Metformin enhances cisplatin cytotoxicity by suppressing Stat3 activity. Chien-Chung Lin, Hsuan-Heng Yeh, Wei-Lun Huang, Jing-Jou Yan, Wu-Wei Lai,

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1 Metformin enhances cisplatin cytotoxicity by suppressing Stat3 activity independently of the LKB1 AMPK pathway Chien-Chung Lin, Hsuan-Heng Yeh, Wei-Lun Huang, Jing-Jou Yan, Wu-Wei Lai, Wen-Pin Su, Helen H. W. Chen, and Wu-Chou Su ONLINE DATA SUPPLEMENT METHODS Western blotting In western blotting, treated and untreated cells were pelleted and washed twice with ice-cold PBS. Cell lysates were obtained after adding lysis buffer containing phosphatase inhibitors (1 mm Na 3 VO 4 and 1 mm NaF) and protease inhibitors (Roche, Indianapolis, IN). The lysates were clarified by centrifugation at 14, rpm for 1 min and the protein concentration was determined using Bradford s assay (Bio-Rad, Richmond, CA). The lysates were then boiled for 5 min with sample buffer, before separation on SDS-polyacrylamide gels. The proteins were transferred to a nitrocellulose filter (Millipore, Billerica, MA) in Tris-glycine buffer and run at 1 V for 1.5 h using an electro-blotter (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The membranes were blocked using PBS buffer containing 5% non-fat milk before incubation with antibodies. Antibody

2 binding was detected by electro-chemiluminescence (Amersham) according to the manufacturer s instructions. β-actin was used as a loading control. Signals were detected by spot densitometry (Alpha EaseFC software from Alpha Innotech CA, USA). Subcutaneous animal tumor models To generate subcutaneous tumors,.1 ml AS2 cell suspensions (5 1 7 cells/ml) were injected subcutaneously into the dorsal skin where palpable tumors developed after one week. The tumor volumes were calculated daily by measuring the greatest longitudinal diameter (length) and the greatest transverse diameter (width) using the modified ellipsoidal formula (15), as follows. Tumor volume =.5 (length width 2 ) Treatment was started when tumors reached 5 1 mm 3. Metformin (5 mg/kg) was given by lavage feeding tube (16) whereas cisplatin was given once a week by intra-peritoneal injection (4 mg/kg), as described previously (17). Immunohistochemistry and quantitative analysis of ki67 The paraffin-embedded animal tumors were fixed in 1% formalin, embedded in paraffin, and cut into 5-mm thick sections. Before immunohistochemistry, the sections were de-paraffinized, re-hydrated, incubated with 3% H 2 O 2 in methanol for 2 min, and subjected to heat-induced antigen retrieval by boiling for 1 min in.1

3 M sodium citrate. The specimens were then blocked with normal goat serum for 3 min and incubated for 2.5 h at room temperature with anti-ki67 antibody (Proteintech Group, Inc., Chicago, IL, USA) at 1:25 dilutions. The negative controls were treated with 5% normal goat serum without primary antibodies. The software program HistoQuest (TissueGnostics) was used to quantify and analyze the immuno-assayed sections (18). Immunofluorescent images and quantitative analysis of Stat3 The animal subcutaneous tumors were soaked in xylene for 1 h to remove the paraffin. Antigen was retrieved by heating the slides in citrate buffer (ph 6.) for 15 min in a pressure cooker. The slides were then blocked by PBS-T containing 5% normal donkey serum for 1 h and incubated overnight at 4 C with anti-stat3 (Novus Biologicals, Littleton, CO) at a 1:25 dilution. An indirect immunofluorescence assay was performed by incubation with secondary antibodies conjugated to anti-mouse Alexa Fluor 488 and anti-rabbit Alexa546 (Invitrogen Corp., Carlsbad, CA, USA). The nuclei were visualized by 4,6-diamidino-2-phenylindole staining (DAPI; Sigma-Aldrich, Saint Louis, MO) while the cytoplasm and cell membranes were visualized by ALDOA, as described previously (19). The fluorophores were excited by laser at 45, 488, or 543 nm and detected using a scanning confocal microscope (FV-1, Olympus). Pixel-by-pixel

4 analysis with the FV-1 program (19) was used to determine the nuclear localization of Stat3. Triple color images were exported as TIFF images.

5 Supplemental Figure Legends Supplemental Figure 1. Phosphorylation levels of Stat3 in CL1-5 cells. After JSI-124 (Stat3 inhibitor) treatment for 8, 24, 48, and 72 h, the phosphorylation of Stat3 was examined by western blotting and quantified by normalization against total Stat3. Values represent the mean ± SEM from at least three experiments. Supplemental Figure 2. Enhanced cytotoxic effects of metformin and JSI in AS2 cells were detected using trypan blue assay. Cell viability of AS2 cells after treatment with different doses of metformin (1 and 2.5 mm), JSI-124 (.5 and.1 M), or AICAR (1 and 2 mm) with cisplatin (.5 M) for 24 h, which were measured using the trypan blue assay. Supplemental Figure 3. Effect of the combined treatment with cisplatin and metformin or JSI on clonogenic survival in AS2 cells. AS2 cells were incubated with cisplatin (1 M/3 h) alone or in combination with metformin (2.5 mm/24 h) or JSI (1 M/1 h) and plated in triplicate. The colony-forming assay was performed after 14 days. Data represent the mean ± SEM obtained from three independent experiments, which were normalized to the values of the control cells. Supplemental Figure 4. Effects of the AMPK inhibitor on the AS2 cell response to metformin treatment. AS2 cells were treated with metformin alone or in

6 combination with the AMPK inhibitor, compound C. The expression and phosphorylation levels of Stat3, AMPK, mtor, 4EBP1, and p7s6k in AS2 cell lines were examined by western blotting and quantified by normalization against the total protein. The values represent the mean ± SEM from at least three experiments. The cell survival rate was measured using the MTT assay. Supplemental Figure 5. Effects of an mtor inhibitor on Stat3 phosphorylation during the AS2 cell response to metformin treatment. AS2 cells were treated with metformin alone or in combination with an mtor inhibitor, rapamycin, for 24 h. The Stat3 expression and phosphorylation levels were examined by western blotting and quantified by normalization against total Stat3. Supplemental Figure 6. Detection of ROS by flow cytometry. The AS2 cells transfected with vector or LKB1 sirna, and those transfected with AMPK shrna (GFP-V-RS plasmid), were treated with cisplatin and/or metformin for 24 h. The cells were trypsinized, washed with serum-free media, incubated in 5 M DCF-DA or 2.5 M HE, and analyzed by flow cytometry using a FACSCalibur system. The FACS results (A) for DCF-DA-stained AS2 cells and (B) for DCF-DA-stained AS2 cells transfected with the vector, LKB1 sirna, and for HE-stained AS2 cells transfected with AMPK shrna show that the levels of ROS attenuation (ROS (cisplatin-metformin) /ROS cisplatin ) were 49%, 59%,

7 and 6%, respectively. Supplemental Figure 7. Blood sugar and body weight measurements of the first animal cohort (n = 16) and GPT, BUN, and CRE levels of the second cohort (n = 12). (A) There were no differences in blood sugar levels of the four groups and the bodyweights only had a borderline difference in the metformin and combined groups (p =.5). (B) To examine the toxicity of the combined therapy in an animal model, 12 mice were separated into three groups: control group (received water + ip PBS), metformin group (oral metformin, 5 mg/kg + ip PBS), and cisplatin + metformin group (oral metformin, 5 mg/kg + ip cisplatin, 4 mg/kg). AS2 cell suspensions were injected subcutaneously into the dorsal skin where palpable tumors developed within one week. Treatments began when tumors reached 5 1 mm 3. On day 24, the mice were sacrificed due to the large tumor burden and sera were collected to determine GPT, BUN, and CRE levels, which were within the normal ranges in the three groups. Supplemental Figure 8. Analysis of the Ki67 intensity of tumors in the four groups. The intensities of Ki67 in the animal tumors from the four groups were analyzed using the TissueFAXs (TissueGnostics) software while the percentage of Ki67-positive cells on each slide was quantified using HistoQuest (TissueGnostics). The number of Ki67-positive cells on each slide was divided

8 by the total number of cells on each slide using hematoxylin-positive counterstaining. Statistical analyses were conducted using the one-way ANOVA (Tukey s post-test) (***p <.1, compared with control). Supplemental Figure 9. Immunofluorescence using laser-scanning confocal microscopy to determine the Stat3 activity. Immunofluorescent analysis was used to determine the expression and distribution of Stat3 (green, mainly in the nucleus after phosphorylation) and ALDOA (red, mainly in the cytoplasm and cell membrane), while the nuclei were labeled with DAPI (blue) in normal lung tissue and in tumors from the four groups using isotype IgG as the negative control. Immunofluorescent analysis was used to assess a confocal series of fluorescence and differential interference contrast (DIC) images, which were obtained simultaneously at 2-s intervals using a 6 oil immersion objective on an FV1 confocal microscope (Olympus)..Supplemental Figure 1. Metformin inhibited Stat3 phosphorylation and activated AMPK phosphorylation at different doses. The Stat3 and AMPK expression and phosphorylation levels in AS2 after metformin treatment for 4 h, which were analyzed by western blotting.

9 Supplemental figure 1. CL1-5 pstat Stat3 8hr 24hr 48hr hr - + JSl µm Actin 4~ I JSI 2.5 µm D Control... T... ~.!.E 3 s l *** T *** E - r.. Q. 2 CD C> c ns -5 1 ::S! u hr 24hr 48hr 72hr

10 Supplemental figure 2. AS2 Survival (%) Ocis-.SµM E2)JSl-.5µM JSl-.1µM IZZI Cis-.SµM+ JSl-.SµM Cis-.SµM+ JSl-.1 µm 14 Cis-.5µM ~Met-1mM 12> DMet-2.SmM llzjcis-.sµm+ Met-1mM cis-.sµm+ Met-2.SmM ~ r=-i I*** - o,... ~..._,,~..a,..._...j...ju.lll_ 14 D Cis-.SµM 12 1ZZJ AICAR 1 mm 1 OO D AICAR 2mM IZ2I cis-.sµm+ AICAR 1mM - cis-.sµm+aicar 2mM o------a...&.~;.i...&---..l...~~

11 Supplemental figure 3. AS2... (2 1 c: CJ i 1 1U... ~ c: :::J... '?!. ocontrol ~ JSI 1µM Deis 1µM cis 1µM+ JSI 1µM D control D Cis 1 µm ~ Met2.5 mm Cis 1µM+ Met 2.5mM c. *~* t--*** ~--!~-***...=::..., I-***

12 Supplemental figure 4. metformin Compound-C 9 - pstat3 Stat3 72 pampk 72 AMPK P-mTOR mtor P756K P-4EBP1 4EBP ] _ c pampk ~:~ lf3-e:-r-b-b p-mtor 2_ 1 Cl p-4ebp CIP-7s6k 1. r:i *** ***. P=9 1. D *** ***. r:i r:i Survival (%) D control ~Met D comp.c comp. C +Met

13 Supplemental figure 5. metformin (mm) rapamycin (nm) p V7 5 Stat3 95.==================~ Stat3 95 actin ii ~~ :JI ~ ~psm~ 11 :: -...

14 Supplemental figure 6. (A) -c: ::I (.,) Q) (_) AS2 _ control - Met-1 mm - Met-2.5mM --Cis-1µM +Met-1 mm --- Cis-1µM+Met-2.5 mm FL 1(DCF-DA) (B) ~ AS2-vector il AS2-LKB1(-) ~ AS2-AMPK(-) _control - cisplatin - cis+met cis+met _control - cisplatin - cis+met _control - cisplatin - cis+met 1' 1' FL 1(DCF-DA) FL1(DCF-DA) 1 ' FL2(HE) c: ~o u QI 111- ib-c... I!:! ~ Ill - c.. en E oo a:::~ AS2-vector 2 cis 15 o cis+met % % AS2-LKB1(-) 12 cis cis+met % 4 1 Yo AS2-AMPK(-) 15 cis o cis+met 6% 5 1 % " 1

15 Supplemental figure 7. (A) Blood sugar (mg/di) 2 control - - c is p la tin metformin -e- cisplatin+metformin day Body weight (gm) control ""'*"" cisplatin metformin -e- cisplatin+metformin 1 5 day o--r---r~""t'"~~--r~""t'"~~--.~-t"~ , ~--~..---~--~~ (B) GPT BUN Cre 6 (mg/di) 1.8 (mg/di) (mg/di) D control D Met I Cis+met

16 Supplemental figure 8. control metformin % 262 ~ % % ao Ki6 7 - Mean I nten sity \. I I I I 4 ~ Ki67 - Mean I nten sity cisplatin - Me.an IM e n.s ltv 223 1~ 25, 21.65% 78.35% - cis+met Me.an l nten.s ity "4 % 25.16% 2!) J... I I II I 4t.> Ki67 - Meain I nten sity I I I I 4 S(l Ki6 7 - Mean I nten sity, *** Ki67+ (%) ~-~A----, r----a----~ r~--.a.--~, 1 ~ +-cisplatin ~ +- cis+met ~

17 Supplemental figure 9. Normal lung control cis met Cis+met

18 Supplemental figure 1. AS2 metformin 4hr (mm) Stat3 72 AMPK actin (IJ!l! g>_gi. (J.s! "C _... ca c:..c: - oc.. LL pstat3 2. ~ ~:~_I I I I c::::j p-ampk *** I I I I D