Plv ef1a ires neo vector

shRNAs targeting mouse Fip200 5′-GCTGAATTTCAGTGCTTAGAA-3′ (TRCN0000084986) and 5′-CCAACTTTAACACAGTCTTAA-3′ (TRCN0000084987) were purchased from Sigma-Aldrich. A scrambled shRNA as control was purchased from Addgene (plasmid #1864). sgRNAs targeting mouse Fip200 (5′-TTCTCTAGAAATAACACTAA-3′; 5′-CTCCATTGACCACCAGAACC-3′), Atg5 (5′-CCCTATAGACCACGACGGAG-3′), Atg7 (5′-GAACGAGTACCGCCTGGACG-3′) and Opn (5′-GCAAATCACTGCCAATCTCA-3′; 5′-GCAGAATCTCCTTGCGCCAC-3′) were respectively cloned into LentiCRISPR v2 vector (Addgene #52961) according to the protocol previously reported^{63 (link)}. sgRNA targeting EGFP (Addgene #86153) was used as control. Spp1 overexpression clone was constructed by RT-PCR from HUVEC RNA and then insertion into pLV-EF1a-IRES-Neo vector (Addgene #85139).
All lentiviral productions were generated by transfecting into HEK293T cells with psPax2, pMD2.G and lentiviral plasmids, as described previously^{64 (link)}. Tsc1^iΔEC cells were then transduced by different lentivirus containing specific sgRNA followed with puromycin selection for one week. The efficiencies of Fip200 knockdown, Fip200, Atg5 and Atg7 knockout or re-overexpression of osteopontin in tumor cells were confirmed by western blot.

The siRNA-targeting region (GCAGATGACTTACAAGCAA) of the human SPRED1 gene (NM_152594.2, CDS 1,335 bp) was designed, synthesized, and ligated with the Tet-pLKO-puro vector (Addgene). First, we observed the growth state of the THP-1 cells and made sure that the exact volume of the venom was absorbed (the ratio of virus number to THP-1 cell number was 100:1), which was added to the prepared medium. With the culture medium removed from the cells, the calculated virus venom plus medium was added to the target cells. Plates were cultured under the following conditions, shaking for a constant mixture of the media while at 37°C and 5% CO₂ for 48 h. Afterward, we selected the detection of related indicators.
The Tet-pLKO-puro vector (Addgene) was used as a non-silencing control or for carrying SPRED1 target shRNA. This is the sequence used for SPRED1 shRNA: GCAGATGACTTACAAGCAA. For SPRED1 expression, the vector was constructed by cloning human SPRED1 cDNA (NM_152594.2) into pLV-EF1a-IRES-NEO vector (Addgene) according to the standard procedure. Lentiviral packaging and transductions were carried out using standard procedure and according to the manufacturer’s instructions. Cells transfected with a virus carrying an empty vector were used as a negative control (control group), and untreated cells were used as a blank control (blank group).

The Tet-pLKO-puro vector (Addgene) was used for non-silencing control or for carrying SPRED1 target shRNA. The sequence for SPRED1 shRNA were: GCAGATGACTTACAAGCAA. For SPRED1 expression, the vector was constructed by cloning human SPRED1 cDNA (NM_152594.2) into pLV-EF1a-IRES-NEO vector (Addgene) according to the standard procedure. Lentiviral packaging and transductions were carried out using standard procedure and according to manufacturer's instructions. The cells transfected with virus carrying empty vector was used as a negative control (Control group) and untreated cells were used as blank control (Blank group).