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Chen et al. Cancer Drug Resist 2024;7:9 https://dx.doi.org/10.20517/cdr.2023.151 Page 3 of 15

circRNA microarray and quantitative real-time polymerase chain reaction, RNA
immunoprecipitation, fluorescence in situ hybridization assays, and luciferase activity
The circRNA microarray, real-time polymerase chain reaction (RT-qPCR), fluorescence in situ
hybridization (FISH), RNA immunoprecipitation (RIP), and luciferase activity experiments were conducted
according to methods described previously and in Supplementary Methods. The primers are presented in
[22]
Supplementary Table 1.

Immunohistochemistry analysis and Western blot
The immunohistochemistry (IHC) procedure was conducted following our previously described
[24]
[23]
protocols . The Western blot (WB) was conducted as previously described .
Cell proliferative and cell invasive capability assay
Cell proliferation was determined using a cell counting kit-8 (CCK-8) and colony formation experiments.
Transwell experiment was performed to determine cell invasive ability. These experiments were performed
following a procedure described previously and in the Supplementary Methods.
[22]
Flow cytometry analysis
Flow cytometry (FC) analysis was performed following the procedure described before and in the
[25]
Supplementary Methods. Information on antibodies is presented in Supplementary Table 2.

Knockdown of circNCOA3 transfection
circNCOA3 stable knockdown lentivirus was obtained from Geneseed Biotech Co., Ltd (Guangzhou,
China). For lentiviral production, HEK293T cells were co-transfected with the lentiviral vectors (or the
control lentiviral vectors) with Lenti-Pac HIV Expression Packaging Mix using Lipofectamine 2000 (Life
Technologies Corporation, Carlsbad, CA, USA). After 48 h, supernatants containing the lentiviral particles
were collected and centrifuged at 500 g for 10 min, followed by filtration using a 0.45 µm filter. MC-38 cells
were then transfected either with sh-circNCOA3 lentivirus or with the control virus (sh-NC). The virus-
transduced cells were identified by GFP fluorescence. Stably transfected cells were selected using puromycin
(2 μg/mL) for 2 weeks and then expanded for subsequent assays.

Tumorigenesis assay
To evaluate the role of circNCOA3 on in vivo tumor growth, the in vivo tumorigenesis assay was carried out
following the method described previously and in the Supplementary Materials. Experimentation on mice
[25]
was approved by the Committee on the Ethics of Animal Experiments of SYSUCC.

Mice xenograft PD-1 antibody treatment
Xenograft experiments were performed using C57BL/6 mice to assess the role of circNCOA3 in influencing
the efficacy of PD-1 antibody treatment. Briefly, a total of 1 × 10 cells (MC38-sh-circNCOA3 or MC38-sh-
6
NC) were implanted subcutaneously in the left flank of 6-week-old C57BL/6 mice. Each mouse population
was randomly divided into two groups and injected intraperitoneally with either PBS or PD-1 antibody (Bio
X Cell, West Lebanon, NH, USA) according to a previously described method .
[26]
Statistical analysis
All the data are presented as mean ±standard deviation unless otherwise noted. The statistical analysis was
conducted by using GraphPad Prism 7 or SPSS 17.0 software. Student’s t-test, one-way ANOVA, chi-
squared test, and Pearson correlation analysis were performed where appropriate. The Kaplan-Meier
method was used to assess OS and PFS using the log-rank test. A P value of < 0.05 was considered
statistically significant.