The human colon cancer HT-29 cell line was obtained from the Pasteur Institute (Tehran, Iran). HT-29 cells were expanded using RPMI-1640 (Gibco) culture medium at standard conditions (37 °C, 5% CO2, 95% relative humidity). The culture medium was supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin/Streptomycin (Gibco), and changed every 3–4 days. Using 0.25% Trypsin-EDTA solution, cells were subcultured. Cells in passages 3–6 were used in this study for different experiments.

To assess the viability of cells, the conventional MTT (Sigma-Aldrich) colorimetric method was used. HT-29 cells (∼ 1 × 104) were placed in each well of 96-well plates (SPL, Korea) and maintained at standard conditions to reach 70–80% confluence. Then, cells were exposed to 60 µM TQ (Cat no: 15,039; Cayman; USA), 15 µM Wnt3a inhibitor (LGK974; Cat no: 14,072; Cayman; USA), and their combination for 48 h according to previously published data [16,17,18]. After treatment, supernatants were removed and 100 µl of 5 mg/ml MTT solution was overlaid on each well. Plates were maintained at 37 °C for 4 hours. In the next step, supernatants were replaced with 200 µl dimethyl sulfoxide (DMSO; Merck). The OD of each well was measured at 630 nm using a microplate reader. In this study, TQ and LGK974 stocking solutions were prepared using DMSO solution and a final concentration of solvent was below 1%.

The expression of genes related to mitophagy [PINK 1 and optineurin (OPTN)], WNT signaling pathway (Axin and c-myc), and VE-cadherin (an angiogenesis factor) was measured by real-time PCR technique. The total RNA of cells was extracted using Trizol Reagent (Cat no: 302-001; RiboExLs). RNA concentration was determined using NanoDrop® ND-1000 (NanoDrop Technologies, USA). The isolated RNAs (1 µg) were reverse transcribed into cDNA using a Takara cDNA Synthesis Kit (Cat no: RR037A). In this study, exon-exon junction spanning primers were designed for the specific detection of target genes (Table 1). The amplification process was performed in a MIC real-time PCR system (BioMolecular Systems, UK) using a program for 40 cycles as follows; denaturation for 10 s at 95 °C, annealing for 30 s at optimized annealing temperature (see Table 1) for each primer, and extension for 20 s at 72 °C. To run the reaction, a solution consisting of 1 µl of diluted (0.5 µM) primers (either forward or reverse sequence), 7 µl SYBR green DNA PCR Master Mix 2X (Ampliqon), 4 µl nuclease-free H2O2, and 1 µl cDNA samples. Fold changes of genes were calculated by the 2−ΔΔCT method normalized to β-actin as a reference gene.

Western blotting was performed to measure protein levels of autophagy. After a 48-hour incubation period, cells were collected and lysed using a lysis buffer solution [1% NP-40, 50 mM Tris-HCl, EDTA, 150 mM NaCl, 0.5% sodium deoxycholate, and 1 mM protease inhibitor cocktail]. Cells were centrifuged at 12,000 g for 30 min at 4 °C. Samples were electrophoresed at 10% SDS-PAGE gel and transferred onto the PVDF membrane. Membranes were blocked with 2% skim milk at RT for one hour and exposed to primary antibodies solution at 4 °C overnight: Beclin-1 (Cat no: sc-48,341; Santa Cruz Biotechnology, Inc.), P62 (Cat no: sc-10,117; Santa Cruz Biotechnology, Inc.), and LC3 (Cat no: 2775; Cell Signaling Technology, Inc.). After several PBST washes, membranes were incubated with the secondary HRP-conjugated anti-IgG antibodies (Cat no: sc-2357; Santa Cruz Biotechnology, Inc.). Target immunoreactive bands appeared on the X-ray films using an ECL solution. Band density was calculated using ImageJ software (NIH) related to an internal housekeeping control protein β-actin (Cat no: sc-47,778 Santa Cruz Biotechnology, Inc.).

HT-29 cells (3 × 105 cells/well) were seeded in six-well plates. On 70–80% confluence, cells from different experimental groups were detached using Trypsin-EDTA and incubated with 1 µg/ml Rhodamine 123 (Cas no: 62,669–70 − 9; Sigma-Aldrich) 37 °C for 40 min. After that, cells were washed twice with PBS, resuspended in 500 µl ice-cold PBS, and analyzed with a flow cytometer system (BD Biosciences, USA) and FlowJo software (Ver. 7.6.1).

Cells were seeded in six-well plates to generate a single-cell monolayer. To induce a scratch line, a sterile 100-µl pipette tip was used. After washing with sterile PBS, cells were treated with 60 µM TQ with or without LGK974 and incubated in a culture medium for another 48 h. Images were taken using an inverted microscope (Optika, Italy) at 0, 24, and 48 h post-wounding, and the distance between the scratch edges was measured using ImageJ software.

Data (mean ± SD) were monitored using One-Way ANOVA with Tukey post hoc analysis. p < 0.05 was considered statistically significant.