Supplementary Fig. S1 Supplementary Figure S1. The co-expression profile among ALDH activity, mem Grp78 and Glut3 in SAS Sphere cells and cisplatin-resistant (cisPt R ) SAS cells was examined by FACS. (A) The co-expression profile among ALDH, CD133 and Glut3 in SAS Sphere cells and cisplatin-resistant (cisPt R ) SAS cells was examined by FACS. Results are means ± SD of triplicate samples from three experiments (***, p < 0.001) (B). A ALDH+ cells (%) SAS-S mem Grp78(%) Glut3(%) SAS cisPt R mem Grp78(%) Glut3(%) B SAS-S CD133(%) Glut3(%) SAS cisPt R CD133(%) Glut3(%) G1G1 G2G2 G3G3 G4G4 G1G1G2G2 G3G3G4G4 G1: Glut3 + mem Grp78 - SAS cisPt R SAS-S G1G1G2G2 G3G3 G4G4 G1G1 G2G2 G3G3G4G4 SAS cisPt R SAS-S G1 G2 G3 G *** SAS cisPt R SAS-S *** SAS cisPt R SAS-S G1 G2 G3 G4 ALDH+ cells (%) ALDH(%) G2: Glut3 + mem Grp78 + G3: Glut3 - mem Grp78 + G4: Glut3 - mem Grp78 - G1: Glut3 + CD133 - G2: Glut3 + CD133 + G3: Glut3 - CD133 + G4: Glut3 - CD133 -

Supplementary Fig. S2 Supplementary Figure S2. Single-cell suspension from parental SAS or sphere SAS cells was stained with (A) CellROX Deep Red reagent and (B) DCF (oxidation-insensitive analog). Then, the intracellular level of ROS in parental SAS or sphere SAS cells was determined by FACS analyses. Supplementary data A CellROX Deep Red Count SAS-PSAS-S 15.7% SAS-P SAS-S Count 15.7% 4.3% CellROX Deep Red 0.7% Count DCF + B Count SAS-PSAS-S 0.7% 0.3%

Supplementary Fig. S3 Supplementary Figure S3. Enrichment of the drug-resistant population after in vitro drug treatment. Image of enrichment of the cisplatin-resistant population cells. Treatment of 5µM cisplatin killed more than 50% of cells at 48 h. The majority of cells died by the fifth day. Over the next 20 days few enlarged cells were seen with flattened, senescent-like morphology. Then the supernatant were replaced with fresh medium containing drugs at 48hr. After two more cycles, sphere cells were seen. cisPt, cisplatin. Supplementary data cis-Pt R cisPt 10 µM 72hr Recover 20 days cisPt 10 µM 72hrSAS-PRecover 20 daysRecover 30 days A

Supplementary Fig. S4 Supplementary data A control H2O2H2O2 Arsenic ROS Low B SAS-S ROS Low ROS Medi ROS High % Sphere-formation efficiency ROS Low ROS Medi ROS High *** Supplementary Figure S4. ROS Low cells were sorted using DCFDA staining from SAS sphere cells. At day 21, the cells were treated with H 2 O 2 or arsenic,respectively, for 72 hours. Representative images of chemical induced differentiation were shown. (A) A single cell of ROS Low, ROS Medi and ROS High under defined serum-free selection medium was plated in each well of 96-well low attachment plates, respectively. After 14 days, counted the number of spheres and calculated the sphere-forming efficiency (B).

Supplementary Fig. S5 Supplementary Figure S5. Differentially expressed genes of reactive oxygen species scavenging in Parental cells and Sphere cells under 2, 3, 5, or 9 weeks of cultivation with defined serum-free selection medium were collected and analyzed. (A) The heat maps of some ROS scavenger genes in parental versus sphere cells. Red and blue indicate high and low expression levels, respectively. SAS-P, SAS parental; OECM1-P, OECM1 parental; SAS-S, SAS Sphere; OECM1-S, OECM1 Sphere; CAT, catalase; PRDX3, peroxiredoxin 3; SOD2, superoxide dismutase 2. Supplementary data A OECM1-P SOD2 CAT PRDX3 SAS-P SAS-Sphere OECM1-Sphere Parental 3 weeks 5 weeks 9 weeks Parental 3 weeks 5 weeks9 weeks

B Supplementary Fig. S6 Supplementary data Supplementary Figure S6. Treatment of sphere cells with catalase inhibitor 3AT reduced catalase activity. (A) SAS-S cells or (B) OECM1-S cells were pretreated with catalase inhibitor (3AT), for 72hr, afterward, we measured the activities of catalase as described in Materials and Methods. A SAS-P SAS-S SAS-S 3AT Catalase specific activity Catalase specific activity OECM1-P OECM1-S OECM1-S 3AT

5.7% 7.6% 6.8% 8.3% 1.5% 21.8% 13.0% SAS-S Control cisPt 2ME 2ME +cisPt 3AT + cisPt 3AT 3AT + 2ME + cisPt Count DCFDA B 6.4% 18.2% 7.0% 68.5% % 5.6% 76.0% SAS-S Control cisPt 2ME 2ME + cisPt 3AT + cisPt3AT 3AT + 2ME + cisPt 10.0%42.9% 11.9% 35.2% 3.5% 8.1% 4.3% 84.1% 4.2% 14.9% 7.0% 74.0% 5.6% 20.8% 13.1% 60.5% 5.3%17.3% 9.7% 67.6% Propidium iodide(PI) + AnnexinV Supplementary Fig. S7 Supplementary data Supplementary Figure S7. Combinatorial treatment of ROS scavenger inhibitor and cisplatin induces apoptosis cell death. (A) SAS-Sphere cells were either singly treated with cisplatin or co-treated with ROS scavenger (2ME and 3AT), for 72hr, afterward, and stained with DCFDA plus (B) Annexin V/Propidium iodide (PI); then, examined by flow cytometry. (C) The amount of CK18 positive cells was determined by staining the drugs treated cells with CK18 antibody, and then examined by flow cytometry (2ME: superoxide dismutase 2; 3AT: 3-Amino-1,2,4-triazole). A Count 56.3% 2ME+3AT+cisPt Control CisPt CK18 + cells (%) C

B Supplementary Fig. S8 Supplementary data A CD44(%) 45.9% 28.0% 33.5% 25.7% 32.9% shLuc shSOD2#1 shSOD2#2 shCAT#1 shCAT#2 Count C D Sphere No shLuc shSOD2 shCAT SAS-S SAS-S shLuc shCAT#1 shCAT#2 shSOD2#1 shSOD2# SAS-S shLuc shCAT#1 shCAT#2 shSOD2#1 shSOD2# SAS-S shLuc shCAT#1 shCAT#2 shSOD2#1 shSOD2#2 ROS Low cells (%) Ck18 cells (%) CD44 cells (%) Colony No shLuc shSOD2 shCAT SAS-S shLuc shCAT shSOD shLuc shCAT shSOD2 Supplementary Figure S8. Knockdown of CAT or SOD2 gene expression diminished spheres-forming capability, stemness marker expression of HN-CICs. The percentages of (A) ROS - cells, (B) CK18 + cells and (C) CD44 + cells in shCAT, shSOD2 and vector control SAS sphere cells were compared by flow cytometry analysis, respectively. SAS sphere cells were first infected with sh-CAT-1, sh-CAT-2, sh-SOD2-1, sh-SOD2-2 or sh-Luc lentivirus, and further cultivated under defined serum-free selection medium. The sphere formation capability (D) and anchorage independent growth ability (E) of SAS sphere cells treated with either sh-Luc or CAT or SOD2-shRNA lentivirus were examined by microscope. E

Supplementary Fig. S9 Supplementary data 22.3% 16.8% PEG-ControlPEG-CAT mem Grp78(%) SAS-S SSC 16.3% PEG-Control PEG-CAT ROS(%) SAS-S SSC 24.6% A C SAS-S PEG-Control PEG-CAT *** ROS Low cells (%) *** SAS-S mem Grp78 cells (%) PEG-Control PEG-CAT 29.6% OECM1-S 3AT 43.8% OECM1-S 54.8% OECM1-S PEG-CAT 47.8% SAS-S 60.6% SAS-S PEG-CAT 41.9% SAS-S 3AT Count D CD44(%) Supplementary Figure S9. Overexpression of CAT in HNCICs promotes stemness properties. (A) SAS-S cells or OECM1-S cells were treated with PEG-CAT (100 U/ml), for 72hr, afterward, the intracellular catalase activities were measured. The percentages of (B) ROS - cells, (C) mem Grp78 + cells and (D) CD44 + cells in treated with PEG-CAT (100 U/ml) or control SAS sphere cells were analyzed by flow cytometry, respectively. (***, p<0.001) *** * B Catalase specific activity SAS-S OECM1-S PEG-Control PEG-CAT

Supplementary Fig. S10 Supplementary data A OECM1-P PEG-CAT 37.8% 17.2% 30.3% 12.0% OECM1-P PEG-Control SAS-P PEG-Control SAS-P PEG-CAT Count ALDH(%) SAS-P OECM1-P PEG-Control PEG-CAT ALDH cells (%) *** Supplementary Figure S10. Overexpression of CAT in HNSCC under defined serum- free selection medium promotes stemness properties. The activity of ALDH in SAS cells treated with PEG-CAT (100 U/ml) or control PEG under further cultivation within defined serum-free medium for 4 days was analyzed by flow cytometry.(***, p<0.001)

Supplementary Fig. S11 Supplementary data Supplementary Figure S11. Cell viability of drugs treated hematopoietic stem cells. (A) Hematopoietic stem cells (HSC) were treated with 5 µM cisplatin, 15 mM 2ME or 25 µM 3AT, respectively, for 72 hours. Cell viability of the treated cells was further determined by MTT assay. HSC, Hematopoietic stem cells. A Cell Viability (%) *** Control cisPt 5 µM HSC *** Control 2ME 15 µM HSC *** Control 3AT 25 mM HSC Cell Viability (%)