SUPPLEMENTARY FILES
Figure S1. Effect of HER2-targeting shRNAs or AEE788 on BT474 cells. A. Top panel:
Immunoblot analysis of BT474 cells 72 hours after lentiviral transduction of shRNAs targeting
HER2 or control shRNAs (LACZ29, LACZ1650). Cells were transduced with the indicated
shRNAs and selected with 2 ug/ml puromycin. Protein lysates were harvested 72 h after
transduction and subjected to immunoblot analysis with the indicated antibodies. Bottom panel:
Relative viability of BT474 cells 6 days after transduction with shRNAs targeting HER2 or
controls. Cells were transduced with the indicated shRNAs and viability was assessed by ATPbased luminescence assay 6 days later. Results are normalized to the shLACZ29 control and
represent the mean and standard deviation of 3 replicates. B. Titration of AEE788 in BT474
cells. BT474 cells were treated with DMSO or increasing concentrations of AEE788. Cell
viability was measured by ATP-based luminescence assay 6 days after drug treatment. Results
are normalized to the DMSO control and represent the mean and standard deviation of 6
replicates.
Figure S2. Schematic illustrating the ORF screen performed. BT474 cells were seeded in
arrayed 384-well format 24 hours prior to transduction with lentivirally-delivered ORFs. Two
days later the cells were either re-transduced with an anti-HER2 shRNA or treated with the
HER2/EGFR tyrosine kinase inhibitor (TKI) AEE788. Viability was read out 6 days later by
ATP-based luminescent cell viability assay.
Figure S3. Immunoblot analyses of V5-tagged ORFs. ORFs scoring in both screens were
subsequently tested in low-throughput in SKBr3 (A) and BT474 (B) cells. Cells were transduced
with lentiviral constructs expressing the indicated ORFs and selected with blasticidin. Protein
lysates were harvested and blots were probed with anti-V5 antibody. Blots were re-probed with
anti--actin as a loading control. C. Transient transfections of 293T cells with pLX304-PIM1
and -PIM2 constructs to confirm size and antibody specificity using anti-PIM1, anti-PIM2, and
anti-V5 antibodies. D. Lysates from BT474 cells transduced with pLX304-PIM1, -PIM2, or LACZ were immunoblotted with antibodies against PIM1, PIM2, or V5, as indicated.
Figure S4. PRKACA-induced resistance to trastuzumab in HER2-amplified cell lines. Cells
were lentivirally-transduced with the indicated ORFs, treated with trastuzumab at the indicated
doses, and cell viability was assessed by ATP-based luminescence assay 6 days later. Results are
normalized to the vehicle control for each ORF and represent the mean and standard deviation of
6 replicates.
Figure S5. Forskolin-induced lapatinib resistance and BAD phosphorylation. A. ZR-75-30 cells
were pre-treated for 1h with 10 uM forskolin. Lapatinib was then added to the cells at increasing
concentrations. Viability was assessed 72 hours later by ATP-based luminescence assay. Results
are normalized to the vehicle control for each ORF and represent the mean and standard
deviation of 3 replicates per condition. B. Forskolin pre-treatment restores BAD phosphorylation
in ZR-75-30 cells in the context of trastuzumab or lapatinib treatment, but does not restore
p70S6K or mTOR phosphorylation. Cells were treated for 24 hours with 10 uM forskolin prior to
the addition of lapatinib 0.1 uM for 24 h. Protein lysates were harvested and subjected to
immunoblot analysis with the indicated antibodies.
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Figure S6. PRKACA expression in human breast cancers before and after anti-HER2 therapy.
Immunohistochemical analysis for PRKACA expression in matched breast cancer samples from
patients at diagnosis, prior to any treatment (panels a, c) and after the development of metastatic
disease and subsequent resistance to anti-HER2 therapy (panels d, e). Magnification 40X.
Figure S7. PRKACA expression in human breast tissue. Immunohistochemical analysis for
PRKACA expression in a breast tissue microarray containing normal breast (a), hyperplastic
breast (b), benign breast tumor (c), Phyllodes sarcoma (d), in situ breast carcinoma (e), and
invasive breast carcinoma (f).
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