Supplementary Figure S1_Cottini

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Zoe Parks
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KMS18 KMS2 RPMI8226 INA6 U266 KMS34 Krps62 1 2 3 4 5

x foi Btz MM1 1 µm H929 PI Btz.2% 2.1% 1.7% 13.6% 1.

2% BTZ (h) 24 24 Cleve PARP Cleve spse 3 H929 MM1

1 MM ells o not unergo poptosis espite pervsive

x immunofluoresene stining of OCIMy5, KMS11,

() Quntittive evlution of γ-h2a.x foi numer.

1 Supplementry Figure S1_Cottini γ-h2a.x Krp OCIMy5 KMS11 Krps62 RPMI8226 INA6-1 µm Cleve C3 γ-h2a.x DAPI Merge OCIMy5 H929 JJN3 UTMC2 KMS11 KMS12PE KMS18 KMS2 RPMI8226 INA6 U266 KMS34 Krps % of ells with > 6 γ-h2a.x foi Btz MM1 1 µm H929 PI Btz.2% 2.1% 1.7% 13.6% 1.2% 2.6%.3% 3.2% 2.7% 36.7% 1.1% 21.2% BTZ (h) Cleve PARP Cleve spse 3 H929 MM1 MM Annexin VFITC Supplementry Fig. 1 MM ells o not unergo poptosis espite pervsive ongoing DNA mge () γ-h2a.x immunofluoresene stining of OCIMy5, KMS11, RMI8226, n INA6 MM ell lines. () Quntittive evlution of γ-h2a.x foi numer. Men vlues ± SD erive from triplite experiments re shown. () Immunofluoresene stining for leve spse 3Alex 488, γ-h2a.xalex 568, n DAPI in ells n one representtive ptient (MM1). As positive ontrol, were trete with 5 nm Bortezomi (Btz) for 24 h. One representtive experiment of three is shown. () Annexin V FITC/PI stining (left pnel) n western lot nlysis of leve spse 3 (C3)/leve PARP in n H929 MM ell lines n in two MM ptient ells (right pnel), efore n fter tretment with 5 nm Bortezomi (Btz) for 24 h. One representtive experiment of three is shown.

Supplementry Figure S2_Cottini ABL1 Doxo 2 Doxo N N

x α-tuulin Histone H3 1 µm Doxo Time (h) DAPI HeL

2 ABL1 loliztion in the nuleus of MM ells, ut not of

HeL ells were inute for 2 h with or 1 µm oxoruiin

Left pnel: Western lot with totl lystes for γ-h2a.

2 Supplementry Figure S2_Cottini ABL1 Doxo 2 Doxo N N C Merge C ABL1 γ-h2a.x α-tuulin Histone H3 1 µm Doxo Time (h) DAPI HeL MM3 1 µm 2 µm MM4 1 µm Supplementry Fig. 2 ABL1 loliztion in the nuleus of MM ells, ut not of HeL ells () ABL1 ellulr loliztion in HeL ell line. HeL ells were inute for 2 h with or 1 µm oxoruiin (Doxo). Left pnel: Western lot with totl lystes for γ-h2a.x n with nuler (N) n ytosoli (C) frtions for ABL1, α-tuulin, n Histone H3 re shown. Right pnel: Immunofluoresene for ABL1 n DAPI fter 2 h inution with or 1 µm Doxo. One representtive experiment of two is shown. () Immunohistohemil ABL1 stining in MM ptient smples. Upper pnel, ptient 3 (MM3), 1X n 4X mgnifition. Lower pnel, ptient 4 (MM-4), 25x mgnifition.

Supplementry Figure S3_Cottini Ku55933 1 µm Time (h) ABL1 α-tuulin H929 Nuleus 6 24 Cytosol 6 24 Ku55933 2 µm Ku55933 1 µm Time (h) Nuleus 1 2 1 2 p84 pchk2 CHK2 pchk2 Nuleolin Doxo 4 nm Nuleus

3 ABL1 nuler loliztion is riven y ATM n JNK phosphoryltion in MM ells () H929 MM ells were trete with or 1 µm ATM kinse inhiitor Ku55933 for 624 h.

3 Supplementry Figure S3_Cottini Ku µm Time (h) ABL1 α-tuulin H929 Nuleus 6 24 Cytosol 6 24 Ku µm Ku µm Time (h) Nuleus p84 pchk2 CHK2 pchk2 Nuleolin Doxo 4 nm Nuleus Cytosol γ-h2a.x Doxo 4 nm patm ABL1 pjnk Histone H3 α-tuulin e 1.1% 3.9% Doxo 3.7% 19.5% Doxo imtini 5.1% 1.6% P <.1 PI 7.4% 32.1% 2.3% Annexin VFITC Supplementry Fig. 3 ABL1 nuler loliztion is riven y ATM n JNK phosphoryltion in MM ells () H929 MM ells were trete with or 1 µm ATM kinse inhiitor Ku55933 for 624 h. Cellulr frtiontion ws performe, n ell lystes were lotte with ABL1, α-tuulin, n p84 (s loing ontrol) n pchk2 (Thr68) n totl CHK2 (s ontrol of Ku55933 tivity). One representtive experiment of two is shown. () Western lot for pchk2 (Thr68) n nuleolin in ells trete with or 21 µm Ku55933 for 1 or 2 h. One representtive experiment of two is shown. () Western lot of γ-h2a.x, patm(ser1981), n pjnk (Thr183/Tyr185) in untrete ells or fter inution with 4 nm Doxo for 72 h. One representtive experiment of two is shown. () Western lot for ABL1, Histone H3, n α-tuulin in ells, efore n fter oxoruiin (Doxo; 4 nm) tretment. One representtive experiment of two is shown. (e) Annexin VFITC/PI stining n MTT sorne ssy in ells fter 48 h tretment. Dt re men vlues ± SD of triplites of two ifferent experiments.

Supplementry Figure S4_Cottini PBMCs #1 #2 #3 Doxo 4 nm γ-h2a.

α-tuulin PBMCs UTMC2 #1 #2 #3 Doxo 4 nm Supplementry Fig.

4 Supplementry Figure S4_Cottini PBMCs #1 #2 #3 Doxo 4 nm γ-h2a.x PBMCs #1 #2 #3 Nuleus Cytosol Nuleus Cytosol Nuleus Cytosol Doxo 4 nm ABL1 Histone H3 α-tuulin PBMCs UTMC2 #1 #2 #3 Doxo 4 nm Supplementry Fig. 4 Peripherl loo mononuler ells (PBMCs) response to inue DNA mge () Western lot nlysis for γ-h2a.x in PBMCs isolte from three helthy onors (#1, #2, #3), untrete or fter inution with 4 nm Doxo for 24 h. () Annexin VFITC/PI in PBMCs, untrete or fter inution with 4 nm n 8 nm Doxo for 48 h. () Western lot for ABL1, Histone H3, n α-tuulin in PBMCs, untrete or fter oxoruiin (Doxo; 4 nm) tretment for 2 h. () Western lot for in, UTMC2, n PBMCs, untrete or fter inution with 4 nm Doxo for 2472 h.

5 Supplementry Figure S5_Cottini 1 8 * * 15 * * Expression levels 6 4 Expression levels NPC (22) MGUS (44) SMM (12) MM (239) CL (6) NPC (22) MGUS (44) SMM (12) MM (239) CL (6) * 5 Expression levels 6 5 Expression levels MGUS (5) MM (25) 3.5 MGUS (11) MM (133) PCL (1) e KMS12PE MCr OPM1 OPM2 293T EGFP 98kD 64kD Supplementry Fig. 5 is onsistently own-regulte in MM ells () expression in tset GSE2658 n GSE59, s in Figure 3, ut using proe sets _t (left pnel) n _t (right pnel). () expression in sujets with MGUS n MM otine from EMTAB372, proe set _t. () expression in sujets with MGUS, MM, n Plsm Cell Leukemi (PCL) otine from the GSE13591 tset. *: P <.5; : P <.1; *: P <.1. () Western lot nlysis of in MM ell lines. (e) mrna levels mesure y qpcr nlysis n lulte s elt elt Ct vlues on pnel of MM ell lines.

Supplementry Figure S6_Cottini KMS2 4.8% LZ 19.3% KMS18 5.1% LZ 23.3% Fol hnge 6 5 4 3 2 1 48 h 6 5 4 4.2% 25.7 % 3 2 1 LZ LZ PI 72 h Fol hnge PI 48 h 72 h 7.5% 37.1% Fol hnge UTMC2 1.2 1.8.6.4.2 shrna #1 shrna #3 SSCA P <.

6 Effets on ell numer n poptosis fter moultion of expression in MM ells () reexpression in KMS2 n () KMS18 MM ells using AMAXA eletroportion.

6 Supplementry Figure S6_Cottini KMS2 4.8% LZ 19.3% KMS18 5.1% LZ 23.3% Fol hnge h % 25.7 % LZ LZ PI 72 h Fol hnge PI 48 h 72 h 7.5% 37.1% Fol hnge UTMC shrna #1 shrna #3 SSCA P <.1 72 h LZ UTMC2 98 kd 64 kd SSCA e RPMI8226 f LZ UTMC2 LZ RPMI8226 PI 2.6% 3.6% 4.5% 4.6% SSCA g LZ 5.2% 41.4% P <.1 PI SSCA Supplementry Fig. 6 Effets on ell numer n poptosis fter moultion of expression in MM ells () reexpression in KMS2 n () KMS18 MM ells using AMAXA eletroportion. qpcr (elt elt Ct) nlysis n PI stining to etet e ells fter gting on GFPpositive ells re shown. Dt shown re representtive of three inepenent experiments. () silening in UTMC2 MM ell line using lentivirl elivery system. Left pnel: mrna levels ssesse with qpcr (elt elt Ct ); right pnel: Cell eth is mesure with Annexin VFITC/PI stining. The perentge of e ells is the sum of Annexin V PI, Annexin V, PI n Annexin V, PI ells. Men vlues ± SD of triplites of two ifferent experiments. () reexpression in UTMC2 MM ell line trnsfete using AMAXA eletroportion (t shown re representtive of two inepenent experiments). levels were evlute y western lot nlysis. The two ns present in the western lot orrespon to wil type (64 kd) n EGFP (98 kd). Right pnel: ell numer evlute y ell ounting with trypn lue exlusion in UTMC2. (e) Cell numer evlute y ell ounting with trypn lue exlusion in RPMI8226 MM ell line fter reexpression using AMAXA eletroportion. (f) PI stining in UTMC2 n RPMI8226 MM ells fter trnsfetion s in pnel () n (e) t 72 h fter trnsfetion. (g) reexpression in ells using AMAXA eletroportion. Left pnel: Cell viility is evlute y MTT sorne ssy. Men vlues ± SD of triplites of two experiments re shown. Right pnel: PI stining ws use to etet e ells fter gting on GFPpositive ells t 72 h.

Supplementry Figure S7_Cottini * * * P <.

Imtini Imtini IP: p73 IB: Supplementry Fig.

MTT sorne ssy in KMS2 n trnsfete ells, s esrie in Fig.

Perentge of vile ells ws lulte normlizing to sorne vlues of LZ mso.

() Western lot nlysis for p73 n BAX in KMS18 MM ells trnsfete with plenti4egfp or LZ.

infete with speifi shrna or with srmle shrna (right pnel).

7 Supplementry Figure S7_Cottini * * * P <.1 KMS2 KMS18 KMS2 UTMC2 LZ p73 BAX KMS2 e KMS2 LZ Input (2 µg) Unoun Boun p53 p63 Imtini Imtini Imtini IP: p73 IB: Supplementry Fig. 7 Role of ABL1 n enggement of p73 in meite poptosis in MM ells () Cell viility mesure y MTT sorne ssy in KMS2 n trnsfete ells, s esrie in Fig. 4 n 4 respetively fter tretment with 1 µm Imtini initite t y of trnsfetion. Perentge of vile ells ws lulte normlizing to sorne vlues of LZ mso. Men vlues ± SD of triplites of two experiments re shown. () Western lot nlysis for p73 n BAX in KMS18 MM ells trnsfete with plenti4egfp or LZ. () p73 mrna levels in KMS2 MM ells trnsfete with EGFP or LZ (left pnel), n UTMC2 ells infete with speifi shrna or with srmle shrna (right pnel). 72 h t fter trnsfetion n infetion, respetively. Men vlues ± SD of triplites of two experiments re shown. () Western lot nlysis for p53 n p63 in KMS2 MM ells trnsfete with plenti4egfp or LZ, 48 h fter trnsfetion. (e) Coimmunopreipittion etween p73 n in KMS2trnsfete ells fter 48h tretment with or 1 µm Imtini.

Supplementry Figure S8_Cottini DAPI Merge plko.

1 1, 1, 5 5 _low _high _low _high Btz 2nM Time (h) 1 2 4 Supplementry Fig.

Left pnel: Western lot with nuler (N) n ytosoli (C) frtions for,, α-tuulin, n Histone H3.

() qpcr for mrna levels in fter silening. One representtive experiment of two is shown.

Top n ottom qurtiles were selete (14 iniviuls ffete y MM for eh group) n expression ws

8 Supplementry Figure S8_Cottini DAPI Merge plko.1 Nuleus #5 Cytosol #5 Histone H3 α-tuulin 1 µm 1,5 P <.1 1,5 P <.1 P <.1 1, 1, 5 5 _low _high _low _high Btz 2nM Time (h) Supplementry Fig. 8 n regultion in MM ells () ellulr loliztion in, fter silening. Left pnel: Western lot with nuler (N) n ytosoli (C) frtions for,, α-tuulin, n Histone H3. Right pnel: Immunofluoresene for fter silening in ells. () qpcr for mrna levels in fter silening. One representtive experiment of two is shown. () Left pnel: GSE2658 gene expression tset ws sorte oring to expression (proe set 21185_s). Top n ottom qurtiles were selete (14 iniviuls ffete y MM for eh group) n expression ws ompre using oxnwhisker plots. Right pnel: Levels of expression for (proe set _t) in the sme groups. () Western lot for n in fter inution with 2 nm Bortezomi (Btz) for 1, 2, n 4 h. One representtive experiment of two is shown.

Supplementry Figure S9_Cottini plko.1 #1 plko.1 #3 plko.1 #4 PI Annexin VFITC H929 Nuleus H929 Cytosol ptripz 63 ptripz 9 ptripz 92 plko.1 Histone H3 α-tuulin e H929 H929 P <.

() Western lot for,, n in ells infete with inuile lentivirl vetors, n selete with 2 µg mlˉ1 puromyin for 48 h. Cells were then expose for 72 h to 2 µg mlˉ1 oxyyline.

One representtive experiment of two is shown. () Western lot with nuler (N) n ytosoli (C) frtions for,, α-tuulin, n Histone H3 in H929 MM ell lines.

9 Supplementry Figure S9_Cottini plko.1 #1 plko.1 #3 plko.1 #4 PI Annexin VFITC H929 Nuleus H929 Cytosol ptripz 63 ptripz 9 ptripz 92 plko.1 Histone H3 α-tuulin e H929 H929 P <.1 7AAD Annexin VPE Supplementry Fig. 9 Effets of silening in n H929 ells () Annexin VFITC/PI stining in srmle n silene ells. Dt shown re representtive of two inepenent experiments. () Western lot for,, n in ells infete with inuile lentivirl vetors, n selete with 2 µg mlˉ1 puromyin for 48 h. Cells were then expose for 72 h to 2 µg mlˉ1 oxyyline. One representtive experiment of three is shown. () Western lot for,, n in H929 ells trnsfete with plko.1 ontining srmle sequene or shrna #5 trgeting. One representtive experiment of two is shown. () Western lot with nuler (N) n ytosoli (C) frtions for,, α-tuulin, n Histone H3 in H929 MM ell lines. One representtive experiment of two is shown. (e) Left pnel: Cell numer evlute y ell ounting in triplites with trypn lue exlusion. Men vlues ± SD of triplites of two ifferent experiments. Right pnel: Annexin VPE/7AAD stining fter gting on GFPpositive ells in silene H929 ells. Dt shown re representtive of two inepenent experiments.

10 Supplementry Figure S1_Cottini KMS18 KMS2 KMS18 KMS2 7AAD 94.27% 87.54% 93.96% 94.28% 7AAD Annexin VPE Annexin VPE C KD Control KD P <.1 Supplementry Fig. 1 is require for meite growth inhiitory effets in MM ells () Western lot for n in KMS18 n KMS2 ells trnsfete with plko.1 ontining srmle sequene or shrna #5 trgeting. () Annexin VPE/7AAD stining in KMS18 n KMS2 ells fter gting on GFPpositive ells t 72 h. () silening using n inuile shrna (plko.1tet #4) y lentivirl infetion, followe y silening (shrna #5 ginst ) in ells. Left pnel: Western lot for, n. Right pnel: Cell ounting in triplites.

Supplementry Figure S11_Cottini P <.1 H929 P <.1 KD P <.1 Alone K59R wt HATg MyTg Supplementry Fig.

with vetor oing for pj3h wil type (wt) or pj3h K59R mutnt (K59R mutnt).

Cells trnsfete with srmle shrnas re use s internl ontrol.

11 Supplementry Figure S11_Cottini P <.1 H929 P <.1 KD P <.1 Alone K59R wt HATg MyTg Supplementry Fig. 11 kinse tivity is require for intivtion () Annexin VFITC/PI stining in silene for n then trnsfete with vetor oing for pj3h wil type (wt) or pj3h K59R mutnt (K59R mutnt). () H929 ells were otrnsfete with plko.1 shrna #5 ginst lone, or together with wt or K59R mutnt. Cells trnsfete with srmle shrnas re use s internl ontrol. Left pnel: Cell growth evlute y ell ounting with trypn lue exlusion. Mile pnel: Annexin VFITC/PI stining. Right pnel: Western lot for,, HATg, MyTg, n t 48 h fter trnsfetion.

12 Supplementry Figure S12_Cottini MWCL.1 DOHH2 N C N C ABL1 α-tuulin Histone H3 Others Meningiom Pnres Stomh Osteosrom Upper eroigestive Coloretl Esophgus Ovry Bile ut Urinry trt CLC Chonrosrom Soft tissue Gliom Thyroi Liver Kiney Enometrium Brest Melnom Prostte Mesotheliom Ewings srom Meullolstom Hogkin lymphom NHL TALL Multiple myelom Burkitt lymphom Neurolstom SCLC BALL Other leukemis AML DLBCL CML Supplementry Fig. 12 ABL1 ellulr loliztion n expression levels in hemtologil ner ell lines () Suellulr frtiontion of MWCL.1 (Wlenström's mrogloulinemi) n DOHH2 (lymphom) hemtologil ner ell lines. Cell lystes from ytoplsmi (C) n nuler (N) frtions were nlyze y western lot for ABL1 expression. α-tuulin n Histone H3 were use s loing ontrols for C n N frtions, respetively. () mrna expression in soli tumors n hemtologil ner ell lines (BroNovrtis Cner Cell Line Enylopei, t Arevitions: CLC (Nonsmllell lung rinom), NHL (NonHogkin lymphom), TALL (Tell ute lympholsti leukemi), SCLC (smllell lung rinom), BALL (Bell ute lympholsti leukemi), AML (ute myeloi leukemi), DLBCL (Diffuse lrge Bell lymphom), n CML (hroni myeloi leukemi).

13 Supplementry Figure S13_Cottini Jurkt OCI/AML3 LZ LZ 7AAD 7AAD Annexin VPE Annexin VPE Supplementry Fig. 13 Effet on poptosis following reexpression in Jurkt n OCI/AML3 ells Annexin VPE/7AAD stining to etet poptoti ells fter gting on GFPpositive in Jurkt n OCI/AML3 ells fter overexpression. Dt shown re representtive of two inepenent experiments.

14 ellulr loliztion in Jurkt ells, fter silening.

14 Supplementry Figure S14_Cottini Jurkt Nuleus Cytosol DAPI Merge Histone H3 α-tuulin 5 µm Supplementry Fig. 14 ellulr loliztion in Jurkt ells, fter silening. () Western lot with nuler (N) n ytosoli (C) frtions for,, α-tuulin, n Histone H3 in Jurkt ells. () Immunofluoresene for fter silening in Jurkt ells.

15 Supplementry Figure S15_Cottini Supplementry Fig. 15 Effets of knokown with speifi shrnas on STK3 mrna levels qpcr nlysis for n STK3 mrna in srmle n silene ells.

Cos7 (3TP) (K): TGFβ1(h): (K)

Cos7 (3TP) (K): TGFβ1(h): (K) IP#2: IP#1: Totl Lystes luiferse tivity (K): 6-4 - (K): luiferse tivity luiferse tivity (K): 2 1 RL-: - + + + + + Sm4-3F: + - + + + + MYC-Sm3: - - - - + + TβRI-HA(T204D): - - - + - + α-ha Luiferse Ativity