Supplementary Information
Rossi et al.
“ HECT E3 ligase ITCH inhibitors ”
Supplementary Methods
1
Materials
DMEM, RPMI 1640, BSA and FBS were purchased from Flow Laboratories (Milan, Italy).
Tissue culture plasticware was from Becton-Dickinson (Lincoln Park, NJ, USA).
Gemcitabine (Gemzar®) was a gift of Lilly, mitomycin-C was a gift of Kyowa Hakko Kirin.
Cell culture
Our experimental models consist of human breast MDA-MB-231, MDA-MB-436, MDA-MB-
468, and MCF-7 cancer cell lines, human prostate carcinoma PC3, DU145 and LnCaP cell lines, human bladder carcinoma T24, RT112, HT-1376 and MCR cell lines (American Type
Tissue culture Collection, Rockville, Maryland). MDA 231, MDA 436, MDA 468, DU145,
T24, HT-1376 and MCR cells were grown in DMEM, and MCF-7, PC3, LnCaP and RT112 cells were grown in RPMI 1640, both supplemented with heat-inactivated 10% FBS, 20 mM HEPES, 100
μg/ml penicillin, 100 μg/ml streptomycin, 1% L-glutamine and 1% sodium pyruvate. Cells were grown in a humidified atmosphere of 95% air/5% CO2 at 37°C.
Cell viability assay
Cells were seeded in serum-containing media in 96-well plates at the density of
1.5×10 3 cells/well for DU145 and T24 cells, 2.5×10 3 cells/well for MDA-MB-231, MDA-MB-436,
MDA-MB-
468, PC3, LnCaP and RT112, 5×10 3 cells/well for MCF-7, HT-1376 and MCR.
After 24 hours of incubation at 37°C, cells were treated with increasing concentrations of clomipramine or norclomipramine (from 0.3 to 160 µM) and of gemcitabine (GEM) or mytomicin C (MIT) (from 0.004 to 2µg/ml), either as single agents or in combination, for 48 and 72 hours. Cell viability was assessed by adding MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide] solution in phosphate-buffered saline (PBS) to a final concentration of 5 mg/mL. The plates were then incubated at 37°C for an additional 4 hours, and the MTT-formazan crystals were solubilized in 1N isopropanol/hydrochloric acid
10% solution at 37°C on a shaking table for 20 minutes. The absorbance values of the solution in each well were measured at 570 nm using a Bio-Rad 550 microplate reader
(Bio-Rad Laboratories, Milan, Italy). Percentage cell viability was cal culated as 100% ×
(absorbance of the treated wells – absorbance of the blank control wells) / (absorbance of the negative control wells – absorbance of the blank control wells). All MTT experiments were performed in triplicate and repeated at least 3 times. Half maximum inhibitory concentration (IC50) values are presented as mean ± standard deviation (SD).
Drug combination studies
This study allowed the effects of the pharmacological combination between each of the
ITCH inhibitors, clomipramine and norclomipramine, and the conventional

Supplementary Information
Rossi et al.
“ HECT E3 ligase ITCH inhibitors ”
2 chemotherapeutic gemcitabine and mitomycin C on the proliferation of the bladder carcinoma cell lines HT-1376 and RT112. Cells were seeded in 96-well plates at the previously indicated density. After 24 h of incubation at 37°C, cells were treated at the drugs range of concentrations described above. In details we have employed different molar ratios between the two drugs in different sequences of administration (simultaneous,
ITCH inhibitor 24h before the cytotoxic agent or cytotoxic agent 24h before the ITCH inhibitor). Assessment of synergy was performed quantitating drug interaction by CalcuSyn computer program (Chou and Talalay , Biosoft , Oregon , USA). Combination index (CI) values <1, 1, and >1 indicate synergy, additivity and antagonism, respectively.
Labeling of autophagic vacuoles with MDC .
To quantify the induction of the autophagic process in RT112 and HT-1376 cells treated with the various compounds as previously indicated.
Cells were incubated with 50
μM
MDC in PBS at 37°C for 15 min. After incubation, cells were washed with PBS, and immediately analyzed by flow cytometry. All fluorescences were analyzed with a
FACScalibur flow cytometer (Becton Dickinson). The fluorescent emissions were collected through a 530 nm band pass filter (FL1 channel). At least 10 000 events were acquired in log mode. For the quantitative evaluation of MDC, CellQuest software (Becton Dickinson) was used to calculate mean fluorescence intensities (MFIs). The MFIs were calculated by the formula (MFI treated/MFI control), where MFI treated is the fluorescence intensity of cells treated with the various compounds and MFI control is the fluorescence intensity of untreated and unstained cells. Values reported in the figures are the me ans±S.D.s from three independent experiments.
Flow cytometric analysis of apoptosis.
Annexin V-FITC (fluorescein isothiocyanate) was used in conjunction with a vital dye,
Propidium Iodide (PI), to distinguish apoptotic (Annexin V-FITC positive, PI negative) from necrotic (Annexin V-FITC positive, propidium iodide positive) cells. Briefly, cells were incubated with Annexin-V
–FITC (MedSystems Diagnostics, Vienna, Austria) and propidium iodide (Sigma, St. Louis, MO, USA) in a binding buffer (10 mM Hepes, pH 7.4,
150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2) for 10 min at room temperature, washed and resuspended in the same buffer. Analysis of apoptotic cells was performed by flow cytometry (FACScan, Becton Dickinson). For each sample, 2×10 4 events were acquired. Analysis was carried out by triplicate determination on at least three separate experiments.

Supplementary Information
Rossi et al.
“ HECT E3 ligase ITCH inhibitors ”
Supplementary figure legends
3
Supplementary Figure S1.
Evalaution of the effects of ITCH inhibition on autophagy of human cancer cells. Norclomipramine/MIT- ( a b ) and clomipramine/GEM- ( c d ) induced autophagy evaluated by flow cytometric analysis of autophagosomes formation
(MDC incorporation). RT112 cells were treated with norclomipramine 0.55 µM and/or MIT
0.006 µg/ml in sequential administration, norclomipramine 24h before MIT. HT-1376 cells were treated with clomipramine 4.1 µM and GEM 0.10 µg/ml, in sequential administration, clomipramine 24h before GEM. The mean fluorescence intensities (MFIs) were calculated, as described in ‘Supplementary Methods’. CTR N.S. (non-treated, non-stained); CTR
(nontreated). Values are the means of three independent experiments (±S.D.).
Supplementary Figure S2. Effects of the clomipramine/GEM combination on apoptosis on HT-1376.
Apoptosis was evaluated by FACS analysis, after cell labeling with propidium iodide (PI) and FITC-Annexin V. HT-1376 cells were not treated ( a ) or treated with clomipramine ( b ) and GEM ( c ) alone or in combination ( d ) in sequential administration, clomipramine 24h before GEM. The lower left quadrants of each panels show the viable cells, which exclude PI and are negative for FITC-Annexin V binding. The upper left quadrants contain the non-viable, necrotic cells, negative for FITC-Annexin V binding and positive for PI uptake. The lower right quadrants represent the cells in early apoptosis that are FITC-Annexin V positive and PI negative. The upper right quadrants represent the cells in late apoptosis, positive for both FITC-Annexin V binding and for PI uptake. The experiments were performed at least three times with similar results.
Supplementary Figure S3. Effects of the norclomipramine/MIT combination on apoptosis on RT112.
Apoptosis was evaluated by FACS analysis, after cell labeling with propidium iodide (PI) and FITC-Annexin V. RT112 cells were not treated ( a ) or treated with norclomipramine ( b ) and MIT ( c ) alone or in combination ( d ) in sequential administration, clomipramine 24h before GEM. The lower left quadrants of each panels show the viable cells, which exclude PI and are negative for FITC-Annexin V binding. The upper left quadrants contain the non-viable, necrotic cells, negative for FITC-Annexin V binding and positive for PI uptake. The lower right quadrants represent the cells in early apoptosis, that are FITC-Annexin V positive and PI negative. The upper right quadrants represent the cells in late apoptosis, positive for both FITC-Annexin V binding and for PI uptake. The experiments were performed at least three times with similar results.
Supplementary Figure S4. Effects of ITCH inhibition on cell growth of breast cancer cells. Percent of growth inhibition induced by increasing concentrations of norclomipramine (•) and clomipramine (○) after 48h and 72h of treatment in human breast cancer cells MDA 231 (A-B), MDA 468 (C-D), MDA 436(E-F) and MCF7 (G-
H),respectively, evaluated by MTT assay and expressed as percentage of untreated cells.
Each point is the mean of at least four different replicate experiments (±S.D.).
Supplementary Figure S5.
Effects of ITCH inhibition on cell growth of prostate cancer cells. Percent of growth inhibition induced by increasing concentrations of

Supplementary Information
Rossi et al.
“ HECT E3 ligase ITCH inhibitors ”
4 norclomipramine (•) and clomipramine (○) after 48 and 72h of treatment in human prostate carcinoma cells PC3 (A-B), DU145 (C-D) and LNCaP (E-F), respectively, evaluated by
MTT assay and expressed as percentage of untreated cells. Each point is the mean of at least four different replicate experiments
(±S.D.).
Supplementary Figure S6.
Effects of ITCH inhibition on cell growth of bladder cancer cells. Percent of growth inhibition induced by increasing concentrations of norclomipramine (•) and clomipramine (○) after 48h and 72h of treatment in human bladder cancer cells T24 (A-B), RT112 (C-D), HT-1376 (E-F) and MCR (G-H), respectively, evaluated by MTT assay and expressed as percentage of untreated cells. Each point is the mean of at least four different replicate experiments (±S.D.).