T1300 100

Targeting three different sites of TRIM37 (shown in Table 1), interference sequences were synthesized (T1300-100, Solarbio) and shRNA was constructed by double chain annealing. Subsequently, each of the shRNAs was inserted into the AgeI/EcoRI sites of the pLKO.1-puro vector to construct pLKO.1-puro-shTRIM37-1, −2, and −3. For TRIM37 overexpression plasmids, the full length 2895 bp coding DNA sequence (CDS) region of TRIM37 (BC036012.1) was synthesized by Genewiz Company (Shanghai, China). Then, TRIM37 CDS was inserted into a pLVX-puro vector at the EcoR I/BamH I sites. After sequence confirmation, (Invitrogen, Thermo Fisher Scientific, Inc., USA), pLKO.1-puro-shTRIM37 (1000 ng) or pLVX-puro-TRIM37 (1000 ng) was co-transfected with viral packaging plasmids, psPAX2 (100 ng) and pMD2G (900 ng) (Addgene, Inc., Cambridge, MA, USA) using Lipofectamine 2000^TM into 293T cells. After 48 hours of transfection, viral particles in supernatants were obtained by ultracentrifugation.Table 1

TRIM37 Interference Sequences

Name	Sequences
TRIM37 target site 1 (1799–1817)	GGAGAAGATTCAGAATGAA
TRIM37 target site 2 (2119–2137)	CCAGTAGTTTACTAGACAT
TRIM37 target site 3 (2730–2748)	GCCTTGATACATGGCAGTA

Primary fibroblasts were isolated from granulation tissue of slow-healing wounds. The tissues were first washed 3 times with PBS containing 1% penicillin and streptomycin, before the heavily polluted granulation tissues and blood vessels were removed. The tissues were cut into pieces in a petri dish using ophthalmic scissors, and then digested in 0.1% collagenase and 0.1% bovine serum albumin (1: 2; C0130, Sigma) at 37°C on a shaker (T1300-100, Solarbio) for 2 h. The reaction was terminated by addition of 10% fetal bovine serum (FBS; 16000-044, GIBCO), and the tissue suspension was filtered using a 200-molybdenum filter. The filter fluid was centrifuged for 10 min at 1000 rpm, and the pellet was resuspended in DMEM medium (SH30243.01; HyClone) containing 10% FBS and 1% penicillin and streptomycin. Finally, the cell suspension was seeded in 6-well plates and cultured in a CO₂ incubator (5% CO₂, Thermo Forma 3111; Thermo) at 37°C. The culture medium was refreshed after the first 48 h in vitro, and was subsequently changed every 2 to 3 days according to the cell morphology and growth. Subculturing was performed when the cell confluence reached 80%.

The Cell Counting Kit-8 (CCK-8, SAB, CP002) was applied to assess cell proliferation rate. NFATc1-silenced DU145 or PC-3 cells were digested with 0.25% trypsin (Solarbio, T1300-100). Cell suspension was seeded at a density of 3×10³ cells/well in 96-well culture plates with 3 identical wells as duplicate wells, followed by incubated in a 5% CO₂ humidified incubator at 37°C overnight. The next day, 100 μl of CCK-8 solution (CCK-8: serum-free medium=1: 10) was added and incubated for 1 h. The optical density (OD) of the absorbance at 450 nm was measured by a microplate reader (Perlong, Beijing). NFATc1-silenced PC-3 cells infected with oe-c-myc or oePKM2 lentivirus were seeded in 96-well plates at equal numbers, and cell proliferation was measure at 0, 24, 48, and 72 h.

Human GBM cell lines (U87 and U373; ATCC, Manassas, VA, USA) were placed in DMEM/4.0 mM l-glutamine medium (SH30243.01; HyClone, Logan, UT, USA) with fetal bovine serum [10% (v/v); 16000-044; Gibco, Carlsbad, CA, USA], as well as 100 U/ml penicillin and 0.1 mg/ml streptomycin (P1400-100; Solarbio, Beijing, P.R. China) at 37°C under 5% CO₂ atmosphere. Cells in logarithmic growth were digested using trypsin (T1300-100; Solarbio) and suspended as 3 × 10⁴ cells/ml in culture medium.
Three siRNA sequences targeting TMEM168 (GenBank No. NM_001287497.1) were designed as shown in Table 1. Cells were transfected with siRNAs (20 μmol/L) via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to a reported study18 (link). After 48 h, silencing efficacy of TMEM168 gene was detected by RT-PCR and Western blot, with the best knockdown effect being seen in Table 1 (Label 1), which was chosen in our following study. A scramble siRNA was identified as a negative control (siRNA-NC).
To study whether the Wnt/β-catenin pathway was involved, lithium chloride (LiCl), a Wnt activator inhibitor, was used for treatment. In our study, U87 cells with siRNA-TMEM168 (siTMEM168) or siRNA-NC (siNC) transfection were treated with LiCl [10 mmol/L; L130113; Aladdin (http://www.aladdin-e.com)] or vehicle [phosphate-buffered saline (PBS)], and then cells were cultured normally as mentioned above.

The cell proliferation of BC cells was evaluated using the Cell Counting kit-8 (CCK-8). ZR-75-30 and BT474 cells in logarithmic growth phase were digested with 0.25% trypsin (Solarbio, T1300-100, Beijing, China) and then counted under a microscope (Shanghai Caikang Optical Instrument Co., XDS-500C) to prepare 3×10⁴ cells/ml of cell suspension. Each 100 μl/well of cell suspension was inoculated into 96-well culture plates and cultured overnight at 37°C in a 5% CO₂ incubator. The next day, the cells were treated with 100 μl of gradient concentrations of Kaempferol (10, 25, 50, and 100 μmol/l). After 0, 24, 48, and 72 h, 100 μl of CCK-8 solution (CCK-8: serum-free RPMI-1640 medium=1: 10, SAB Biotherapeutics, Inc., CP002) was added to incubate for 1 h. The optical density of the absorbance at 450 nm was evaluated at 0, 24, 48, and 72 h using a microplate reader (Perlong, DNM-9602, Beijing, China).

GC cells (HGC27, MKN45 and AGS) were grown to the logarithmic phase, trypsinized (T1300-100, Solarbio) and cultured overnight in 6-well plates with 3 × 10⁵ cells/well. After treatment for 48 hours, the cells were trypsinized to collect in a centrifuge tube for apoptosis detection (Annexin V-FITC Apoptosis Detection Kit, C1063, Beyotime). About 1 × 10⁵ resuspended cells were incubated with 195 μL of Annexin V-FITC binding solution, followed by 15 min incubation with 5 µL of Annexin V-FITC at 4°C in the dark. Thereafter, at 4°C in the dark, cells were incubated for 5 min in 5 µL PI staining solution. A tube without Annexin V-FITC and PI was used as a negative control. Subsequently, flow cytometry was used to detect cell apoptosis (FCM; Accuri C6, BD Biosciences).