Figure 7: KIAA1199 promotes EGF-dependent signalling.

(a) Defective EGF-dependent signalling on KIAA1199 deficiency in cervical cancer cells. Serum-starved control or KIAA1199-depleted CaSki cells (shRNA KIAA1199#2) were untreated or stimulated with EGF (100 ng ml⁻¹) from 5 to 30 min. The resulting cell extracts were subjected to anti-FLAG (negative control) or -EGFR IPs followed by anti-Src, -c-Cbl and -EGFR WBs (top three panels). Crude cell extracts were also subjected to WB analyses using the indicated antibodies. (b) Optimal EGF-dependent Ras activation requires KIAA1199 in CaSki cells. Ras activity on stimulation with EGF (10 ng ml⁻¹) was assessed in control or KIAA1199-depleted cells. Ras⁺ and His-Ras were used as positive controls. An anti-pan-RAS WB was carried out for normalization purposes (‘input’, bottom panel). (c) Defective HER2 expression on KIAA1199 deficiency in cervical cancer cells. Extracts from control or KIAA1199-depleted CaSki cells were subjected to WB analyses using the indicated antibodies. (d) Defective MEK1 activation on stimulation by NRG-1 in KIAA1199-deficient cervical cancer cells. Serum-starved control or KIAA1199-deficient CaSki cells were untreated or stimulated with NRG1 (100 ng ml⁻¹) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses, using the indicated antibodies. (e) KIAA1199 is dispensable for EGFR dimer formation on EGF stimulation in cervical cancer-derived cells. Serum-starved CaSki cells were unstimulated or treated with EGF (100 ng ml⁻¹) for the indicated periods of time. Cells were subsequently cross-linked using 1 mM of bis(sulfosuccinimidyl) suberate (BS³) as chemical cross-linker. On the left, extracts were subjected to anti-EGFR WB analyses to detect both EGFR monomers and dimers. On the right, quantification of the dimer/monomer/HSP90 ratio in both control and KIAA1199-depleted cells subjected to EGF stimulations or not. The value of the dimer/monomer/HSP90 ratio in control and unstimulated cells was set to 1 and values obtained in other experimental conditions were relative to that. Data from three independent experiments (means±s.d.) are shown. (f) KIAA1199 differentially controls the expression of ErbB ligands in CaSki cells. Total RNAs from control or KIAA1199-depleted (shRNA KIAA1199#1 or shRNA KIAA1199#2) CaSki cells were subjected to real-time PCR to assess mRNA levels of the indicated ErbB ligands. The abundance of transcripts in control cells was set to 1 and their levels in KIAA1199-depleted cells were relative to that after normalization with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data from three independent experiments performed in triplicates (means±s.d.) are shown (Student’s t-test, P-values: ***<0.001; P-values: **<0.01; P-values: *<0.05). (g) KIAA1199 is dispensable for TGFβ-dependent SMAD3 phosphorylation in cervical cancer cells. Control or KIAA1199-depleted CaSki cells were untreated or stimulated with TGFβ (5 ng ml⁻¹) for the indicated periods of time and WB analyses were carried out on the resulting cell extracts.