Block ittm adenoviral rnai expression system

m-TERC53 and m-TERC53r expressing vectors were constructed with BLOCK-iT^TM U6 RNAi Entry Vector Kit (Invitrogen) according to the supplier’s instruction, and the virus were produced, amplified and purified with BLOCK-iT^TM Adenoviral RNAi Expression System (Invitrogen) according to the supplier’s instruction. Mice were maintained under standard housing conditions, and anaesthetized with Isoflurane (3 mL/h for 10 min to induce anaesthetization, 1.2 mL/h to maintain the anaesthetized state). Mice were placed in the stereotaxic apparatus and a small hole was drilled at each bilateral injection location. 0.2 μL virus (0.1 OD) was injected per site using a Hamilton microsyringe (0.02 μL/min) into the dorsal DG using the following coordinates: anterioposterior = − 2.2 mm from bregma; lateral = ± 1.5 mm; ventral = 2.3 mm. The skin incision was closed carefully after adenoviral injection to minimize inflammation. Injection needles were left in place for 5 min before and after injection to ensure right location and even distribution of the virus.

Full‐length PALB2 cDNA was cloned into pRDI lentiviral expression vector between ClaI and EcoRI site. The lentivirus was expressed in 293T cells using FuGENE6 Transfection Reagent (Roche Applied Sciences, Penzberg, Germany). MDA‐MB‐231 cells were infected with the virus in the presence of 8 μg·mL⁻¹ polybrene and were selected with puromycin. BLOCK‐iT^TM Adenoviral RNAi Expression System (Invitrogen, Carlsbad, CA, USA) was used to transiently express short hairpin RNA (shRNA) in MDA‐MB‐231 cells. The PALB2 RNAi sequences were determined by using BLOCK‐iT^TM RNAi Designer (Invitrogen) and was cloned into the pENTRTM/U6 vector. The following two shRNAs were selected for human PALB2: sense 5′‐CAC CGC TTG CGA AGA TGT AGT TTC TCG AAA GAA ACT ACA TCT TCG CAA GC‐3′ and antisense 5′‐AAA AGC TTG CGA AGA TGT AGT TTC TTT CGA GAA ACT ACA TCT TCG CAA GC‐3′; and sense 5′‐CAC CGC AGC AGC AAT CTT GAC TTC TCG AAA GAA GTC AAG ATT GCT GCT GC‐3′, anti‐sense 5′‐AAA AGC AGC AGC AAT CTT GAC TTC TTT CGA GAA GTC AAG ATT GCT GCT GC‐3′.

For the individual knockdowns of mouse SHARPIN, HOIL-1L, and HOIP, we generated each shRNA expressing recombinant adenoviruses using the BLOCK-iT^TM U6 RNAi Entry Vector Kit and the BLOCK-iT^TM Adenoviral RNAi Expression System (Invitrogen). The top and bottom strands used to generate each of the shRNA expressing recombinant adenoviruses are as follows: SHARPIN shRNA, top strand 5′-CACCGCGGAAGCTGCAATTGATAGCCGAAGCTATCAATTGCAGCTTCCGC-3′, bottom strand 5′-AAAAGCGGAAGCTGCAATTGATAGCTTCGGCTATCAATTGCAGCTTCCGC-3′; HOIL-1L shRNA, top strand 5′-CACCGCCTATCTCTACCTGCTGTCACGAATGACAGCAGGTAGAGATAGGC-3′, bottom strand 5′-AAAAGCCTATCTCTACCTGCTGTCATTCGTGACAGCAGGTAGAGATAGGC-3′; HOIP shRNA, top strand 5′-CACCGGTCTTCTCAGCTCTCCAATACGAATATTGGAGAGCTGAGAAGACC-3′, bottom strand 5′-AAAAGGTCTTCTCAGCTCTCCAATATTCGTATTGGAGAGCTGAGAAGACC-3′.