Key Resource Table.
| Reagents or resource | Source | Identifier |
|---|---|---|
| Recombinant DNAs | ||
| pLJM1-EGFP | Addgene (Sabatini Lab) | Cat#19313 |
| pULTRA | Addgene (Moore Lab) | Cat#24129 |
| Tet-pLKO-Puro | Addgene (Weiderschain lab) | Cat#21915 |
| pcDNA3.1-HA | Addgene (Laur lab) | Cat#128034 |
| H. sapience HA-Foxo3a WT | Addgene (Greenberg lab) | Cat#1787 |
| pLJM1-mCx37FL-EGFP (m = Mus musculus) | This study | N/A |
| pLJM1-mCx37TA-EGFP | This study | N/A |
| pLJM1-mCx37ΔCT-EGFP | This study | N/A |
| pLJM1-mCx37Tail-EGFP | This study | N/A |
| pLJM1-mCx37S275A-EGFP | This study | N/A |
| pLJM1-mCx37S319A-EGFP | This study | N/A |
| pLJM1-mCx37S321A-EGFP | This study | N/A |
| pULTRA-HA-mCx37FL | This study | N/A |
| pULTRA-HA-mCx37TA | This study | N/A |
| pULTRA-HA-mCx37ΔCT | This study | N/A |
| pULTRA-HA-mCx37Tail | This study | N/A |
| pULTRA-HA-mCx37S275A | This study | N/A |
| pULTRA-HA-mCx37S319A | This study | N/A |
| pULTRA-HA-mCx37S321A | This study | N/A |
| Antibodies | ||
| Cx37/GJA4 | Abcam | Cat# ab181701 |
| CX37/GJA4 | Thermo Fisher Scientific | Cat# 40-4300 |
| p27 KIP 1 | Abcam | Cat# ab193379 |
| p27 KIP 1 | Cell Signaling Technology | Cat# 3686 |
| p21 (CIP1/WAF1) | Abcam | Cat# ab218311 |
| p57 KIP2 | Cell Signaling Technology | Cat# 2557 |
| c-MYC | Novus Biologicals | Cat# NB100-1642 |
| Foxo3a | Cell Signaling Technology | Cat# 2497 |
| Phospho-FoxO3a (Ser253) | Cell Signaling Technology | Cat# 9466 |
| Phospho-FoxO3a (Ser294) | Cell Signaling Technology | Cat# 5538 |
| Phospho-FoxO3a (Ser425) | Cell Signaling Technology | Cat# 64616 |
| FoxO1 | Cell Signaling Technology | Cat# 2880 |
| FoxO4 | Cell Signaling Technology | Cat# 9472 |
| P44/42 MAPK (Erk1/2) | Cell Signaling Technology | Cat# 9102 |
| Phospho p44/42 MAPK (Erk1/2) | Cell Signaling Technology | Cat# 9106 |
| GFP-tag | Origene | Cat# R1091TR |
| HA-tag | R&D Systems | Cat# MAB060 |
| VE-Cadherin | R&D Systems | Cat# AF938 |
| Alpha Tubulin (YOL1/34) | Novus Biologicals | Cat# NB100-1639 |
| Beta-Actin | Cell Signaling Technology | Cat# 4967 |
| DNA cloning primers | ||
| Construct | Forward (5′ to 3′) | Reverse (5′ to 3′) |
| pLJM1-mCx37TA-EGFP | CGTCAGATCCGCTAGCGCCACCATGGGCGACTGG | CATGGTGGCGACCGGTCACATACTGCTTCTTGGATGC |
| pLJM1-mCx37ΔCT-EGFP | CGTCAGATCCGCTAGCGCCACCATGGGCGACTGG | CATGGTGGCGACCGGTGGGTCCCTCGCCCATGGG |
| pLJM1-mCx37Tail-EGFP | CGTCAGATCCGCTAGCGCCACCATGGTCAGCCGG | CATGGTGGCGACCGGTCACATACTGCTTCTTGGATGC |
| Annealed HA-oligo in pULTRA vector | CCGGTGCCACCATGGTGTACCCATACGATGTTCCAGATTACGCTT | CTAGAAGCGTAATCTGGAACATCGTATGGGTACACCATGGTGGCA |
| pULTRA-HA-mCx37FL | TCCCGGGCCTTCTAGAATGGGCGACTGGGGCTTC | CGCCGGAGCCGGATCCCTACACATACTGCTTCTTGGATGC |
| pULTRA-HA-mCx37ΔCT | TCCCGGGCCTTCTAGAATGGGCGACTGGGGCTTC | TCCCGGGCCTTCTAGAATGGGCGACTGGGGCTTCC |
| pULTRA-HA-mCx37Tail | AGATTACGCTTCTAGATGTCGGTGTGTCAGCCGG | CGCCGGAGCCGGATCCCTACACATACTGCTTCTTGGATGC |
| GJA4-T154A | GCTCATGGGTGCCTATGTGGTCA | GCCCCACGAATCCGAAGA |
| GJA4-S275A | CGAGGGACCCGCTTCCCCACCGT | CCCATGGGGAGGTAGAAGAAAACCTG |
| GJA4-S319A | TGGCCGAAAGGCCCCTAGCCGCC | CCCTGGGGAGCTGTGTTTACAAATGGGG |
| GJA4-S321A | AAAGTCCCCTGCCCGCCCCAACAGCTCTG | CGGCCACCCTGGGGAGCT |
| Human GjA4 shRNA1 | CCGGGAAGCAGAAGATAACACCTCTCTCGAGAGAGGTGTTATCTTCTGCTTCTTTTTG | AATTCAAAAAGAAGCAGAAGATAACACCTCTCTCGAGAGAGGTGTTATCTTCTGCTTC |
| Human GjA4 shRNA2 | CCGGGGGTGACGAGCAATCAGATTTCTCGAGAAATCTGATTGCTCGTCACCCTTTTTG | AATTCAAAAAGAAGCAGAAGATAACACCTCTCTCGAGAGAGGTGTTATCTTCTGCTTC |
| Human Foxo3a shRNA1 (CDS) | Sigma-Aldrich; Mission shRNA | TRC Clone ID: Version 1 TRCN000010335 |
| Human Foxo3a shRNA2 (3′UTR) | Sigma-Aldrich; Mission shRNA | TRC Clone ID: Version 2 TRCN0000235491 |
| siRNA-CDKN1B | Dharmacon Ltd. | Cat# L- 040178-00 |
| siRNA-control | Qiagen | Cat# 1022076 |
| qRT-PCR primers | ||
| Human genes | Forward (5′ to 3′) | Reverse (5′ to 3′) |
| ACTB (β-Actin) | TCACCCACACTGTGCCCATCTACGA | CAGCGGAACCGCTCATTGCCAATGG |
| CDKN1B (p27) | ATCACAAACCCCTAGAGGGGCA | GGGTCTGTAGTAGAACTCGGG |
| GJA4 (Cx37) | ACACCCACCCTGGTCTACC | CACTGGCGACATAGGTGCC |
| FOXO3A | TTCAAGGATAAGGGCGACAGCAAC | CTGCCAGGCCACTTGGAGAG |
| c-MYC | CTTCTCTCCGTCCTCGGATTCT | GAAGGTGATCCAGACTCTGACCTT |
| MEN1 (MENIN) | CAGGGGCCAGACAGTCAATG | GGTGGGCTCCAGCTCCTCTA |
| GJA5 (Cx40) | CCGTGGTAGGCAAGGTCTG | ATCACACCGGAAATCAGCCTG |
| HEY2 | GCCCGCCCTTGTCAGTATC | CCAGGGTCGGTAAGGTTTATTG |
| EFNB2 | TATGCAGAACTGCGATTTCCAA | TGGGTATAGTACCAGTCCTTGTC |
| NR2F2 | GGACCACATACGGATCTTCCAA | ACATCAGACAGACCACAGGCAT |
| EPHB4 | CGCACCTACGAAGTGTGTGA | GTCCGCATCGCTCTCATAGTA |
| Reagents and kits | ||
| NaveniFlex MR (PLA Kit) | Cambridge Bioscience | Cat# NF.MR.100 (Suppl. Navinci Diagnostics AB) |
| Dextran, rhodamine B, 70,000 MW, neutral | Invitrogen | Cat# D1841 |
| Lucifer yellow CH, lithium Salt | Invitrogen | Cat# L453 |
| MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) | Invitrogen | Cat# M6494 |
| Edu Proliferation Assay Kit (iFlour 647) | Abcam | Cat# ab222421 |
| X-rhod-1, AM Cell permeable Ca2+ assay | Thermo Fisher Scientific/molecular probe | Cat# X14210 |