Key reagents and resource
| Reagent or Resource . | Source . | Identifier . |
|---|---|---|
| Antibodies | ||
| Rabbit anti-Sun2 | Christian Schlieker lab (Yale University) (Rose et al., 2014) | N/A |
| Rabbit anti-Sun2 | Christopher Carroll lab (Yale University) | N/A |
| Rabbit anti-LBR | Christopher Carroll lab (Yale University) | N/A |
| Rabbit anti-Sun1 | Atlas antibodies | Cat# HPA008346, RRID:AB_1080462 |
| Mouse anti-Lamin A/C | Santa Cruz Biotechnology | Cat# sc-376248, RRID:AB_10991536 |
| Rabbit anti-Lamin B1 | Abcam | Cat# ab16048, RRID:AB_443298 |
| Rabbit anti-emerin | Proteintech | Cat# 10351-1-AP, RRID:AB_2100056 |
| Rabbit anti-Lipin 1 | Millipore Sigma | Cat# ABS400 (discontinued) |
| Rabbit anti-NRF2 | Abcam | Cat# ab62352, RRID:AB_944418 |
| Mouse anti-mNeonGreen | ChromoTek | Cat# 32f6-100, RRID:AB_2827566 |
| Mouse anti-HA | Enzo | Cat# ENZ-ABS120 |
| Rabbit anti-HA | Cell Signaling Technologies | Cat# 3724; RRID: AB_1549585 |
| Rabbit anti-Flag | Sigma-Aldrich | Cat# F3165, RRID:AB_259529 |
| Mouse anti-tubulin alpha | Millipore | Cat# 05-829, RRID:AB_310035 |
| Bacterial and virus strains | ||
| E. coli BL21(DE3) cells | New England Biolabs | Cat# C2527 |
| Chemicals, peptides, and recombinant proteins | ||
| Propranolol | Millipore Sigma | Cat# 537075 |
| Puromycin | Thermo Fisher | Cat# A1113803 |
| TOFA | Cayman Chemicals | Cat# 10005263 |
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (chloroform) | Avanti | Cat# 850457C |
| 18:1 (Δ9-Cis) PC (DOPC) | Avanti | Cat# 850375C |
| 18:1 (Δ9-Cis) PE (DOPE) | Avanti | Cat# 850725C |
| 18:1 DG (DAG) | Avanti | Cat# 800811C |
| 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti | Cat# 850355C |
| Cholesterol | Sigma-Aldrich | Cat# C3045 |
| Pierce Glutathione Agarose | Thermo Fisher Scientific | Cat# 16100 |
| Digitonin | Sigma-Aldrich | Cat# D5628 |
| Cycloheximide | Cell Signaling | Cat# 2112 |
| 3-Indoleacetic acid (IAA) | Sigma-Aldrich | Cat# I3750 |
| ProLong Gold Antifade Mountant with DAPI | Thermo Fisher Scientific | Cat# P36935 |
| Experimental models: Cell lines | ||
| U2OS | Slack lab (Harvard Medical School) | N/A |
| U2OS CTDNEP1^EGFP | This study | N/A |
| U2OS mNeonGreen-NLS-2xC1ab (INM-DAG biosensor) stable | This study | N/A |
| DLD-1 OsTIR-1 stable | (Holland et al., 2012) | N/A |
| DLD-1 OsTIR-1 stable CTDNEP1EN-mAID-HA | This study | N/A |
| HEK293T | Breslow lab (Yale University) | N/A |
| Oligonucleotides | ||
| siGENOME non-targeting siRNA Pool #2 | Dharmacon | Cat# D-001206-14-05 |
| CTDNEP1 SMARTpool siRNA | (Merta et al., 2021) Dharmacon | Cat# M-017869-00-0005 |
| Lipin 1 custom single siRNA, no modifications: 5′-GAAUGGAAUGCCAGCUGAA-3′ | (Brohée et al., 2015; Sołtysik et al., 2021) Dharmacon | N/A |
| Silencer select siRNA against human LMNA | Thermo Fisher | Cat# AM16708; assay ID 144426 |
| BTRC&FBXW11 custom single siRNA (targeting both genes), no modifications: 5′-GUGGAAUUUGUGGAACAUC-3′ | (Kim et al., 2015) Dharmacon | N/A |
| qPCR primer: Hs Sun2 forward: 5′-TGACGTGCCTGACGTATGG-3′ | Primer Bank (Wang et al., 2012) | PrimerBank ID 313760642c1 |
| qPCR primer: Hs Sun2 reverse: 3′-AAATGTGGCGATGAGTCTCTG-5′ | Primer Bank | PrimerBank ID 313760642c1 |
| qPCR primer: Hs CTDNEP1 forward: 5′-CATTTACCTTCTGCGGAGGC-3′ | (Merta et al., 2021) | N/A |
| qPCR primer: Hs CTDNEP1 reverse: 3′-CACCTGGGCTAGCCGATTC-5′ | (Merta et al., 2021) | N/A |
| qPCR primer: Hs 36B4 forward: 5′-AACATGCTCAACATCTCCCC-3′ | (Neuhaus et al., 2011) | N/A |
| qPCR primer: Hs 36B4 reverse: 3′-CCGACTCCTCCGACTCTTC-5′ | (Neuhaus et al., 2011) | N/A |
| Recombinant DNA | ||
| CTDNEP1-GFP HR template | This study | N/A |
| pSPCas9(BB)-2A-Puro (PX459) v2.0 | (Ran et al., 2013) | Cat #62988; RRID: Addgene_62988 |
| pX459 CTDNEP1_HR7 | This study | N/A |
| POM121-mCherry | (Dultz et al., 2008) | Euroscarf #P30554 |
| sfCherry2(1-10) | (Feng et al., 2017) | RRID:Addgene_82602 |
| sfCherry2(11)_H2B | (Feng et al., 2017) | RRID:Addgene_82605 |
| pcDNA CTDNEP1-sfCherry2(1-10) | This study | N/A |
| Flag-sfCherry2(1-10)-NEP1R1 | This study | N/A |
| sfCherry2(11)-EGFP-H2B | This study | N/A |
| mNeonGreen-NLS | This study | N/A |
| mNeonGreen-NLS-1xC1ab | This study | N/A |
| mNeonGreen-NLS-2xC1ab (INM-DAG biosensor) | This study | N/A |
| mNeonGreen-NLS-2xC1ab (2xWG) | This study | N/A |
| pCMV-VSV-G | (Matsudaira et al., 2017) | N/A |
| pCG-Gal-Pol | (Matsudaira et al., 2017) | N/A |
| pMXs-IP-EGFP | (Matsudaira et al., 2017) | N/A |
| pMXs-IP-mNeonGreen-NLS-2xC1ab | This study | N/A |
| CTDNEP1-mAID-HA wild-type | This study | N/A |
| CTDNEP1-mAID-HA D67E | This study | N/A |
| pRK5 Flag-mLipin 1β (mouse) wild-type | (Peterson et al., 2011) | RRID: Addgene_32005 |
| pRK5 Flag-mLipin 1β (mouse) D712/D714E | This study | N/A |
| Flag-hLipin 1βRR (human, resistant to siLipin 1) wild-type | This study | N/A |
| Flag-hLipin 1βRR (human, resistant to siLipin 1) D714E | This study | N/A |
| Flag-DGAT1 (mouse) wild-type | (Sołtysik et al., 2021; Xu et al., 2012) | N/A |
| Flag-DGAT1 (mouse) H426A | This study | N/A |
| mScaret-I-DGAT1 (mouse) wild-type | This study | N/A |
| mScaret-I-DGAT1 (mouse) H426A | This study | N/A |
| Venus-DGAT2 (human) wild-type | (Sołtysik et al., 2021; Xu et al., 2012) | N/A |
| Venus-DGAT2 (human) H163A | This study | N/A |
| GFP-Sec61β | Gia Voeltz lab | N/A |
| GFP-CHMP7 under CMVΔ5 | (Gu et al., 2017) | RRID: Addgene_97006 |
| Sun2 (human)-mNeonGreen | This study | N/A |
| Sun1 (human)-mNeonGreen | This study | N/A |
| Sun1(1-219)-Sun2(213-717)-mNeonGreen | This study | N/A |
| Sun2(1-209)-Sun1(220-716)-mNeonGreen | This study | N/A |
| Sun1(1-138)-Sun2(126-717)-mNeonGreen | This study | N/A |
| Sun2(1-180)-Sun1(191-716)-mNeonGreen | This study | N/A |
| Sun2(1-150)-Sun1(156-716)-mNeonGreen | This study | N/A |
| pGEX-6P-1 GST-AH | This study | N/A |
| pGEX-6P-1 GST-AH (LVW/A) | This study | N/A |
| AH-mNeonGreen | This study | N/A |
| AH-mNeonGreen-NLS | This study | N/A |
| AH(LVW/A)-mNeonGreen-NLS | This study | N/A |
| Sun1(156-190) (Sun1-AH)-mNeonGreen-NLS | This study | N/A |
| HA-AH-mNeonGreen-NLS | This study | N/A |
| Sun2 (LVW/A)-mNeonGreen | This study | N/A |
| Sun2 (2xSA)-mNeonGreen | This study | N/A |
| Sun2 (2xSA- LVW/A)-mNeonGreen | This study | N/A |
| pMK292 (mAID-mCherry2-NeoR) | (Natsume et al., 2016) | RRID:Addgene_72830 |
| CTDNEP1-mAID-HA HR template | This study | N/A |
| Software and algorithms | ||
| FIJI | (Schindelin et al., 2012) | https://imagej.net/Fiji |
| GraphPad Prism 9 | GraphPad Software | https://www.graphpad.com/scientificsoftware/prism/ |
| SnapGene | GSL Biotech LLC | https://www.snapgene.com/ |
| ImageJ file name encrypter | Astha Jaiswal, Holger Lorenz | https://imagej.net/plugins/blind-analysis-tools |
| ImageJ StarDist | (Schmidt et al., 2018) | https://imagej.net/plugins/stardist |
| TMHMM | (Krogh et al., 2001) | https://services.healthtech.dtu.dk/service.php?TMHMM-2.0 |
| PEP-FOLD3 | (Lamiable et al., 2016; Thévenet et al., 2012) | https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/ |
| HeliQuest | (Gautier et al., 2008) | https://heliquest.ipmc.cnrs.fr/ |
| MemBrain | (Feng et al., 2022) | http://www.csbio.sjtu.edu.cn/bioinf/MemBrain/ |
| AmphipaSeeK | (Combet et al., 2000; Sapay et al., 2006) | https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_amphipaseek.html |
| Clustal Omega | (Madeira et al., 2022; Sievers et al., 2011) | https://www.ebi.ac.uk/Tools/msa/clustalo/ |
| ConSurf | (Ashkenazy et al., 2016; Ashkenazy et al., 2010) | https://consurf.tau.ac.il/ |
| Python version 3.8 | Python Software Foundation | https://www.python.org/ |
| Anaconda | Anaconda Inc | https://www.anaconda.com/products/distribution |
| Reagent or Resource . | Source . | Identifier . |
|---|---|---|
| Antibodies | ||
| Rabbit anti-Sun2 | Christian Schlieker lab (Yale University) (Rose et al., 2014) | N/A |
| Rabbit anti-Sun2 | Christopher Carroll lab (Yale University) | N/A |
| Rabbit anti-LBR | Christopher Carroll lab (Yale University) | N/A |
| Rabbit anti-Sun1 | Atlas antibodies | Cat# HPA008346, RRID:AB_1080462 |
| Mouse anti-Lamin A/C | Santa Cruz Biotechnology | Cat# sc-376248, RRID:AB_10991536 |
| Rabbit anti-Lamin B1 | Abcam | Cat# ab16048, RRID:AB_443298 |
| Rabbit anti-emerin | Proteintech | Cat# 10351-1-AP, RRID:AB_2100056 |
| Rabbit anti-Lipin 1 | Millipore Sigma | Cat# ABS400 (discontinued) |
| Rabbit anti-NRF2 | Abcam | Cat# ab62352, RRID:AB_944418 |
| Mouse anti-mNeonGreen | ChromoTek | Cat# 32f6-100, RRID:AB_2827566 |
| Mouse anti-HA | Enzo | Cat# ENZ-ABS120 |
| Rabbit anti-HA | Cell Signaling Technologies | Cat# 3724; RRID: AB_1549585 |
| Rabbit anti-Flag | Sigma-Aldrich | Cat# F3165, RRID:AB_259529 |
| Mouse anti-tubulin alpha | Millipore | Cat# 05-829, RRID:AB_310035 |
| Bacterial and virus strains | ||
| E. coli BL21(DE3) cells | New England Biolabs | Cat# C2527 |
| Chemicals, peptides, and recombinant proteins | ||
| Propranolol | Millipore Sigma | Cat# 537075 |
| Puromycin | Thermo Fisher | Cat# A1113803 |
| TOFA | Cayman Chemicals | Cat# 10005263 |
| 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (chloroform) | Avanti | Cat# 850457C |
| 18:1 (Δ9-Cis) PC (DOPC) | Avanti | Cat# 850375C |
| 18:1 (Δ9-Cis) PE (DOPE) | Avanti | Cat# 850725C |
| 18:1 DG (DAG) | Avanti | Cat# 800811C |
| 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti | Cat# 850355C |
| Cholesterol | Sigma-Aldrich | Cat# C3045 |
| Pierce Glutathione Agarose | Thermo Fisher Scientific | Cat# 16100 |
| Digitonin | Sigma-Aldrich | Cat# D5628 |
| Cycloheximide | Cell Signaling | Cat# 2112 |
| 3-Indoleacetic acid (IAA) | Sigma-Aldrich | Cat# I3750 |
| ProLong Gold Antifade Mountant with DAPI | Thermo Fisher Scientific | Cat# P36935 |
| Experimental models: Cell lines | ||
| U2OS | Slack lab (Harvard Medical School) | N/A |
| U2OS CTDNEP1^EGFP | This study | N/A |
| U2OS mNeonGreen-NLS-2xC1ab (INM-DAG biosensor) stable | This study | N/A |
| DLD-1 OsTIR-1 stable | (Holland et al., 2012) | N/A |
| DLD-1 OsTIR-1 stable CTDNEP1EN-mAID-HA | This study | N/A |
| HEK293T | Breslow lab (Yale University) | N/A |
| Oligonucleotides | ||
| siGENOME non-targeting siRNA Pool #2 | Dharmacon | Cat# D-001206-14-05 |
| CTDNEP1 SMARTpool siRNA | (Merta et al., 2021) Dharmacon | Cat# M-017869-00-0005 |
| Lipin 1 custom single siRNA, no modifications: 5′-GAAUGGAAUGCCAGCUGAA-3′ | (Brohée et al., 2015; Sołtysik et al., 2021) Dharmacon | N/A |
| Silencer select siRNA against human LMNA | Thermo Fisher | Cat# AM16708; assay ID 144426 |
| BTRC&FBXW11 custom single siRNA (targeting both genes), no modifications: 5′-GUGGAAUUUGUGGAACAUC-3′ | (Kim et al., 2015) Dharmacon | N/A |
| qPCR primer: Hs Sun2 forward: 5′-TGACGTGCCTGACGTATGG-3′ | Primer Bank (Wang et al., 2012) | PrimerBank ID 313760642c1 |
| qPCR primer: Hs Sun2 reverse: 3′-AAATGTGGCGATGAGTCTCTG-5′ | Primer Bank | PrimerBank ID 313760642c1 |
| qPCR primer: Hs CTDNEP1 forward: 5′-CATTTACCTTCTGCGGAGGC-3′ | (Merta et al., 2021) | N/A |
| qPCR primer: Hs CTDNEP1 reverse: 3′-CACCTGGGCTAGCCGATTC-5′ | (Merta et al., 2021) | N/A |
| qPCR primer: Hs 36B4 forward: 5′-AACATGCTCAACATCTCCCC-3′ | (Neuhaus et al., 2011) | N/A |
| qPCR primer: Hs 36B4 reverse: 3′-CCGACTCCTCCGACTCTTC-5′ | (Neuhaus et al., 2011) | N/A |
| Recombinant DNA | ||
| CTDNEP1-GFP HR template | This study | N/A |
| pSPCas9(BB)-2A-Puro (PX459) v2.0 | (Ran et al., 2013) | Cat #62988; RRID: Addgene_62988 |
| pX459 CTDNEP1_HR7 | This study | N/A |
| POM121-mCherry | (Dultz et al., 2008) | Euroscarf #P30554 |
| sfCherry2(1-10) | (Feng et al., 2017) | RRID:Addgene_82602 |
| sfCherry2(11)_H2B | (Feng et al., 2017) | RRID:Addgene_82605 |
| pcDNA CTDNEP1-sfCherry2(1-10) | This study | N/A |
| Flag-sfCherry2(1-10)-NEP1R1 | This study | N/A |
| sfCherry2(11)-EGFP-H2B | This study | N/A |
| mNeonGreen-NLS | This study | N/A |
| mNeonGreen-NLS-1xC1ab | This study | N/A |
| mNeonGreen-NLS-2xC1ab (INM-DAG biosensor) | This study | N/A |
| mNeonGreen-NLS-2xC1ab (2xWG) | This study | N/A |
| pCMV-VSV-G | (Matsudaira et al., 2017) | N/A |
| pCG-Gal-Pol | (Matsudaira et al., 2017) | N/A |
| pMXs-IP-EGFP | (Matsudaira et al., 2017) | N/A |
| pMXs-IP-mNeonGreen-NLS-2xC1ab | This study | N/A |
| CTDNEP1-mAID-HA wild-type | This study | N/A |
| CTDNEP1-mAID-HA D67E | This study | N/A |
| pRK5 Flag-mLipin 1β (mouse) wild-type | (Peterson et al., 2011) | RRID: Addgene_32005 |
| pRK5 Flag-mLipin 1β (mouse) D712/D714E | This study | N/A |
| Flag-hLipin 1βRR (human, resistant to siLipin 1) wild-type | This study | N/A |
| Flag-hLipin 1βRR (human, resistant to siLipin 1) D714E | This study | N/A |
| Flag-DGAT1 (mouse) wild-type | (Sołtysik et al., 2021; Xu et al., 2012) | N/A |
| Flag-DGAT1 (mouse) H426A | This study | N/A |
| mScaret-I-DGAT1 (mouse) wild-type | This study | N/A |
| mScaret-I-DGAT1 (mouse) H426A | This study | N/A |
| Venus-DGAT2 (human) wild-type | (Sołtysik et al., 2021; Xu et al., 2012) | N/A |
| Venus-DGAT2 (human) H163A | This study | N/A |
| GFP-Sec61β | Gia Voeltz lab | N/A |
| GFP-CHMP7 under CMVΔ5 | (Gu et al., 2017) | RRID: Addgene_97006 |
| Sun2 (human)-mNeonGreen | This study | N/A |
| Sun1 (human)-mNeonGreen | This study | N/A |
| Sun1(1-219)-Sun2(213-717)-mNeonGreen | This study | N/A |
| Sun2(1-209)-Sun1(220-716)-mNeonGreen | This study | N/A |
| Sun1(1-138)-Sun2(126-717)-mNeonGreen | This study | N/A |
| Sun2(1-180)-Sun1(191-716)-mNeonGreen | This study | N/A |
| Sun2(1-150)-Sun1(156-716)-mNeonGreen | This study | N/A |
| pGEX-6P-1 GST-AH | This study | N/A |
| pGEX-6P-1 GST-AH (LVW/A) | This study | N/A |
| AH-mNeonGreen | This study | N/A |
| AH-mNeonGreen-NLS | This study | N/A |
| AH(LVW/A)-mNeonGreen-NLS | This study | N/A |
| Sun1(156-190) (Sun1-AH)-mNeonGreen-NLS | This study | N/A |
| HA-AH-mNeonGreen-NLS | This study | N/A |
| Sun2 (LVW/A)-mNeonGreen | This study | N/A |
| Sun2 (2xSA)-mNeonGreen | This study | N/A |
| Sun2 (2xSA- LVW/A)-mNeonGreen | This study | N/A |
| pMK292 (mAID-mCherry2-NeoR) | (Natsume et al., 2016) | RRID:Addgene_72830 |
| CTDNEP1-mAID-HA HR template | This study | N/A |
| Software and algorithms | ||
| FIJI | (Schindelin et al., 2012) | https://imagej.net/Fiji |
| GraphPad Prism 9 | GraphPad Software | https://www.graphpad.com/scientificsoftware/prism/ |
| SnapGene | GSL Biotech LLC | https://www.snapgene.com/ |
| ImageJ file name encrypter | Astha Jaiswal, Holger Lorenz | https://imagej.net/plugins/blind-analysis-tools |
| ImageJ StarDist | (Schmidt et al., 2018) | https://imagej.net/plugins/stardist |
| TMHMM | (Krogh et al., 2001) | https://services.healthtech.dtu.dk/service.php?TMHMM-2.0 |
| PEP-FOLD3 | (Lamiable et al., 2016; Thévenet et al., 2012) | https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/ |
| HeliQuest | (Gautier et al., 2008) | https://heliquest.ipmc.cnrs.fr/ |
| MemBrain | (Feng et al., 2022) | http://www.csbio.sjtu.edu.cn/bioinf/MemBrain/ |
| AmphipaSeeK | (Combet et al., 2000; Sapay et al., 2006) | https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_amphipaseek.html |
| Clustal Omega | (Madeira et al., 2022; Sievers et al., 2011) | https://www.ebi.ac.uk/Tools/msa/clustalo/ |
| ConSurf | (Ashkenazy et al., 2016; Ashkenazy et al., 2010) | https://consurf.tau.ac.il/ |
| Python version 3.8 | Python Software Foundation | https://www.python.org/ |
| Anaconda | Anaconda Inc | https://www.anaconda.com/products/distribution |