Hit compounds that passed hit selection criteria in the high-throughput screen. (a) Hit compounds identified in the screen. (b) High-throughput screen expression levels of marker genes c-fos and EGR1, normalized by control gene ATP5B, in the presence of 50 μM hit compounds and PDGF.

ATA targets the extracellular domain of PDGFR, not the transmembrane or cytoplasmic portions of the receptor. (a) Schematic representation of TEL/PDGFRβ, PER, and EKR. RTK, receptor tyrosine kinase; TK, tyrosine kinase. (b) ATA does not target the transmembrane or cytoplasmic portions of PDGFR. Western analysis of total lysates of Ba/F3 cells expressing TEL/PDGFRβ fusion protein. Cells were treated with ATA. pPDGFRβ, PDGFRβ, and tubulin indicate antibodies against phospho-PDGFRβ, total PDGFRβ, and total tubulin, respectively. (c) ATA targets the extracellular domain of PDGFR. Western analysis of total PER-PC12 cell lysates. Cells were serum-starved overnight and treated with ATA and PDGF. pEGFR, EGFR, and tubulin indicate antibodies against phospho-EGFR, total EGFR, and total tubulin, respectively.

ATA does not fully abrogate phosphorylation of constitutively active PER. (a) Western analysis of total MEL501 cell lysates. Cells were transfected with PER plasmid, serum-starved overnight and treated with ATA, PDGF, and SCF. pEGFR, p-cKIT, cKIT, and tubulin indicate antibodies against phospho-EGFR, phospho-cKIT, total cKIT, and total tubulin, respectively. (b) Western analysis of total MCF7 cell lysates. Cells were transfected with PER plasmid, serum-starved overnight and treated with ATA and PDGF. pEGFR, EGFR, and tubulin indicate antibodies against phospho-EGFR, total EGFR, and total tubulin, respectively.

Basic Violet 3, Ethyl Violet, and Victoria Pure Blue BO exhibit less specific patterns of receptor inhibition than ATA. (a-c) Basic Violet 3 (BV3), Ethyl Violet (EV), and Victoria Pure Blue BO (VPB) inhibit SCF-activated cKIT. Western analysis of total MEL501 cell lysates. Cells were serum starved overnight and treated with BV3 (a), EV (b), or VPB (c) and SCF. p-cKIT and cKIT indicate antibodies against phospho-cKIT and cKIT, respectively. (d-f) BV3, EV, and VPB inhibit activated EGFR. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with BV3 (d), EV (e), or VPB (f) and EGF. pEGFR and EGFR indicate antibodies against phospho-EGFR and total EGFR, respectively. (g-i) BV3, EV, and VPB inhibit activated IGF1R. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with BV3 (g), EV (h), or VPB (i) and IGF. pIGFR and IGFR indicate antibodies against phospho-IGFR and total IGFR, respectively.

PDGFR/ERK activation signature for high-throughput screening. (a) Genes whose expression is correlated with ERK activation by PDGFR. Genes (in rows) sorted by their expression in samples (columns) with or without U0126, apigenin, and PDGF. Red indicates high relative expression, blue low expression. (b) RT-PCR of signature genes in four sample wells: two lanes (replicas) per condition. TIP5 cells were serum starved overnight and then treated with PDGF. (c) Screening schema overview. SBE, single-base extension.

ATA abrogates phosphorylation of activated ERK, MEK and PDGFR. (a) ATA and aluminon, but not quinacrine dihydrochloride, abrogated PDGF-mediated phosphorylation of ERK. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with ATA, aluminon, and quinacrine dihydrochloride in the presence of PDGF. pERK and ERK indicate antibodies against phospho-ERK and total ERK, respectively. DMSO, dimethyl sulfoxide. (b) ATA abrogates phosphorylation of MEK. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with ATA and PDGF. pMEK and MEK indicate antibodies against phospho-MEK and total MEK, respectively. (c) ATA abrogates phosphorylation of PDGFR. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with ATA and PDGF. pPDGFRβ and PDGFRβ indicate antibodies against phospho-PDGFRβ and total PDGFRβ, respectively. (d) Wash-out experiment: PDGFR phosphorylation remains inhibited upon removal of ATA. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and then incubated with ATA. After ATA was removed by washing, cells were induced with PDGF. pPDGFRβ and PDGFRβ indicate antibodies against phospho-PDGFRβ and total PDGFRβ, respectively.

ATA failed to inhibit activated EKR, IGF1R, or EGFR, but inhibited SCF-mediated activation of cKIT. (a) ATA does not inhibit activated EKR. Western analysis of total TIP5 cell lysates. Cells were transfected with EKR plasmid, serum-starved overnight and treated with ATA, EGF and PDGF. p-cKIT, pPDGFRβ, PDGFRβ, and tubulin indicate antibodies against phospho-cKIT, phospho-PDGFRβ, total PDGFRβ, and total tubulin, respectively. (b) ATA does not inhibit activated IGF1R. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with ATA and IGF. pIGFR and IGFR indicate antibodies against phospho-IGFR and total IGFR, respectively. (c) ATA does not inhibit activated EGFR. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with ATA and EGF. pEGFR and EGFR indicate antibodies against phospho-EGFR and EGFR, respectively. (d) ATA inhibits SCF-activated cKIT. Western analysis of total MEL501 cell lysates. Cells were serum starved overnight and treated with ATA and SCF. p-cKIT and cKIT indicate antibodies against phospho-cKIT and total cKIT, respectively.

ATA analogues inhibiting PDGFR. Western analysis of total TIP5 cell lysates. Cells were serum starved overnight and treated with (a) 5,5'-methylenedisalicylic acid (MDA), (b) Basic Violet 3 (BV3), (c) Ethyl Violet (EV), or (d) Victoria Pure Blue BO (VPB) and PDGF. pPDGFRβ and PDGFRβ indicate antibodies against phospho-PDGFRβ and total PDGFRβ, respectively.

Ethyl Violet and Victoria Pure Blue BO inhibit cytoplasmic TEL/PDGFR. Western analysis of total lysates of Ba/F3 cells expressing TEL/PDGFRβ fusion protein. Cells were treated with either (a) Ethyl Violet (EV) or (b) Victoria Pure Blue BO (VPB). pPDGFRβ and PDGFRβ indicate antibodies against phospho-PDGFRβ and total PDGFRβ, respectively.