DDR2 controls the epithelial-mesenchymal-transition-related gene expression via c-myb acetylation upon matrix stiffening

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1 Supplementary information controls the epithelialmesenchymaltransitionrelated gene expression via cmyb acetylation upon matrix stiffening Daehwan Kim, Eunae You, Jangho Jeong, Panseon Ko, JungWoong Kim and Sangmyung Rhee* Department of Life Science, ChungAng University, Seoul 6974, Republic of Korea These two authors contributed equally to this work. *To whom correspondence should be addressed: Tel.: Fax: sangmyung.rhee@cau.ac.kr

2 Relative expression Relative expression Fold expression a (bp) 5 PAG (kpa) b DDR1 5 DDR GAPDH PAG (kpa) PAG (kpa) c d Collagen coated PAG (kpa) Gelatin coated PAG (kpa) Serum (bp) 5 NC G NC G (bp) 5 NC (kpa) 5 GAPDH 5 GAPDH G G (kpa) PAG PAG No S S Supplementary Figure 1. Expression of transcripts is dependent on matrix rigidity. (a,b) RTPCR (a) and qrtpcr (b) analyses of expression levels of DDR1,, and GAPDH in H199 cells incubated on a PAG of various stiffness or on glass (G). (c) RTPCR analysis of the expression levels of and GAPDH in H199 cells incubated on a PAG of various stiffness or glass coated with collagen or gelatine. (d) RTPCR analysis of expression levels of and GAPDH in H199 cells incubated on a PAG in the absence or presence of serum. Band densities were measured and normalised to GAPDH expression levels under the same conditions. NC, negative control. Data represent the mean of three independent experiments ± SEM. P <.1.

3 TF? TF promoter 78 candidates 1 st selection : Searching program (threshold.9) nd selection : Relevance for lung cancer 8 candidates (STAT1b, PEA3, NFkB1, FOXA1, cmyb, ELF1, LEF1, crel) Supplementary Figure. The experimental scheme employed to analyse transcription factors that regulate promoter activity in lung cancer. Seventyeight candidates are listed in Supplementary Table 1, and eight candidates (STAT1b, PEA3, NFκB1, FOXA1, cmyb, ELF1, LEF1, and crel) were eventually selected.

4 Relative Luciferase Activity GFP GFPcMyb GFPLEF1 p3 bgal Supplementary Figure 3. Coexpression of cmyb with p3 induces promoter activity. Relative expression of WT promoter driven luciferase reporters in cmyb, LEF1, or p3overexpressing H199 cells. Data represent the mean of three independent experiments ± SEM. P <.1.

5 Cell number (x1 4 ) 1 cmyb KD cmyb KD 8 * (days) Supplementary Figure 4. Inhibition of proliferation in cmyb knockdown H199 cells is reversed by overexpression. Quantification of proliferation of H199 cells incubated on a stiff matrix for the indicated periods. The cells were trypsinized and counted. Data represent the mean of three independent experiments ± SEM. * P <.5, P <.1.

6 cmyb KD KD GM Fold change of spheroid area cmyb KD KD GM a Day Day 4 b Factin Ki67 DAPI Merged.5 * * Supplementary Figure 5. A knockdown of cmyb and inhibits cancer invasion in a 3D environment. H199 cells expressing mock (with or without 5 mm GM61), anti, or anti cmyb shrna were incubated for days to form spheroids as described in the Methods section. Collagenembedded spheroids were further incubated for 4 days, fixed, and stained with phalloidin, an antiki67 antibody, and DAPI. The spheroids were examined by phase contrast microscopy (a) and fluorescence microscopy (b). GM: GM61. Scale bars: μm (a) and 5 μm (b). Data represent the mean of three independent experiments ± SEM. * P <.5, * P<.1.

7 Number of invading cells Absorbance (55 nm) Cell number (x1 4 ) Fold expression Fold expression a b.5.5 kpa 4 kpa 1. cmyb KD * * c.3 DDR1 cmyb KD MYB DDR1 4 cmyb KD. * 3 * (days) (days) d cmyb KD Supplementary Figure 6. The knockdown of cmyb inhibits proliferation and invasiveness of A549 cells. (a) qrtpcr analysis of DDR1 and on.5 and 4kPa PAGs in A549 cells. (b) qrtpcr of MYB, DDR1 and on 4 kpa PAGs in mock shrna and cmyb KD A549 cells. (c) Quantification of cell proliferation in mock or anti cmyb shrna expressing A549 cells. Graphs show absorbance of crystal violet (left) and the number of counted cells (right). (d) Quantification of invasion. Data represent the mean of three independent experiments ± SEM. * P<.5, P<.1, * P<.5.

8 Fold ratio of transcript level Fold ratio of EP3 transcript level Fold ratio of MYB transcript level a.5 kpa 4 kpa ITGB1 KD #1 ITGB1 KD # 4 kpa b (kda) Integrin b1 perk ERK pmlc MLC atubulin (kpa) c Mo * * Mo #1 # ITGB1 KD d.5 kpa 4 kpa.5 kpa 4 kpa.5 kpa 4 kpa Mo * * Mo #1 # ITGB1 KD e Mo Mo #1 # ITGB1 KD Supplementary Figure 7. Cellular contractility on a stiff matrix regulates the expression of and EP3 via integrin b1. (a) A phase contrast image of H199 cells transfected with mock sirna or anti integrin β1 sirna (#1 and #) on.5 and 4kPa PAGs. Scale bar, 5 μm. (b) Western blot analysis of mock or anti integrin β1 sirna transfected H199 cells. (c,d,e) qrtpcr analysis of expression of (c), EP3 (d), and MYB (e) in mock and integrin β1 knockdown cells. qrtpcr values were normalised to GAPDH. Mo:. Data represent the mean of three independent experiments ± SEM. * P<.5, P<.1, * P<.5.

9 LEF1 cmyb GFP.5 kpa 4 kpa GFP DAPI Merged GFP DAPI Merged Supplementary Figure 8. Cellular localisation of cmyb and LEF1 does not depend on matrix stiffness. Fluorescent images of H199 cells transfected with plasmids encoding GFP, GFPcMyb, or GFPLEF1 and incubated on a PAG. Nuclei were stained with DAPI. Scale bar, 1 mm.

10 Figure 1c pfak perk pmlc atub FAK ERK MLC Figure a DDR1 atub Figure b Figure d (long) atub atub perk ERK p1

11 Figure 3b Figure 4e and 4g atub atub GFP DDR1 pmlc MLC Figure 5d Figure 5f p3 p3 AcK cmyb DDR1 atub pro

12 Figure 5a Figure 6c Figure 6e cmyb LEF1 AcK (of cmyb) cmyb atub cmyb atub AcK (of LEF1) perk perk ERK ERK Supplementary Figure 9. Full length blots. Red dotted lines show the cropping locations.

13 Supplementary Table 1. Seventyeight transcription factors that are predicted to bind to the promoter region. No Name Description 1 FOXP3 Forkhead box P3 RXR Retinoid X receptor alpha 3 NR3C1 Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) 4 RAR Retinoic acid receptor beta 5 GR Glucocorticoid receptor alpha 6 C/EBP CCAAT/enhancer binding protein alpha 7 C/EBP CCAAT/enhancer binding protein beta 8 POUF POU class homeobox 9 cmyb Transcription factor cmyb 1 FOXA1 Forkhead box A1 11 FOXA Forkhead box A 1 XBP1 Xbox binding protein 1 13 TFIID Transcription factor II D 14 cets1 Vets erythroblastosis virus E6 oncogene homolog 1 15 cets Vets erythroblastosis virus E6 oncogene homolog 16 IRF1 Interferon regulatory factor 1 17 NFATC1 Nuclear factor of activated Tcells, cytoplasmic, calcineurindependent 1 18 NFATC Nuclear factor of activated Tcells, cytoplasmic, calcineurindependent 19 AP A Transcription factor APalpha A p53 Tumor protein p53 1 AR Androgen receptor GATA1 GATA binding protein 1 (globin transcription factor 1) 3 GATA GATA binding protein (globin transcription factor ) 4 STAT1 Signal transducer and activator of transcription 1beta 5 STAT4 Signal transducer and activator of transcription 4 6 STAT5A Signal transducer and activator of transcription 5a 7 ATF3 Activating transcription factor 3 8 NFI/CTF Nuclear factor ICCAATbinding transcription factor 9 NF1 Neurofibromin 1 3 SRY Sexdetermining region Y 31 TCF4E Transcription factor 7 like, T cell specific, HMG box 3 VDR Vitamin D (1,5 dihydroxyvitamin D3) receptor

14 33 PXR1 Pregnane X receptor 34 cjun Jun protooncogene 35 cfos FBJ murine osteosarcoma viral oncogene homolog 36 HNF1A HNF1 homeobox A 37 HNF1B HNF1 homeobox B 38 YY1 YY1 transcription factor 39 POUF1 POU class homeobox 1 4 TEAD TEA domain family member 41 RBPJ Recombination signal binding protein for immunoglobulin kappa J region 4 PEA3 Phosphatidylinositol4phosphate 5kinase and related FYVE fingercontaining proteins signal transduction mechanisms 43 COUP TF1 Nuclear receptor subfamily, group F, member 1 44 LEF1 Lymphoid enhancerbinding factor 1 45 NF B1 Nuclear factor of kappa light polypeptide gene enhancer in Bcells 46 EF1 EF transcription factor 1 47 SP1 Sp1 transcription factor 48 MAZ MYCassociated zinc finger protein (purinebinding transcription factor) 49 TBP TATA box binding protein 5 MEFA Myocyte enhancer factor A 51 HOXD9 Homeobox D9 5 HOXD1 Homeobox D91 53 NFY Nfy Histonelike transcription factor NFY protein family (IC) 54 Elk1 Member of ETS oncogene family 55 EBF Transcription factor COE1like 56 Ik1 IKAROS family zinc finger 1 (Ikaros) 57 ELF1 E74like factor 1 (ets domain transcription factor) 58 PU.1 SPI1 (PU 1) spleen focus forming virus (SFFV) proviral integration oncogene 59 WT1 Wilms tumor 1 6 Egr3 Early growth response 3 61 AhR:Arnt Aryl hydrocarbon receptor nuclear translocator 6 PPAR Peroxisome proliferatoractivated receptor alpha 63 TCF4 Transcription factor 4 64 IRF Interferon regulatory factor 65 USF Upstream transcription factor, cfos interacting 66 Pax Paired box

15 67 Nkx.1 Homeobox protein Nkx.1 68 Sox5 sex determining region Ybox 5 69 HSF1 Heat shock transcription factor 1 7 HSF Heat shock transcription factor 71 CP Transcription factor CP 7 NFE Nuclear factor, erythroid 73 MZF1 Myeloid zinc finger 1 74 Evi1 Ecotropic viral integration site 1 75 crel Vrel avian reticuloendotheliosis viral oncogene homolog 76 NRF NfE related factor 77 Lyf1 Lymphoid transcription factor LyF1 78 Pbx1 PreBcell leukemia homeobox 1