Pumvc

Doxcyclin (Dox) and Jak inhibitor (Jaki) were purchased from Merck Millipore (Billierica, MA, USA). CHIR99021 and PD0325901 were purchased from SelleckChem (Houston, TX, USA). The LIF neutralizing antibody (LIFAb) was from R&D Systems. The retro- and lenti-viral vectors including pMXs-Nanog, and FUW- M2rtTA, and the viral packaging plasmids PUMVC, psPAX2 and pCMV-VSV-G (Stewart et al., 1992 (link)) were all obtained from Addgene (Cambridge, MA, USA). FUW-TetO-Esrrb and pMXs-Stat3C were described previously (Tang et al., 2012 (link), 2014 (link)). Nr5a2 cDNA was PCR amplified using primers (forward primer: 5′-AGTTAATTAAGGATCCATGTCTTCTAATTCAGATACTGGGG-3′ and reverse primer: 5′-ACTGTGCTGGCGGCCGCTTATGCTCTTTTGGCATGCAAC-3′) and cloned into linearized pMXs vectors (Cell Biolabs, San Diego, CA, USA) using the In-Fusion kit (Clontech Inc., Mountain View, CA, USA). Lenti- and retro-viruses were prepared with 293T cells according to the protocol from Addgene and filtered with 0.8 μm filters.

Stock pMX-RFP pseudovirus and pBABE-CMV-RFP pseudovirus were generated by co-transfecting 293T cells with 10 μg of pMX-RFP/pBABE-CMV-RFP, 9 μg of pUMVC and 1 μg pMD2.G (Addgene #12259) in 10 cm dishes. The medium was changed 6–8 h after transfection, and the supernatant was collected after 48–72 h. The supernatant was filtered through a 0.45 μm membrane to clear the cell debris, and then collected for infection or concentrated using polyethylene glycol (PEG) 8000 at a 10% final concentration with rolling overnight at 4°C, followed by centrifuging at 3500 g for 30 min. 293T cells (1 × 10⁵ cells) were infected with 200 μl of concentrated pMX-RFP pseudovirus and pBABE-CMV-RFP pseudovirus supernatants in 96-well plates. The cells were washed twice with phosphate-buffered saline 4–6 h after infection and transferred to 24-well plates with fresh medium. Cells were harvested 48–72 h after infection for fluorescence-activated cell sorting (FACS).

293 T cells were plated onto six-well plates at 2.5 × 10⁶ cells/plate. The next day, retroviral constructs, PUMVC and pCMV-VSVG (Addgene) plasmids were co-transfected into 293 T cell using Fugene 6 reagent (Promega, Madison, WI, USA). Cell culture media containing retrovirus were harvested at 48 and 72 hours post-transfection and filtered through a 0.8 μ m filter. The viruses were stored in −70 °C before use.

MCF7 cells were transfected with a mixture of packaging retroviral plasmids pCMV-VSV-G (cat. No. 8454, Addgene, Watertown, MA, USA), pUMVC (cat. No. 8449, Addgene, Watertown, MA, USA), and EGFRwt transfer plasmid (cat. No. 11011, Addgene, Watertown, MA, USA), or control plasmid (cat. No. 27490; Addgene, Watertown, MA, USA) using the calcium-phosphate transfection protocol [40 (link)]. Two days later, supernatants of conditioned media were filtered using 0.45 µm PES filters and used for cell transduction either immediately or following ultracentrifugation. MCF7 cells were transduced by target or control retroviral particles using spinoculation protocol [41 (link)] in the presence of polybrene (8 µg/mL). The transduced cells were then supplied with puromycin (1 µg/mL) 2 days after transduction to discard the non-transduced cells. EGFR expression was analyzed by flow cytometry using anti-EGFR antibodies 1:100 (#MA5-13319; Invitrogen, Rockford, IL, USA), specified to extracellular domain of EGFR receptor. MCF7 cells with EGFR-high phenotype were sorted (SONY SH800S sorter, San Jose, CA, USA, 100 µm nozzle chip) into adhesive 24-wells cell culture plate (Eppendorf^TM; Hamburg, Germany) and cultivated under standard conditions.

The cell line was generated using MACS separation (Miltenyi biotec, autoMACS Pro Separator, #130-092-545, autoMACS Columns #130-021-101) of PDGFRb+ cells that were isolated from the healthy part of the kidney cortex after nephrectomy as previously described in ref. ^{43 (link)}. The following antibodies were used for staining the cells and MACS procedure: PDGFRb (R&D #MAB1263 antibody, dilution 1:100) and anti-mouse IgG1-MicroBeads solution (Miltenyi, #130-047-102). The cells were cultured in DMEM media (Thermo Fisher #31885) added 10% FCS and 1% penicillin/streptomycin for 14 days. For immortalization (SV40-LT and HTERT) the retroviral particles were produced by transient transfection of HEK293T cells using TransIT-LT (Mirus). Amphotropic particles were generated by co-transfection of plasmids pBABE-puro-SV40-LT (Addgene #13970) or xlox-dNGFR-TERT (Addgene #69805) in combination with a packaging plasmid pUMVC (Addgene #8449) and a pseudotyping plasmid pMD2.G (Addgene #12259) respectively. Using Retro-X concentrator (Clontech) 48 h post-transfection the particles were concentrated. For transduction, the target cells were incubated with serial dilutions of the retroviral supernatant (1:1 mix of concentrated particles containing SV40-LT or rather hTERT) for 48 h. At 72 h after transfection, the infected PDGFRb+ cells were selected with 2 μg/ml puromycin for 7 days.

HEK293T cells were utilized to produce the retrovirus carrying either GFP-fused wt-lamin A or progerin gene. Viral production was achieved by the assembly of a retroviral packaging system that contains the packaging plasmid pUMVC (Addgene #8449), the envelope plasmid pCMV-VSV-G (Addgene #8454), and the target plasmid—pBABE-puro-GFP-wt lamin A (Addgene #17662) or pBABE-puro-GFP-progerin (Addgene #17663). Viral supernatants were collected after 48, 72, and 96 h of transfection, and were added on ECs in the presence of polybrene at a concentration of 8 μg/ml. The infection medium was replaced with regular DMEM after 4 h of infection. Drug selection began 48 h after the transduction, and the selection process continued for 10 days until 95% of cells were GFP positive.