Supplementary Fig. 6: Fzd9b-5GS-APEX2 cell line validation and EGFR mediates Wnt9a–Fzd9b signalling. | Nature Cell Biology

Supplementary Fig. 6: Fzd9b-5GS-APEX2 cell line validation and EGFR mediates Wnt9a–Fzd9b signalling.

From: EGFR is required for Wnt9a–Fzd9b signalling specificity in haematopoietic stem cells

Supplementary Fig. 6

a, Immunoblot for streptavidin after treatment of cells with vehicle, biotin-phenol, H2O2 or both, representative of five independent experiments. See also Supplementary Fig. 7. b, Immunoblots of cells for Fzd9b, FLAG (APEX2) and actin; representative of three independent experiments. See also Supplementary Fig. 7. c, Immunofluorescence for FLAG showing APEX2 localization on the membrane; representative of three independent experiments. Scale bar, 30 µm. d, Quantification of the HEK293T cell STF assay with zebrafish Wnt9a conditioned medium, WT Fzd9b and Fzd9b-5GS-APEX2; n = 3 biological replicates each; statistical analysis was done by ANOVA compared to Wnt9a conditioned medium alone. e, Fold enrichment of GO terms for biological processes identified from the top 5% of changed proteins in Fzd9b-APEX2 HEK293T cells treated with Wnt9a. The P values are listed next to each bar; n = 3 biological replicates; see the Methods for analysis and statistics. f, Normalized intensity of the top three transmembrane proteins detected in the APEX assay; n = 3 biological replicates. g, Immunoblot for EGFR in siControl-treated or siEGFR-treated HEK293T cells; representative of three independent experiments. See also Supplementary Fig. 7. h, Quantification of the HEK293T cell STF assay with human WNT9A and FZD9 with siEGFR or siControl; n = 3 biological replicates. i, WISH for cmyb at 40 hpf; quantified in j; n = 10 biological replicates each. Scale bar, 30 µm. k,l, STF assay with zebrafish Wnt9a and Fzd9b (k; n = 8 biological replicates each) or human WNT9A and FZD9 (l; n = 3 biological replicates each), treated with the selective EGFR inhibitor AG1478. m, Expression of runx1 and gata2b in vehicle and EGFR inhibitor zebrafish treated from 15–36 hpf; n = 4 biological replicates each. n, Expression of known EGFR ligands as transcripts per million (TPM) in HEK293T cells as reported from RNA-sequencing data sets42. The red dashed line indicates the commonly accepted cut-off for expression levels by TPM; n = 6 biological replicates from 2 experiments. o, Quantification of the HEK293T cell STF assay in Fzd9b-expressing cells cultured in serum-free conditions with neutralizing antibody for HBEGF as indicated; n = 3 biological replicates each. In all graphs, the dots represent biological replicates from a single experiment (except for panel n, which is pooled from two experiments), the bars represent the mean and the error bars represent the standard deviation. Statistical analyses were done by ANOVA compared to controls as indicated. All STF assays were repeated independently with a similar trend.

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