Gv141 vector

The full lengths of CCNG1 (Table 1) were synthesized by Biosune (Shanghai, China) and inserted into Sgf1/MIu1 sites of a PLenti‐C‐Myc/DDKvector (OriGene, 10069). PLenti‐C‐Myc/DDK vector was designated as the mock control. The vector pCMV‐p53mt135 (631922) was bought from Conetech, USA.
Plasmid GV141‐NICD3 (Notch3 intracellular domain plasmid; amino acids 1663 to 2312) was constructed by cloning the NICD3‐coding region (NM_000435‐P1) to GV141 vector (Genechem, CON106). GV141 null vector was designated as the mock control. PCR primers used for amplification of the full‐length cDNAs are shown in Table 1. Ultimately, shRNAs were used to knockdown CCNG1 and Notch3 pathways. Empty pLKO.1 vectors (Addgene, 10878) were used as controls (shRNA sequences displayed in Table 2).
For stable infection, Lentivirus expressing CCNG1 and Plko.1‐shRNA (CCNG1, Notch3, P53 proteins), packaged with psPAX2 (Addgene, 12260) and pMD2G (Addgene, 12259), were produced in HEK293T cells with Lipofectamine 2000 (Invitrogen, 11668019) according to a protocol (Appendix S1). After transfection by Lentivirus for 24 hours, the cells were selected in medium containing 2 μg/mL puromycin (Merck Millipore; Burlington, MA, USA) for two weeks. Stable expression cells were obtained and expanded for further studies.

siRNAs targeting FZD2 were designed and synthesized by Ribobio (Guangzhou, China). For the silencing of FZD2, siRNAs were transfected into SK-BR-3 and MDA-MB-231 cells in accordance with the instruction manual for Lipofectamine 3000 (#L3000015, Invitrogen, Carlsbad, CA, USA). The full-length cDNA sequence of FZD2 was amplified and inserted into GV141 vector (Genechem, China) for overexpressing FZD2. The levels of FZD2 mRNA and protein were separately checked by qRT-PCR and western blot. Sequences for all siRNAs are as follows:

si-FZD2-1	CATCCTATCTCAGCTACAA
si-FZD2-2	CCATCATGCCCAACCTTCT
si-FZD2-3	CCCGATGGTTCCATGTTCT

Transient transfections of negative controls, miR‐92b‐3p mimics, and inhibitor were performed by FECT according to the protocols. The reagents were all purchased from Ribobio Co. miR‐92b‐3p overexpression lentiviruses and lentiviral construct expressing luciferase were purchased from Genechem and used in the experiments. For inhibition, siRNAs against SOX4 were synthesized by Genepharma and transfected by DharmaFECT transfection reagents (T‐2001‐03, Thermo). The SOX4 overexpression plasmids were constructed with the GV141 vector by GeneChem and transfected into HUVECs using X‐tremeGENE HP DNA transfection reagent (43940400, Roche). Exosomes were transfected with miR‐92b‐3p inhibitor using Exo‐Fect Reagent (EXFT10A‐1, SBI).

miR-148b-3p mimics, miR-148b-3p inhibitor, mimic control and inhibitor control were obtained RiboBio Co. (Guangzhou, Guangdong, China). KIT-coding sequences without the 3′-UTR were cloned into the GV141 vector by GeneChem (Shanghai, China) (called pGV141-KIT). The psiCHECK-2 vector (C8021) was purchased from Promega (Madison, WI, USA). The psiCHECK-2-KIT-wt, psiCHECK-2-KIT-mut-1378, and psiCHECK-2-KIT-mut-1639 were constructed by Bios Biological (Wuhan, Hubei, China). miR-148b-3p agomir and miR agomir NC were generated by GenePharma Co., Ltd. (Shanghai, China). Lipofectamine 3000 (L3000015, Invitrogen, Grand Island, NY, USA) was used to transfect cells with the desired genes or plasmids.

The molecular clone protocol was done according to the previous study^{35 (link)}. The full-length of human HSP90AA1 (NM_001017963.3), hHNRNPK (NM_002140.5), and hPPARγ (NM_015869.5) open reading frames were amplified and connected with flag label (hHSP90AA1-Flag, hHNRNPK-Flag and hPPARγ-Flag) and then inserted into GV141 vector (GeneChem, Shanghai, China). The primer sequences of hHSP90AA1 were as follows: forward: 3′-ACGGGCCCTCTAGACTCGAGCGCCACCATGCCCCCGTGTTCGGGCGGGGACGGC-5′, reverse: 3′-AGTCACTTAAGCTTGGTACCGAGTCTACTTCTTCCATGCGTGATGTGTC-5′; hHNRNPK, forward: 3′-ACGGGCCCTCTAGACTCGAGCGCCACCATGGAAACTGAACAGCCAGAAG-5′, reverse: 3′-AGTCACTTAAGCTTGGTACCGAGAATCCTTCAACATCTGCATACTGCTTCACACTG-5′; hPPARγ, forward: 3′-ACGGGCCCTCTAGACTCGAGCGCCACCATGGGTGAAACTCTGGGAGATTC-5′, reverse: 3′-AGTCACTTAAGCTTGGTACCGAGTACAAGTCCTTGTAGATCTCCTGC-5′. The product was cut with XhoI/KpnI (New England BioLabs, Inc., Ipswich, MA, USA). The full-length of hHSP90AA1 was inserted into GV314 vector and used to construct the Lentiviral hHSP90AA1 (LV-hHSP90AA1) plasmid.