Az505

BAY-498(SMYD2 inhibitor), AZ505(SMYD2 inhibitor), Cisplatin(CDDP), Vinorelbine(NVB), Paclitaxel (Taxol), and Vincristine sulfate(VCR) was obtained from MedChem Express (Princeton, NJ, USA). The primary antibodies against SMYD2, p53, Cleaved-PARP, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA), and the primary antibodies against p53^K370Me was purchased from Immunoway Technology (Plano, TX, USA). The pcDNA3-p53 vector was obtained from Addgene.

Antibodies to Kim-1, p21 and H3K36me3 were purchased from Abcam Inc. (Cambridge, United Kingdom). Antibodies to cleaved Caspase 3, Cyclin D1, SMYD2, p-p53, p53, p-STAT3, STAT3, p-NFκBp65, and Tubulin were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to PCNA, F4/80, MCP-1, ICAM-1 and TUNEL were purchased from Servicebio (Wuhan, China). Antibodies to NGAL was purchased from R and D SYSTEMS (Minneapolis,United States). Antibodies to GAPDH were purchased from Arigo Biolaboratories Corp. (Shanghai, China). Antibodies to BAX was purchased from Absin Bioscience Inc. (Shanghai, China). Antibodies to Histone-H3 was purchased from Merck (Darmstadt, Germany). Antibodies to NFκBp65 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to BCL-2 was purchased from Proteintech Group Inc. (Chicargo, United States). SMYD2 siRNA and scramble siRNA were purchased from GenePharma Co.Ltd. (Shanghai, China). AZ505 was purchased from MedChemExpress (New Jersey, United States). Cisplatin and all other chemicals were purchased from Sigma (St. Louis, MO).

For SMYD2 inhibition, cells were treated with AZ505 (HY-15226), AZ506 (HY-134828), LLY-507 (HY-19313), or BAY598 (HY-19546) (all from Medchemexpress, Monmouth Junction, NJ, USA). These inhibitors were dissolved in DMSO. Cytotoxicity analysis was performed at 24 and 48 h post-treatment via the Lactate dehydrogenase (LDH) assay using the Cytotoxicity Detection Kit (11644793001; Sigma-Aldrich) according to the manufacturer’s protocol. Supernatants from cells cultured in medium with 0.1% Triton X-100 were used as the positive control and the cell culture medium was used as the negative control. Experiments were performed in triplicate wells at least two times and representative data are shown. The percentage of dead cells was calculated as follows: $$\mathrm{cell} \mathrm{death} \left(\mathrm{LDH}\right) = \frac{{\left(\mathrm{LDH}\right)}_{\mathrm{sample}}-{\left(\mathrm{LDH}\right)}_{\mathrm{Negative} \mathrm{control}}}{{\left(\mathrm{LDH}\right)}_{\mathrm{Positive} \mathrm{control}}-{\left(\mathrm{LDH}\right)}_{\mathrm{Negative} \mathrm{control}}}\times 100$$

In the xenograft model, 2 × 10⁶ HT-29 cells were implanted into the flanks of Rag1^−/− mice via subcutaneous injection (n = 8/group). MC-38 cells were injected subcutaneously (1 × 10⁶ cells) into the flanks of B6/J mice (n = 8/group) or into the colon mucosa (1 × 10⁴ cells) of C57BL/6J mice (n = 5/group). Tumor size was measured at serial time points using a digital caliper and the tumor volume was calculated using V = (length × width × height)/2, or scored as previously described [41 (link), 42 (link)]. Each experiments was replicated independently in other experimental models and using other cell lines as described above.
For SMYD2 inhibition in vivo, 100 µg/tumor AZ505 (HY-15226; MedchemExpress) was injected directly into tumors every two days using colonoscopy (n = 5/group). Tumor size was blindly scored at day 5 and day 10. In a different experiment, 50 mg/kg BAY-598 (HY-19546; MedchemExpress) was intraperitoneally injected once daily into mice bearing HT-29 xenografts (n = 6/group). Tumors were measured at serial time points using a digital caliper as mentioned above.