*4.9. Gene Expression Analysis*

To assess the gene expression of GCB and Terr and their combination, total RNA extraction from cells was performed using the easy-BLUE Kit® (Qiagen Inc., Valencia, CA, USA). Reverse transcription was undertaken to construct a cDNA library from different treatments using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The archived cDNA libraries were then subjected to quantitative real-time PCR reactions [72] using SYBR-green fluorophore (Fermentas Inc., Glen Burnie, MD, USA). Primer sequences were as shown in Table 3.

**Table 3.** Primer sequences of target genes were used for the qPCR analysis.


*4.10. Metabolomics Analysis*

4.10.1. Sample Processing for NMR Spectroscopy

The lyophilized extracellular cell media were mixed with the internal reference 3- (Trimethylsilyl)-1- propane sulfonic acid-d6 sodium salt (DSS-d6, dissolved in methanol-d4, 10 mM) to reach the final concentration of 1 mM. From each sample, 600 μL was placed in 5 mm NMR tubes for NMR analyses. Three biological replicates from each sample were analyzed [73].

#### 4.10.2. NMR Measurement

The 1H-NMR experiments were carried out using a Bruker NMR spectrophotometer (Bruker Biospin GmbH, Karlsruhe, Germany) operating at 600 MHz and a temperature of 25 ◦C.

#### 4.10.3. NMR Spectral Processing

Metabolite annotation was conducted using ChenomX NMR Suite 8.6 (ChenomX Inc., Edmonton, AB, Canada), and phase and baseline corrections were performed initially. The identification was then verified by Human Metabolome Database (http://www.hmdb. ca/) accessed on 1 April 2021, Madison Metabolomics Consortium Database (http:// mmcd.nmrfam.wisc.edu) accessed on 1 April 2021. The metabolites with corresponding concentrations were subjected to multivariate analysis [73].

#### 4.10.4. Multivariate Analysis

Metabolite data from all replicates were then imported to MetaboAnalyst 5.0 platform (http://www.metaboanalyst.ca/) accessed on 5 May 2021, for multivariate analysis [74]. Hierarchical cluster analyses were performed to visualize the grouping resulting from the difference between the metabolites released from the HCT-116 cells treated with terrein, GCB, and GCB + Terr under both normoxic and hypoxic conditions. Partial least squares discriminant analysis (PLS-DA) was performed to visualize the grouping tendencies in the samples with many variables. Significant metabolites were determined from variable importance in projection scores (VIP) values. VIP values above 1.00 were significant [75].
