1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-3-3σ alleles. (B) PCR results showing 14-3-3σ allele excision in mammary tumors from ErbB2 KI /14-3-3σ +/+, ErbB2 KI /14-3-3σ F/+, and ErbB2 KI /14-3-3σ F/F mice. (C) Representative H&E-stained and Masson s Trichromestained mammary tumor sections from ErbB2 KI /14-3-3σ +/+, ErbB2 KI /14-3-3σ F/+, and ErbB2 KI /14-3-3σ F/F mice. Upper panel were H&E pictures taken at lower magnification; middle panel were H&E pictures taken at higher magnification; lower panel were Masson s Trichrome staining pictures with collagen stained blue, cytoplasm red, and nucleus black. (D) Quantitative RT-PCR results showing the activated ErbB2 (Neu) mrna levels of 3 ErbB2 KI /14-3-3σ +/+, 5 ErbB2 KI /14-3-3σ F/+, and 5 ErbB2 KI /14-3-3σ F/F tumor samples. Results were normalized against GAPDH. All experiments were performed in triplicates. Data are represented as mean ± STDEV. (E) Southern blotting on DNA extractions of all samples mentioned above, showing the endogenous erbb2 allele (upper band) and the activated erbb2 allele (lower band). (F) Percentages of nuclei staining positively for Ki67 (black) and TUNEL (white) positive cells from 9 ErbB2 KI /14-3-3σ +/+ and 7 ErbB2 KI /14-3-3σ F/F tumor samples. Data are represented as mean ± STDEV. P-values were generated between the 14-3-3σ KO and the parental ErbB2 KI group. (G) Representative images of Ki67 (upper panel) and TUNEL (lower panel) staining of ErbB2 KI /14-3-3σ +/+ and ErbB2 KI /14-3-3σ F/F samples. Figure S2. Ablation of 14-3-3σ does not affect Akt phorsphorylation in ErbB2-induced tumors (A) Quantifications of phorspho-akt levels of 3 ErbB2 KI /14-3-3σ +/+, 10 ErbB2 KI /14-3-3σ F/+, and 10 ErbB2 KI /14-3-3σ F/F mammary tumor samples normalized with Akt levels. (B) Quantifications of phorspho-akt levels of 1 ErbB2 KI /14-3-3σ +/+, 5 ErbB2 KI /14-3-3σ F/+, and 5 ErbB2 KI /14-3-3σ F/F salivary tumor samples normalized with Akt levels. (C) Quantifications of phorspho-akt levels of 5 NIC /14-3-3σ +/+ and 5 NIC /14-3-3σ F/F
mammary tumor samples normalized with Akt levels. P-values were generated between each 14-3-3σ KO group and the parental ErbB2 KI or NIC group. All immunoblottings were performed in duplicate. 2 Figure S3. ErbB2 KI /14-3-3σ KO mice develop salivary gland tumors and pulmonary metastasis (A) Salivary tumor onset curves for 26 ErbB2 KI /14-3-3σ +/+, 24 ErbB2 KI /14-3-3σ F/+, and 28 ErbB2 KI /14-3-3σ F/F mice. The tumor onset rate was presented as T 50, the age at which 50% of the animal cohorts develop their first palpable tumors. (B) Immunoblots of MAPK and Akt on 1 ErbB2 KI /14-3-3σ +/+, 5 ErbB2 KI /14-3-3σ F/+, and 5 ErbB2 KI /14-3-3σ F/F salivary tumor lysates. β-actin was used as a loading control. (C) Quantifications of phorspho-mapk levels of all 11 samples normalized with MAPK levels. All immunoblotts were performed in duplicate. (D) Percentages of 26 ErbB2 KI /14-3-3σ +/+, and 17 ErbB2 KI /14-3-3σ F/F mice with pulmonary metastases. (E) Average size of total tumors per mouse for all ErbB2 KI /14-3-3σ +/+ and ErbB2 KI /14-3-3σ F/F animals. Data are represented as mean ± S.E.M. P-values were generated between the 14-3-3σ KO and the parental ErbB2 KI group. Figure S4. Mammary tumors of NIC mice with 14-3-3σ ablation have elevated cell proliferation (A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3) 14-3-3σ alleles. (B) PCR results showing 14-3-3σ allele excision in tumors from NIC/14-3-3σ +/+, NIC/14-3-3σ F/+, and NIC/14-3-3σ F/F mice. (C) Representative images of H&E-stained mammary tumor sections from NIC/14-3- 3σ +/+, NIC/14-3-3σ F/+, and NIC/14-3-3σ F/F mice. Upper panel pictures were taken at lower magnification; lower panel pictures were taken at higher magnification. (D) Percentage of Ki67 positive nuclei from 11 NIC/14-3- 3σ +/+ and 13 NIC/14-3-3σ F/F tumor samples. Data are represented as mean ± STDEV. (E) Representative images of Ki67-stained tumors from NIC/14-3-3σ +/+ and NIC/14-3-3σ F/F mice. (F) Percentage of TUNEL positive nuclei of the aforementioned tumor samples. Data are represented as mean ± STDEV. (G) Representative images of TUNEL-stained tumors from NIC/14-3-3σ +/+ and NIC/14-3-3σ F/F mice. P-values were generated between the 14-3-3σ KO and the parental NIC group.
Figure S5. NIC/14-3-3σ KO tumor cells have increased migratory and invasive capacities and a higher lung colonization rate 3 (A) Boyden chamber migration assay of tumor cells from two NIC/14-3-3σ +/+ tumors and two NIC/14-3-3σ F/F tumors, with representative images. 1x10 5 cells were initially applied on each chamber, and cells passing the chamber membranes were counted. (B) Boyden chamber invasion assay of the same tumor cells as in (A), with representative images. The same number of cells was used. Experiments were performed in triplicate. (C) The average number of lung lesions per mouse in nude mice 4 weeks after tail vein injections. Mammary tumors from NIC/14-3-3σ +/+ mice (ID# 8995 and 8997) and NIC/14-3-3σ F/F mice (ID# 7930 and 7931) were dissociated and for each tumor, 4 nude mice received injections of 5 x 10 5 cells. Data are represented as mean ± STDEV. Data that is significantly different from the control are indicated by asterisks (**P <0.01). Figure S6. NIC/14-3-3σ KO mammary tumor cells exhibit loss of membrane localization of Par3 in 2D and 3D cultures (A) Representative immunofluorescent images of established NIC/14-3-3σ +/+ and NIC/14-3-3σ F/F tumor cells in monolayer cultures. Par3 was stained green, 14-3-3σ was stained red, and nuclei were counterstained with DAPI (blue). (B) Representative immunofluorescent images of the aforementioned cells in 3D Matrigel cultures with the same staining as in (A). All experiments were performed in triplicate. Figure S7. Re-expression 14-3-3σ in NIC/14-3-3σ KO tumor cells partially restores Par3 membrane localization in 2D and 3D cultures (A) Representative immunofluorescent images of established NIC/14-3-3σ F/F tumor cells with empty vector control (EV), and the cells stably expressing 14-3-3σ and HA-14-3-3ζ cells on plastic. Par3 was stained green, 14-3-3σ was stained red, and nuclei were counterstained with DAPI (blue). (B) Representative
immunofluorescent images of the same established tumor cells in 3D Matrigel cultures with the same staining as in (A). All experiments were performed in triplicate. 4 Figure S8. 14-3-3σ deficient NIC tumor cells have normal membrane localization of β-catenin (A) Representative immunofluorescent images of established NIC/14-3-3σ +/+ and NIC/14-3-3σ F/F tumor cells in monolayer cultures. β-catenin was stained green, E-Cadherin was stained red, and nuclei were counterstained with DAPI (blue). (B) Representative immunofluorescent images of established NIC/14-3-3σ F/F tumor cells with empty vector control (EV), and the cells stably expressing 14-3-3σ and HA-14-3-3ζ cells in monolayer cultures with the same staining as in (A). All experiments were performed in duplicate.
5 Supplemental Experimental Procedures Histology analysis. All tissue samples were fixed in 10% formalin at room temperature for 24h, prior to indefinite 70% ethanol incubation at 4. Samples were then sent to the Histology Core Facility in McGill Goodman Cancer Center for paraffin embedding. Sample sections at 5 μm were obtained and stained with Hematoxylin and Eosin (H&E) or with Hematoxylin, Biebrich scarlet-acid fuchsin and Aniline blue (Masson s Trichrome). Quantitative real time RT-PCR. Tumor RNA was isolated using the RNeasy Midi Kit (Qiagen), following the manufacturer s directions. RNA concentrations were measured using a spectrophotometer (Biochrome).The reverse transcription, PCR and quantification were carried out in a single capillary using the SYBR Green RNA Amplification Kit (Qiagen) with the LightCycler (Roche). For each reaction, 50ng of total RNA was used. All results were normalized against GAPDH levels. Primer sequences used for detecting rat Neu mrna: qneuf: 5 -GTGCTAGACAACCGAGATCCTCAGG-3 ; qneur: 5 -CCCTTCAGGATCTCTGTGAGACTTCG-3. Primer sequences used for detecting mouse GAPDH mrna: qgapdhf: 5 -CATCAAGAAGGTGGTGAAGC-3 ; qgapdhr: 5 -GGGAGTTGCTGTTGAAGTCG-3. Southern analysis. DNA extractions from ES cells, tails, and tumors were resuspended in Tris/EDTA (TE) buffer (10mM Tris-HCl, 1mM EDTA), and concentration was determined using a spectrophotometer (Biochrome). 10µg of DNA was digested overnight at 37 with 100 units of HC-HindIII (Invitrogen). Digested DNA was separated by size by electrophoresis in a 0.6% agarose gel, and then transferred to Hybond- N Nitrocellulose membranes (Amersham). DNA was crosslinked to membranes with ultra violet (UV) light,
6 and prehybridized in a rotation oven (Fisher) at 42 for 4h. 32 P-dCTP labeled probes generated with Random Primer Labeling Kit (Roche) were hybridized to the membrane overnight at 42. Membranes were washed for 15min each with two charges of low stringent wash buffer (2xSSC, 0.1%SDS) and two charges of high stringent wash buffer (0.2xSSC, 0.1%SDS) at 65. Membranes were exposed to films (Amersham) for development of the Southern blot. The probe for examining ErbB2 amplification was a ~600bp ScaI digestion fragment from the FloxNeoNeuNT targeting construct (14). Immunohistochemistry (IHC). Paraffin embedded sections were deparaffinized in 3 changes of xylenes. Sections were heated in 10 mm sodium citrate (ph6), followed by incubation in 3% H 2 O 2 for 20 minutes. Samples were incubated in primary antibodies of Ki67 (1:100; Cedarlane) diluted in PBS/2% BSA for 1 hour at room temperature. Samples were subject to incubation at room temperature with HRP-conjugated secondary antibody (1:1000 in PBS/2% BSA; Jackson Laboratories). Immunoreactivity was visualized using the DAB+ substrate chromagen system (DAKO) and the tissues were counterstained with hematoxylin. For TUNEL IHC staining, ApopTag Fluorescein In Situ Apoptosis Detection Kit (Serological) was used. Slides were subsequently scanned and analyzed using Scanscope (Aperio) and the ImageScope software (Aperio). Nude mice tail vein injection. Four-week-old female nude mice were anaesthetized under sterile conditions. 5x10 5 well dissociated tumor cells suspended in 100μl sterile PBS were slowly injected into the tail veins using a syringe with a 27G needle. Four weeks after injection, lungs of the mice were collected for analysis.