Supporting Information

Butyrate Suppresses Proliferation of Colon Cancer Cells via targeting Pyruvate Kinase M2

and Metabolic Reprogramming

Qingran Li1, †, Lijuan Cao1, †, Yang Tian1, Pei Zhang2, Chujie Ding1, Wenjie Lu1, Chenxi Jia2, Chang Shao1, Wenyue

Liu1, Dong Wang1, Hui Ye1,* and Haiping Hao1,*

1
State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China

Pharmaceutical University, Nanjing, 210009, China

2
National Center for Protein Sciences-Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center,

Beijing Institute of Radiation Medicine, Beijing 102206, China.

*
Correspondence: haipinghao@cpu.edu.cn (H.H.), Tel: 86-25-83271179; cpuyehui@cpu.edu.cn (H.Y.), Tel: 86-25-83271128

†
These authors contributed equally to this work.
Supporting Methods

Reverse Transcription and Real-time PCR

Cells were washed twice by ice-cold phosphate buffered saline (PBS), and RNA was extracted by RNAiso plus

Reagent (Takara, Shiga, Japan) according to the instructions of manufacturer. Purified RNA was quantified, and

reverse-transcripted using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA) to harvest

cDNA. CFX 96 real-time PCR system (Bio-Rad, CA) was used to semi-quantify the expression levels of mRNA for

indicated genes. Amplification was performed by iTaqTM Universal SYBR® green PCR Supermix (Bio-Rad, CA)

following protocol: 95 °C for 30 s, 40 cycles at 95 °C for 30 s, 60 °C for 15 s and 72 °C for 30 s, a stepped

temperature increase from 65 °C to 95 °C was performed to determine the purity of PCR products. Sequences of the

primers for each gene were listed in supplementary Table S6.

Lactate Dehydrogenase Activity Assay

For measurement of lactate dehydrogenase (LDH) activity, a LDH activity assay kit (JianCheng, Nanjing, China)

was used according to the manufacturer’s instructions. Briefly, cell lysate containing 5 μg of total protein was

incubated with reaction buffer, substrate mix and NAD+ for 15 min at 37 °C in 96-well plates. Subsequently, 2,

4-dinitrophenylhydrazine solution was added and allowed for incubation for 15 min. The reaction was quenched by

addition of NaOH solution. The activity of LDH was quantified using a colorimetric plate reader (Synergy H1, Bio-tek,

Winooski, VT) at OD450 nm.

Detection of PKM2 oligomerization

Cells were gently lysed by NP-40 buffer supplemented with 1% protease inhibitor cocktail (Sigma-aldrich, St.

Louis, MO, USA) on ice. Cells lysates were then obtained by centrifugation at 12,000 g for 10 min at 4°C.

Glutaraldehyde (Ourchem, Shanghai, China) was added into the lysate to a final concentration of 0.05% (w/v). The
cell lysate was gently mixed and allowed to crosslink for 30 min at room temperature. The resultant samples were

separated by SDS-PAGE to characterize the extent of PKM2 oligomerization.

Preparation of recombinant PKM2 protein

PKM2 (NM_002654.5) was cloned into GV296 vector at EcoRI / SalI site by GeneChem (Shanghai, China). The

structure of the plasmid was shown in the Fig. S6. The vector was transformed into E.coli (BL21) and subsequently

amplified overnight at 16 °C in LB medium containing 0.5 mM isopropyl β-D-thiogalactoside (IPTG) to induce the

expression of PKM2. Bacteria were collected and lysed in deionized water containing 1% (v/v) protease inhibitor

cocktail (Sigma Aldrich, St. Louis, MO). The His-tagged PKM2 was then purified with His-tag Protein Purification

Kit (Beyotime, Nantong, China) according to the instructions of manufacturer. The identity of purified PKM2 was

confirmed by western blotting experiments.

Supporting Tables

Table S1. Summarized information of antibodies used in this study.

Table S2. Identified proteins from the in-gel digested 60 kDa gel bands from the SDS-PAGE separated DARTS

samples.

Table S3. Detailed information of potential metabolic biomarkers of HCT116 colorectal cancer cells in response to

butyrate treatment.

Table S4. MS/MS spectra of all the peptides included for the relative quantification of PKM and BSA.

Table S5. Quantitative details for the peptides included for the relative quantification of potential target proteins and

BSA.

Table S6. Primer pairs used for real-time PCR in this study.
Supporting Figures

Figure S1. Butyrate inhibited proliferation of colorectal cancer cells. Effect of 6-day prolonged treatment of

butyrate on proliferation of (A) HT29 and (B) LoVo cells. Proliferation rates were determined by CCK-8 (n=5). Data

are represented as mean ± SEM.

Figure S2. Butyrate-treated metabolome of HCT116 cells based on an OPLS-DA model. (A) Score plot of

OPLS-DA model differentiates the metabolome of the butyrate-treated cells (1 mM, treatment lasting 24 hrs) from the

non-treated HCT116 cells. (B) Based on the OPLS-DA model, S-plot validates the metabolites with VIP value > 1 as

potential biomarkers (highlight in blue color). P[1] value displays the relative abundance of ions, while p(corr)[1]

value displays the differences between groups.

Figure S3. Effect of butyrate on BrdU incorporation in colorectal cancer cells. BrdU incorporation shows

interference of 48 hr-butyrate treatment to nucleotide synthesis of (A) LoVo cells and (B) HT29 cells (n=4). Data are

represented as mean ± SEM, **P<0.01, ***P<0.001.

Figure S4. In-gel digestion results show PKM2 became less susceptible to pronase digestion after incubation with butyrate
at increased concentrations. (A) Coomassie blue-stained gel shows that BSA co-eluted with the target proteins at ~60 kDa by
SDS-PAGE. (B) Relative abundances of internal standard BSA (left panel) and the proteins of highest abundances (top 3, right
panel) including ATP synthase subunit alpha (P25705|ATPA_HUMAN) and 60 kDa heat shock protein (P10809|CH60_HUMAN)
identified from the 60 kDa band (n=3). (C) Changes of abundance levels of quantifiable PKM peptides following butyrate
treatment of increased concentrations (n=3). (D) Relative abundance for mRNA of PKM1 and PKM2 was determined via
real-time PCR (n=3). (E) Comparison of PKM1/PKM2 protein expression levels by immunoblotting. (F) Stabilizing effect of
butyrate on PKM2 in HT29 (Left) and LoVo (right) cells. Data are represented as mean ± SEM, ***P<0.001.
Figure S5. Dependence of butyrate’s effect on PKM2 activity. (A) The effect of butyrate treatment on PK activity

of HT29 and LoVo cells. (B) The expression level of LDHA mRNA and intracellular LDH activity were not modulated

by butyrate treatment. (C) Effect of butyrate and/or pyruvate treatment (concentration as indicated, treatment lasting

24 hrs) on Histone H3 acetylation. Data are represented as mean ± SEM, **<0.01, ***P<0.001.
Figure S6. Plasmid structure used for preparation of recombinant PKM2 protein.