (2 μM) treatment for 24 h. The basal and maximum OCR was analyzed ( n = 6 biologically independent samples). c , Flow cytometry analysis of the mitochondrial superoxide (MitoSOX) intensity of Atf4 −/− MEFs stably expressing WT, 5A or F94A forms of ATF4, after DMSO control or AntiA (2 μM) exposure for 48 h. The percentages of MitoSOX-positive cells were analyzed ( n = 3 independent experiments). d , Representative bright field photographs of Atf4 −/− MEFs stably expressing empty vector, WT, 5A or F94A forms of ATF4, grown with or without the antioxidant β-mercaptoethanol (β-ME) or AntiA (2 μM) for 96 h. Mean percentages (± S.E.M) of the survival ratio of cells are indicated ( n = 3 independent experiments). Error bars denote S.E.M. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test (** P < 0.01; *** P < 0.001; N.S., not significant).

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: Staining, Stable Transfection, Expressing, Flow Cytometry, Plasmid Preparation

Journal: bioRxiv

Article Title: Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation

doi: 10.1101/2022.11.14.516427

Figure Lengend Snippet: Left: in response to mitochondrial stress, mTORC1 is activated at the lysosomal surface and EIF2α phosphorylation is mildly increased, leading to moderate increase in ATF4 translation. Meanwhile, activated mTORC1 directly phosphorylates ATF4, leading to increased ATF4 binding to the promoters of mitochondrial UPR (UPR mt ) genes and UPR mt activation. Right: in response to ER stress, mTORC1 activity is suppressed but the EIF2α phosphorylation is dramatically increased, leading to strongly increased translation and nuclear accumulation of ATF4, and subsequent activation of ER UPR (UPR ER ). CQ, chloroquine. ConA/BafA1, Concanamycin A/Bafilomycin A1.

Article Snippet: For western blotting, the antibodies used were: P-EIF2α (Cat. 3597, CST, 1:500), Tubulin (Cat. T5168, Sigma, 1:2,000), P-S6K (Cat. 9205, CST, 1:1,000), S6K (Cat. 9202, CST, 1:1,000), P-S6 (Cat. 2215, CST, 1:1,000), S6 (Cat. 2317, CST,1:1,000), P-4E-BP1 (Cat. 9644, CST, 1:1,000), 4E-BP1 (Cat. 2855, CST, 1:1,000), ASNS (Santa Cruz, Cat. sc-365809, 1:1,000), EIF2α (Cat. 9722, CST, 1:1,000), ATF4 (Cat. 11815, CST, 1:1,000), ATF5 (Cat. ab60126, Abcam, 1:1,000), ATP6V0D1 (Cat. ab202899, Abcam, 1:1,000), Flag-tag (F7425, Sigma, 1:1,000), Myc-tag (Cat. sc-40, Santa Cruz, 1:2,000), mTOR (Cat. 2972, CST, 1:1,000), Phospho-S*P (Cat. 2325, CST, 1:1,000; for detecting P-S166-ATF4), Phospho-ST*P (Cat. 5243, CST, 1:1,000; for detecting P-T173-ATF4), and HRP-labelled anti-rabbit (Cat. 7074, CST, 1:5,000), anti-rabbit (Light-Chain Specific) (Cat. 93702, CST, 1:5,000, for detecting the endogenously immunoprecipitated ATF4 and its phosphorylation) and anti-mouse (Cat. 7076, CST, 1:5,000) secondary antibodies.

Techniques: Binding Assay, Activation Assay, Activity Assay

Journal: Cell reports

Article Title: Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

doi: 10.1016/j.celrep.2022.111371

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) , Cell Signaling Technologies , 2325.

Techniques: Recombinant, Protease Inhibitor, Staining, Cell Isolation, Flow Cytometry, Purification, Fluorescence, Software

Journal: Cell reports

Article Title: Thymidine rescues ATR kinase inhibitor-induced deoxyuridine contamination in genomic DNA, cell death, and interferon-α/β expression

doi: 10.1016/j.celrep.2022.111371

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) , Cell Signaling Technologies , 2325.

Techniques: Recombinant, Protease Inhibitor, Staining, Cell Isolation, Flow Cytometry, Purification, Fluorescence, Software