, AR (L-003400-00), DECR1 (L-009642-00-005), as well as non-targeting siRNA (D-001810-01-20) were purchased from Dharmacon (Dharmacon, Horizon inspired cell solutions, transfection. Cells were seeded in 6-well plates to reach 70% confluence and allowed to attach overnight. ON-TARGETplus smartpool siRNAs against AMPKa (L-005027-00)
, USA) using a nucleofector (Amaxa Biosystems, Lonza, Basel, Switzerland) according to the manufacturer's instructions. Cells were then cultured in the presence of 1 µg ml ?1 of puromycin, Generation of DECR1 stable KO cells. DECR1 deletion was performed using the CRISPR CAS9 gene editing technology
, Final cell number was normalised to the initial cell count obtained at T0. qPCR analysis. RNA was extracted from cells (70-80% confluence) using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with on-column DNase digestion (RNase-Free DNase Set, Cell proliferation. 6-8 × 10 5 cells were seeded in 6-well plates and allowed to attach for 16 h
Membrane was blocked in 5% milk-TBST for 1 h and probed overnight with primary antibodies (see Supplementary Table 2) diluted in 5% BSA-TBST. The membrane was then washed three times with TBST, incubated with respective HRP-conjugated secondary antibodies diluted in 5% milk-TBST, washed another three times with TBST and revealed using the ECL kit, SDS buffer (1% SDS supplemented with protease and phosphatase inhibitors) and protein concentration was determined using the BCA protein assay kit ,
, The next day, coverslips were washed three times with PBS-Tween, incubated with fluorophore-coupled secondary antibodies (Abcam, Cambridge, UK) and washed again three times with PBS. Coverslips were mounted using Diamond Prolong with DAPI, Immunofluorescence. Cells were seeded on coverslips in 24-well plates to reach 50% confluence and allowed to attach overnight
The DNA/protein complexes were washed four times in IP Wash buffer (100 mM Tris-HCl pH 8.0; 500 mM LiCl 1%; Triton X100; 1% deoxycholic acid. After reversal of crosslinking, the immunoprecipitated DNA was purified by a regular DNA extraction protocol and analysed employing RT-qPCR with the SYBR-Green Takara, Chromatin immunoprecipitation (ChIP). Chromatin was prepared with the tru-ChIP? Chromatin Shearing Kit (Covaris, Brighton, UK) according to manufacturer's instructions. Each sample was sonicated for 10 min using Covaris sonicator. ChIP were performed using the IP-Star Compact Automated System ,
, Statistical analysis. Statistical analyses were performed using GraphPad PRISM software v7.05
, 51 (ENZ) organoids examined over two independent biological experiments. Panels d, e: n = 3 independent biological experiments, Data reproducibility. Figure 1: Panels a, b, f, h: representative image from three independent biological experiments. Panel c (top): n = 6 independent biological experiments. Panel c (bottom): n = 55 (LN), 40 (BIC), vol.51
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