Journal: Nucleic Acids Research

Article Title: Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres

doi: 10.1093/nar/gkac1269

Figure Lengend Snippet: TRF2 phosphorylation at S410 increases its binding to TIN2. ( A ) Biotin-Ahx-ISRLVLEEDpSQSTEPSAGLN (TRF2-pS410) and Biotin-Ahx–SRLVLEEDSQSTEPSAGLN (TRF-CTRL) peptides were incubated with nuclear extracts and pulled down by streptavidin beads. Bound proteins were identified by mass spectrometry ( n = 3). Plotted are –log P values of proteins enriched or reduced in condition with TRF2-pS410 peptide. The line delineates the statistical significance (FDR < 0.1). ( B ) Fluorescently-labelled TRF2-pS410 and TRF2-CTRL peptides were titrated with purified TIN2 to a final concentration of 500 nM. Fluorescence anisotropy change was measured and dissociation constant values for unmodified and modified oligopeptides were calculated as described in Methods. ( C ) HEK293 cells stably expressing EGFP-TRF2 were treated with DMSO or with PPM1D inhibitor for 4 h. EGFP-TRF2 was immunoprecipitated from cell extracts with GFP Trap. Proteins were separated by SDS-PAGE and binding of TIN2 was determined by immunoblotting. Numbers at the bottom indicate the TIN2 signal relative to the total immunoprecipitated TRF2 and normalized to the wild-type TRF2. Representative result from three experiments is shown. ( D ) TRF2:TIN2 interaction was determined in parental U2OS and U2OS-PPM1D-KO cells by PLA. Mean PLA foci count is plotted ± SD, n = 500. Statistical significance was evaluated using Mann–Whitney test (**** P < 0.0001). Representative experiment is shown from two independent repeats. ( E ) TRF2:TIN2 interaction was determined in U2OS cells treated with DMSO or PPM1Di by PLA. Mean PLA foci count is plotted ± SD, n = 500. Statistical significance was evaluated using Mann–Whitney test (**** P < 0.0001). Representative experiment is shown from two independent repeats. ( F ) U2OS cells were treated or not with PPM1Di for 24 h, pre-extracted, fixed and stained with TRF2 (m-Santa Cruz) and TIN2 (Rb-Novus) antibodies. Mean TIN2 intensity in TRF2 foci is plotted ± SD, n = 300. Statistical significance was evaluated using Mann–Whitney test (**** P < 0.0001). Representative experiment is shown from two independent repeats. The scale bar represents 10 μm. ( G ) Parental U2OS, U2OS-PPM1D-KO and U2OS-PPM1D-KO stably expressing FLAG-PPM1D cells were treated or not with PPM1Di for 24 h. Cells were pre-extracted, fixed and stained for TIN2 and TRF2. Mean TIN2 intensity in TRF2 foci ± SD is plotted, n = 300. Statistical significance was evaluated using Mann–Whitney test (**** P < 0.0001). Representative experiment is shown from two independent repeats. ( H ) Levels of TRF2 and TIN2 were analyzed in whole cell extracts from the parental U2OS and U2OS-PPM1D-KO cells by immunoblotting. Importin staining was used as a loading control. ( I ) Cells from G were analysed for TRF2 intensity in TRF2 foci. Plotted is mean ± SD, n = 300. ( J ) U2OS cells were treated or not with PPM1Di for 24 h, pre-extracted, fixed and stained with TRF2 and TPP1 antibodies. Mean TPP1 intensity in TRF2 foci normalized to the mean nuclear TPP1 intensity ± SD is plotted, n > 300. Statistical significance was evaluated using Mann–Whitney test (**** P < 0.0001). The scale bars in representative images corresponds to 10 μm and 1 μm respectively. ( K ) Parental U2OS, U2OS-PPM1D-KO cells and U2OS-PPM1D-KO stably expressing FLAG-PPM1D cells were pre-extracted, fixed and stained for TPP1 and TRF2. Mean TPP1 intensity in TRF2 foci normalized to the mean nuclear TPP1 intensity ± SD is plotted, n > 300. Statistical significance was evaluated using Mann–Whitney test (**** P < 0.0001, *** P < 0.001). ( L ) Chromosome spreads from parental U2OS and U2OS-PPM1D-KO cells were hybridized with TAACCC FISH-probe and imaged by 3D-SIM. Plotted is a fraction of telomeres that formed t-loops. More than 203 telomeres were quantified per condition in each experiment ( n = 3). Significance was determined by unpaired t -test.

Article Snippet: The following antibodies were used in this study: TRF2 (ab108997, for WB), TIN2 (ab197894, for WB) from Abcam; TRF2 (NB110-57130, for IF), TIN2 (NBP2-55709, for IF), RAP1 (NBP1-82433, for IF), 53BP1 (NB100-305, for IF) from Novus Biologicals; TRF2 (sc271710, for IF), TIN2 (sc73177, for IF), TPP1 (sc100597, for IF and WB), RAP1 (sc53434, for WB), PPM1D (sc376257, for IF and WB), PPM1D (sc20712, for IF) from Santa Cruz Biotechnology; Phospho-Histone H2A.X (Ser139) (clone D7T2V, #80312), KAP1-S824 (#4127) and PPM1D (clone D4F7, 11901 for WB) from Cell Signaling Technology; γH2AX (05-636, for WB), GFP (11814460001, for WB), FLAG (F1804, for IF), Fk2 (04-263, for IF) from Roche.

Techniques: Phospho-proteomics, Binding Assay, Incubation, Mass Spectrometry, Purification, Concentration Assay, Fluorescence, Modification, Stable Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot, MANN-WHITNEY, Staining, Control

Journal: Cell metabolism

Article Title: FBW7 Mediates Senescence and Pulmonary Fibrosis through Telomere Uncapping.

doi: 10.1016/j.cmet.2020.10.004

Figure Lengend Snippet: Figure 2. FBW7 Mediates TPP1 Multisite Polyubiquitination and Turnover via GSK3b Signaling in Stress Response (A) Effect of various E3 ubiquitin ligase gene KDs on TPP1 in A549 cells exposed to H2O2, BLM, or IR by IB. Cells transfected for 48 h were exposed to BLM (200 mM, 16 h), H2O2 (1 mM), or IR (8 Gy).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 Thermo Cat#A27034 Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 555 Thermo Cat#A27039 Anti-rabbit IgG-Peroxidase antibody produced in goat Sigma Cat#A6154 Anti-Rap1 antibody IMGENEX Cat#IMG-289 Anti-SP-C antibody (for western blot) Abcam Cat#ab90716 Anti-SP-C antibody (for immunofluorescence) Santa Cruz Biotechnology Cat#sc-7706 Anti-T1a antibody Biolegend Cat#127408 Anti-Ter119 antibody Biolegend Cat#116204 Anti-TIN2 antibody GeneTex Cat59B388 Anti-TPP1 antibody (for immunofluorescence) Novus Cat#NBP2-45475 Anti-TPP1 antibody (for western blot) Abcam Cat#ab112050 and ab54685 Anti-TRF1 antibody Cell Signaling Technology Cat#3529 Anti-TRF2 antibody Novus Cat#NB100-56506 Anti-a-SMA antibody Sigma Cat#A2547 Anti-a-Tubulin antibody Cell Signaling Technology Cat#2144 Peroxidase-AffiniPure goat anti mouse IgG Jackson Cat#115-035-062 Bacterial and Virus Strains Cre lentivirus Genomeditech Cat#GM-0220CR02 E. coli (DH5a) TransGen Biotech Cat#CD201-01 pEF-1a-TPP1 lentivirus GenePharma N/A pLMCS-FBW7a lentivirus Invitrogen (Shanghai) N/A pLMCS-FBW7g lentivirus Invitrogen (Shanghai) N/A Chemicals, Peptides, and Recombinant Proteins Bleomycin Sigma Cat#5507 BrdU Sigma Cat#B5002 CHIR99021 Sigma Cat#SML1046 Colcemid Thermo Cat# 15212012 Cycloheximide Sigma Cat#239763 Digoxin Roche Cat#11585614910 Dispase II Sigma Cat#4942078001 DMSO Sigma Cat#D8418 Dynabeads MyOne streptavidin T1 magnetic beads Thermo Cat#65601 Flag-coupled magnetic beads Sigma Cat#M8823 Glutathione-Sepharose 4B Sigma Cat#GE17-0756-01 H2O2 (3%) Sigma Cat#88597 Lipofectamine 2000 Thermo Cat#11668019 Masson Trichrome Sigma Cat#HT153 MG132 Sigma Cat#M8699 Peptides KE BIOCHEM See Table S5 PNA probe Panagene Cat#F1001,F1002 Streptavidin-PE Biolegend Cat#405204 TC-G24 TOCRIS Cat#4353 (Continued on next page) e2 Cell Metabolism 32, 1–18.e1–e9, November 3, 2020

Techniques: Ubiquitin Proteomics, Transfection

Journal: Cell metabolism

Article Title: FBW7 Mediates Senescence and Pulmonary Fibrosis through Telomere Uncapping.

doi: 10.1016/j.cmet.2020.10.004

Figure Lengend Snippet: Figure 4. FBW7 Regulates Telomere Uncapping and Cell Proliferation in Response to H2O2 or IR in a TPP1-Dependent Manner (A) Effect of FBW7 KD on TIFs determined by IF-FISH in HeLa cells transfected with the indicated siRNAs for 48 h. (B and C) Effect of FBW7 KD on telomere length by flow-FISH in HT1080 cells (B) and on telomere numbers by metaphase FISH per chromosome in HeLa cells (C).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 Thermo Cat#A27034 Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 555 Thermo Cat#A27039 Anti-rabbit IgG-Peroxidase antibody produced in goat Sigma Cat#A6154 Anti-Rap1 antibody IMGENEX Cat#IMG-289 Anti-SP-C antibody (for western blot) Abcam Cat#ab90716 Anti-SP-C antibody (for immunofluorescence) Santa Cruz Biotechnology Cat#sc-7706 Anti-T1a antibody Biolegend Cat#127408 Anti-Ter119 antibody Biolegend Cat#116204 Anti-TIN2 antibody GeneTex Cat59B388 Anti-TPP1 antibody (for immunofluorescence) Novus Cat#NBP2-45475 Anti-TPP1 antibody (for western blot) Abcam Cat#ab112050 and ab54685 Anti-TRF1 antibody Cell Signaling Technology Cat#3529 Anti-TRF2 antibody Novus Cat#NB100-56506 Anti-a-SMA antibody Sigma Cat#A2547 Anti-a-Tubulin antibody Cell Signaling Technology Cat#2144 Peroxidase-AffiniPure goat anti mouse IgG Jackson Cat#115-035-062 Bacterial and Virus Strains Cre lentivirus Genomeditech Cat#GM-0220CR02 E. coli (DH5a) TransGen Biotech Cat#CD201-01 pEF-1a-TPP1 lentivirus GenePharma N/A pLMCS-FBW7a lentivirus Invitrogen (Shanghai) N/A pLMCS-FBW7g lentivirus Invitrogen (Shanghai) N/A Chemicals, Peptides, and Recombinant Proteins Bleomycin Sigma Cat#5507 BrdU Sigma Cat#B5002 CHIR99021 Sigma Cat#SML1046 Colcemid Thermo Cat# 15212012 Cycloheximide Sigma Cat#239763 Digoxin Roche Cat#11585614910 Dispase II Sigma Cat#4942078001 DMSO Sigma Cat#D8418 Dynabeads MyOne streptavidin T1 magnetic beads Thermo Cat#65601 Flag-coupled magnetic beads Sigma Cat#M8823 Glutathione-Sepharose 4B Sigma Cat#GE17-0756-01 H2O2 (3%) Sigma Cat#88597 Lipofectamine 2000 Thermo Cat#11668019 Masson Trichrome Sigma Cat#HT153 MG132 Sigma Cat#M8699 Peptides KE BIOCHEM See Table S5 PNA probe Panagene Cat#F1001,F1002 Streptavidin-PE Biolegend Cat#405204 TC-G24 TOCRIS Cat#4353 (Continued on next page) e2 Cell Metabolism 32, 1–18.e1–e9, November 3, 2020

Techniques: Transfection

Journal: Cell metabolism

Article Title: FBW7 Mediates Senescence and Pulmonary Fibrosis through Telomere Uncapping.

doi: 10.1016/j.cmet.2020.10.004

Figure Lengend Snippet: Figure 5. TPP1 KD Causes Telomere Shortening, AEC2 Stem Cell Senescence, and Pulmonary Fibrosis in Mice (A–G) TPP1 or FBW7 KD by weekly intratracheal shRNA lentiviruses for 4 times, before IB (A), FACS (B and C), examinations of lung function (D–F), and fibrotic marker HYP content (G). (H–J) Masson’s trichrome staining (H), telomere Q-FISH of 300–500 AEC2 cells (I), and IF for SPC-positive AEC2 cells (J) and HP1g-positive AEC2 cells (insets in J) in lung sections from 3 animals per group (20 fields per lung). *p < 0.05 versus control shRNA. (K) qPCR for FBW7, SPC, p21, a-SMA, and Col1a1 gene expression in the lung with TPP1 or FBW7 deficiency.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 Thermo Cat#A27034 Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 555 Thermo Cat#A27039 Anti-rabbit IgG-Peroxidase antibody produced in goat Sigma Cat#A6154 Anti-Rap1 antibody IMGENEX Cat#IMG-289 Anti-SP-C antibody (for western blot) Abcam Cat#ab90716 Anti-SP-C antibody (for immunofluorescence) Santa Cruz Biotechnology Cat#sc-7706 Anti-T1a antibody Biolegend Cat#127408 Anti-Ter119 antibody Biolegend Cat#116204 Anti-TIN2 antibody GeneTex Cat59B388 Anti-TPP1 antibody (for immunofluorescence) Novus Cat#NBP2-45475 Anti-TPP1 antibody (for western blot) Abcam Cat#ab112050 and ab54685 Anti-TRF1 antibody Cell Signaling Technology Cat#3529 Anti-TRF2 antibody Novus Cat#NB100-56506 Anti-a-SMA antibody Sigma Cat#A2547 Anti-a-Tubulin antibody Cell Signaling Technology Cat#2144 Peroxidase-AffiniPure goat anti mouse IgG Jackson Cat#115-035-062 Bacterial and Virus Strains Cre lentivirus Genomeditech Cat#GM-0220CR02 E. coli (DH5a) TransGen Biotech Cat#CD201-01 pEF-1a-TPP1 lentivirus GenePharma N/A pLMCS-FBW7a lentivirus Invitrogen (Shanghai) N/A pLMCS-FBW7g lentivirus Invitrogen (Shanghai) N/A Chemicals, Peptides, and Recombinant Proteins Bleomycin Sigma Cat#5507 BrdU Sigma Cat#B5002 CHIR99021 Sigma Cat#SML1046 Colcemid Thermo Cat# 15212012 Cycloheximide Sigma Cat#239763 Digoxin Roche Cat#11585614910 Dispase II Sigma Cat#4942078001 DMSO Sigma Cat#D8418 Dynabeads MyOne streptavidin T1 magnetic beads Thermo Cat#65601 Flag-coupled magnetic beads Sigma Cat#M8823 Glutathione-Sepharose 4B Sigma Cat#GE17-0756-01 H2O2 (3%) Sigma Cat#88597 Lipofectamine 2000 Thermo Cat#11668019 Masson Trichrome Sigma Cat#HT153 MG132 Sigma Cat#M8699 Peptides KE BIOCHEM See Table S5 PNA probe Panagene Cat#F1001,F1002 Streptavidin-PE Biolegend Cat#405204 TC-G24 TOCRIS Cat#4353 (Continued on next page) e2 Cell Metabolism 32, 1–18.e1–e9, November 3, 2020

Techniques: shRNA, Marker, Staining, Control, Gene Expression

Journal: Cell metabolism

Article Title: FBW7 Mediates Senescence and Pulmonary Fibrosis through Telomere Uncapping.

doi: 10.1016/j.cmet.2020.10.004

Figure Lengend Snippet: Figure 6. Peptidomimetic TELODIN Inhibits FBW7 Binding to TPP1 and Telomere Uncapping in Human Lung Epithelial Cells (A–C) Structure of TELODIN. FBW7 F-box and WD40 domains with the TELODIN motif colored in purple (PDB: 2OVP) (A), TELODIN in FBW7 WD40 domain (B), and synthetic TELODIN equilibrated conformation under a 300 ns MD simulation (C).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 Thermo Cat#A27034 Anti-rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 555 Thermo Cat#A27039 Anti-rabbit IgG-Peroxidase antibody produced in goat Sigma Cat#A6154 Anti-Rap1 antibody IMGENEX Cat#IMG-289 Anti-SP-C antibody (for western blot) Abcam Cat#ab90716 Anti-SP-C antibody (for immunofluorescence) Santa Cruz Biotechnology Cat#sc-7706 Anti-T1a antibody Biolegend Cat#127408 Anti-Ter119 antibody Biolegend Cat#116204 Anti-TIN2 antibody GeneTex Cat59B388 Anti-TPP1 antibody (for immunofluorescence) Novus Cat#NBP2-45475 Anti-TPP1 antibody (for western blot) Abcam Cat#ab112050 and ab54685 Anti-TRF1 antibody Cell Signaling Technology Cat#3529 Anti-TRF2 antibody Novus Cat#NB100-56506 Anti-a-SMA antibody Sigma Cat#A2547 Anti-a-Tubulin antibody Cell Signaling Technology Cat#2144 Peroxidase-AffiniPure goat anti mouse IgG Jackson Cat#115-035-062 Bacterial and Virus Strains Cre lentivirus Genomeditech Cat#GM-0220CR02 E. coli (DH5a) TransGen Biotech Cat#CD201-01 pEF-1a-TPP1 lentivirus GenePharma N/A pLMCS-FBW7a lentivirus Invitrogen (Shanghai) N/A pLMCS-FBW7g lentivirus Invitrogen (Shanghai) N/A Chemicals, Peptides, and Recombinant Proteins Bleomycin Sigma Cat#5507 BrdU Sigma Cat#B5002 CHIR99021 Sigma Cat#SML1046 Colcemid Thermo Cat# 15212012 Cycloheximide Sigma Cat#239763 Digoxin Roche Cat#11585614910 Dispase II Sigma Cat#4942078001 DMSO Sigma Cat#D8418 Dynabeads MyOne streptavidin T1 magnetic beads Thermo Cat#65601 Flag-coupled magnetic beads Sigma Cat#M8823 Glutathione-Sepharose 4B Sigma Cat#GE17-0756-01 H2O2 (3%) Sigma Cat#88597 Lipofectamine 2000 Thermo Cat#11668019 Masson Trichrome Sigma Cat#HT153 MG132 Sigma Cat#M8699 Peptides KE BIOCHEM See Table S5 PNA probe Panagene Cat#F1001,F1002 Streptavidin-PE Biolegend Cat#405204 TC-G24 TOCRIS Cat#4353 (Continued on next page) e2 Cell Metabolism 32, 1–18.e1–e9, November 3, 2020

Techniques: Binding Assay