KEY RESOURCES TABLE.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Mouse monoclonal anti-β-actin | Sigma-Aldrich | Cat#1978; RRID: AB_476692 |
| Mouse monoclonal anti-tubulin β3 (TUJ1) | Biolegend | Cat#801202; RRID: AB_10063408 |
| Chicken polyclonal anti-GFP | Thermo Fisher Scientific | Cat#A10262; RRID: AB_2534023 |
| Rabbit polyclonal anti-Lin28a | Cell Signaling Technology | Cat#3978S; RRID: AB_2297060 |
| Rabbit polyclonal anti-Lin28b (mouse preferred) | Cell Signaling Technology | Cat#5422S; RRID: AB_10697489 |
| Rabbit polyclonal anti-hLin28b | Cell Signaling Technology | Cat#4196S; RRID: AB_2135047 |
| Rabbit monoclonal anti-pS6 (Ser235/236) | Cell Signaling Technology | Cat#4858S; RRID: AB_916156 |
| Rabbit monoclonal anti-pAkt (Ser473) | Cell Signaling Technology | Cat#4060S; RRID: AB_2315049 |
| Rabbit monoclonal anti-pGSK3β (Ser9) | Cell Signaling Technology | Cat#5558S; RRID: AB_10013750 |
| Rabbit monoclonal anti-DYKDDDDK tag (Flag) | Cell Signaling Technology | Cat#14793S; RRID: AB_2572291 |
| HRP-linked horse anti-rabbit IgG | Cell Signaling Technology | Cat#7074S; RRID: AB_2099233 |
| HRP-linked horse anti-mouse IgG | Cell Signaling Technology | Cat#7076S; RRID: AB_330924 |
| Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) cross-adsorbed |
Thermo Fisher Scientific | Cat#A-11001; RRID: AB_2534069 |
| Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) cross-adsorbed |
Thermo Fisher Scientific | Cat#A-11004; RRID: AB_2534072 |
| Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) cross-adsorbed |
Thermo Fisher Scientific | Cat#A-21235; RRID: AB_2535804 |
| Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) cross-adsorbed |
Thermo Fisher Scientific | Cat#A-11008; RRID: AB_143165 |
| Alexa Fluor 488-conjugated goat anti-chicken IgY (H+L) | Thermo Fisher Scientific | Cat#A-11039; RRID: AB_2534096 |
| Bacterial and Virus Strains | ||
| DH5α competent cells | Thermo Fisher Scientific | Cat#18265–017 |
| AAV2-GFP | SignaGen Laboratories | Cat#SL100812 |
| AAV2-Lin28a-Flag (codon optimized) | SignaGen Laboratories | N/A |
| AAV2-shPten | Vigene Biosciences | N/A |
| Chemicals, Peptides, and Recombinant Proteins | ||
| Type I Collagense | Thermo Fisher Scientific | Cat#17018–017 |
| Dispase II | Thermo Fisher Scientific | Cat#17105–041 |
| MEM | Thermo Fisher Scientific | Cat#11090–081 |
| DMEM/F12 (1:1) | Thermo Fisher Scientific | Cat#11330–032 |
| Opti-MEM reduced serum medium | Thermo Fisher Scientific | Cat#31985–070 |
| GlutaMAX-I | Thermo Fisher Scientific | Cat#A12860–01 |
| HBSS | Thermo Fisher Scientific | Cat#13175–095 |
| Penicillin-Streptomycin | Thermo Fisher Scientific | Cat#15140–122 |
| Fetal bovine serum | GE Healthcare Life Sciences | Cat#SH30071.03HI |
| Poly-D-lysine | Sigma-Aldrich | Cat#P6407 |
| Laminin | Thermo Fisher Scientific | Cat#23017–015 |
| 5-Fluoro-2′-deoxyuridine | Sigma-Aldrich | Cat#F0503 |
| Uridine | Sigma-Aldrich | Cat#U3750 |
| Lipofectamine 2000 | Thermo Fisher Scientific | Cat#11668–027 |
| Doxycycline diet | Bio-Serv | Cat#F3893 |
| Fluoroshield histology mounting medium | Sigma-Aldrich | Cat#F6182 |
| Alexa Fluor 594-conjugated cholera toxin subunit B | Thermo Fisher Scientific | Cat#C-22842 |
| Goat serum | Sigma-Aldrich | Cat#G9023 |
| Tetrahydrofuran | Sigma-Aldrich | Cat#186562 |
| Triton X-100 | Sigma-Aldrich | Cat#T9284 |
| Benzyl alcohol | Sigma-Aldrich | Cat#305197 |
| Benzyl benzoate | Sigma-Aldrich | Cat#B6630 |
| RIPA buffer | Sigma-Aldrich | Cat#R0278 |
| Protease inhibitor cocktail | Sigma-Aldrich | Cat#11836153001 |
| Phosphatase inhibitor cocktail | Sigma-Aldrich | Cat#4906845001 |
| Blotting-grade blocker | Bio-Rad | Cat#1706404 |
| EcoRI-HF restriction endonuclease | New England Biolabs | Cat#R3101S |
| NotI-HF restriction endonuclease | New England Biolabs | Cat#R3189S |
| BamHI-HF restriction endonuclease | New England Biolabs | Cat#R3136S |
| EcoRV-HF restriction endonuclease | New England Biolabs | Cat#R3195S |
| T4 DNA ligase | New England Biolabs | Cat#M0202S |
| Critical Commercial Assays | ||
| Mouse neuron nucleofector kit | Lonza | Cat#VPG-1001 |
| miRNeasy mini kit | QIAGEN | Cat#217004 |
| Transcriptor first strand cDNA synthesis kit | Roche | Cat#04897030001 |
| LightCycler 480 SYBR green I master | Roche | Cat#04707516001 |
| Endofree plasmid maxi kit | QIAGEN | Cat#12362 |
| Experimental Models: Cell Lines | ||
| CAD (Cath.-a-differentiated) cells | Gift from Dr. William Snider | N/A |
| Experimental Models: Organisms/Strains | ||
| CF-1 mice | Charles River Laboratories | Strain#023 |
| CD-1 IGS mice | Charles River Laboratories | Strain#022 |
| C57BL/6J mice | The Jackson Laboratory | Stock#000664 |
| hLin28b mice | Gift from Dr. George Daley | Stock#023911 |
| M2rtTA mice | Gift from Dr. Angelika Doetzlhofer | Stock#006965 |
| Oligonucleotides | ||
| For all primers, see Table S1 | This paper | N/A |
| siRNA targeting Lin28a (for target sequence, see Table S2) |
Dharmacon | Cat#L-051530–01-0005 |
| siRNA targeting Lin28b (for target sequence, see Table S2) |
Dharmacon | Cat#L-063393–01-0005 |
| Non-targeting siRNA control (for target sequence, see Table S2) |
Dharmacon | Cat#L-001810–10-20 |
| Fluorescent siRNA control | Sigma-Aldrich | Cat#SIC003 |
| let-7a microRNA mimic | Dharmacon | Cat#C-310503–07-0005 |
| let-7b microRNA mimic | Dharmacon | Cat#C-310504–05-0005 |
| Non-mammalian targeting microRNA control (cel-miR-67) |
Dharmacon | Cat#CN-001000–01-05 |
| let-7 microRNA family inhibitor (for target sequence, see Table S2) |
QIAGEN | Cat#YFI0450006 |
| Recombinant DNA | ||
| pCMV-GFP | Addgene | Cat#11153 |
| pMSCV-mLin28A | Addgene | Cat#26357 |
| pCMV-Lin28a | This paper | N/A |
| Lin28a-Flag open reading frame (codon optimized) | Integrated DNA Technologies | N/A |
| pAAV-Ef1a-EYFP | Gift from Dr. Hongjun Song | N/A |
| pAAV-Lin28a-Flag | This paper | N/A |
| pAAV-shPten | Gift from Dr. David Turner and Dr. Kevin Park |
N/A |
| Software and Algorithms | ||
| LightCycler 480 software, release 1.5 | Roche | Cat#04994884001 |
| AxioVision, release 4.8 | Zeiss | N/A |
| GraphPad Prism 7 | GraphPad Software | N/A |
| LSM 510 software | Zeiss | N/A |
| Office 2016 | Microsoft | N/A |
| ImageJ | NIH | N/A |
| Adobe Illustrator | Adobe | N/A |
| Amaxa Nucelfector II | Lonza | N/A |
| Glass capillaries | World Precision Instruments | Cat#TW100–4 |
| Picospritzer III | Parker Hannifin | Cat#051–0500-900 |
| Flat tweezertrode | BTX | Cat#45–0524 |
| ECM 830 Electro Square Porator | BTX | N/A |
| Dumont #5 fine forceps | Fine Science Tools | Cat#11254–20 |
| LightCycler 480 Instrument II | Roche | Cat#05015243001 |