Sirnas

RNAi was performed by electroporating siRNAs as previously reported (Watanabe et al., 2014 (link)) with minor modifications. Briefly, 30 animals were electoporated as above either with a ‘scrambled’ siRNA (4 µM, diluted in water) used as control, or with a mixture of three siRNAs (Eurofins Genomics) targeted against p62/SQSTM1 or WIPI2 (final concentration 4 µM in water, see Table S5 for sequences). The procedure was repeated every other day as indicated.

Transfection of siRNAs was performed as described earlier (Klanert et al., 2019). Briefly, siRNAs (Eurofins, Luxembourg City, Luxembourg) were transfected using the Neon® transfection system (Thermo Fisher Scientific) with the Neon® transfection system 100 µl kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Therefore, 5 × 10⁶ cells were centrifuged at 170 rcf for 8 min and then resuspended in 100 µl buffer R. After the addition of 300 pmol of siRNAs, cells were transfected by applying one pulse with 1,700 V and 20 ms. A mock transfection and a nontargeting “scrambled” siRNA (AllStars Negative Control siRNA; Qiagen, Venlo, The Netherlands) were included as controls. Cells were allowed to recover in 10 ml of the respective media in a 50‐ml TPP® Tubespin bioreactors for 1.5−2 hr post‐transfection without shaking at 37°C, humidified air and 7% CO₂. Subsequently, cultures were shaken at 220 rpm. For the repeated transfections of cells with siRNAs, this procedure was repeated every 4th day. All siRNA sequences are provided in Table S2.

The procedure for transient transfection of RAW264.7 cells with siRNAs targeting group IVC phospholipase A₂ (cytosolic phospholipase A₂γ; cPLA₂γ) was adapted from Valdearcos et al. [51 (link)]. Briefly, 3 × 10⁵ cells were transfected with siRNAs (20 nM) (sequence: 5′-(GGAGGAGAGAGAGGAAGAGAA)TT-3′; Eurofins Genomics, Ebersberg, Germany) in the presence of 5 µl/mL Lipofectamine^TM RNAiMAX (Invitrogen) under serum-free conditions for 5 h. Afterward, 5% serum was added and the cells were maintained at normal culture conditions for 24 h. A scrambled siRNA was used as a negative control (sequence: 5′-UGGUUUACAUGUCGACUAA-3′).

The anti-p53 antibodies Pab421, Pab246, CM5 and 1C12 were purchased from Millipore (Darmstadt, Germany), Vector laboratories (Burlingame, CA, USA) and Cell signaling (Danvers, MA, USA), respectively. From Santa Cruz (Dallas, TX, USA), we obtained the antibodies against Oct-4 (C-10) Nanog (C-4), c-Jun (H79) and PCNA (PC10). Antibodies against acetylated and phosphorylated p53 (K379, S6, S15, S392) were from Cell signaling. Antibodies against GAPDH (6C5), PARC (PO69) and α-7 (MCP72) were from Hytest (Turku, Finland), BioLegend (San Diego, CA, USA) and Enzo (Loerrach, Germany), respectively. Antibodies against β-Actin and Histone H3 were purchased from Abcam (Cambridge, UK) and the antibody against MdmX (MDMX82) was from Sigma-Aldrich.
siRNAs were purchased from Eurofins (Ebersberg, Germany). Sequences are available on request. siRNA transfections were performed using Macsfectin (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer's instructions.

siRNAs were purchased from Eurofins Genomics (sequences shown in Supplementary Table 1).
The AllStars negative control siRNA (Qiagen, 1027281) was used as negative control.
Lipofectamine RNAi-MAX (Invitrogen) was used for siRNAs transfections in antibiotic-free medium according to the manufacturer's instructions. Cells were transfected in reverse using 20nM siRNA. The TEAD1/3/4 and YAP/TAZ silencing was performed by transfecting the siRNAs in MDA-MB-231 and MDA-MB-157 cell lines for 72 hours.
GTSE1 and E2F1 silencing was carried out transfecting cells with siRNAs for 48 hours.