Fig 2: Intestinal homeostasis is disrupted in the absence of Aux(A) Dl and Pros in control intestines (3 days).(B and C) Dl and Pros in esgts>auxRNAi intestines (white and yellow arrowheads, respectively).(D) Dl and Pros in control intestines (white and yellow arrowheads, 7 days).(E and F) Dl and Pros in esgts>auxRNAi intestines.(G) Dl and Pros in esgts>auxRNAi, aux-flag-GFP intestines.(H) Quantification of Dl+ cells in control and esgts>auxRNAi intestines. Means ± SDs are shown. n = 15. ****p < 0.0001.(I) Quantification of the percentage of Dl+ cells in control and esgts>auxRNAi intestines. Means ± SDs are shown. n = 15. ****p < 0.0001.(J) Quantification of Pros+ cells in control and esgts>auxRNAi intestines. Means ± SDs are shown. n = 15. ****p < 0.0001.(K) Quantification of the percentage of Pros+ cells in control and esgts>auxRNAi intestines. Means ± SDs are shown. n = 15. ****p < 0.0001.(L) Dl-lacZ (white arrowheads) in control intestines. Dl-lacZ channel is shown separately in black and white.(M) Dl-lacZ + cells in esgts>auxv22 intestines (white arrowheads).(N) Quantification of Dl-lacZ+ cells in control and esgts>auxRNAi intestines. Means ± SDs are shown. n = 10. ****p < 0.0001.(O) Dl and Pros in FRT MARCM clones 6 days ACI (white arrowheads). Dl/Pros channel is shown separately in black and white.(P) Dl and Pros in auxv22 ISC MARCM clones (6 days ACI, white arrowheads).(Q) Quantification of the size of control and auxv22 ISC MARCM clones. Means ± SDs are shown. n = 15. ****p < 0.0001.(R) Dl and Pros in auxF596X mosaic clones (6 days ACI, white arrowheads and circles). GFP+ and Dl+ cells outside the mosaic clones are indicated by blue arrowheads. Dl/Pros and GFP channels are shown separately in black and white and the clones are outlined with yellow dotted lines.(S) Quantification of the number of Dl+ cells per clone in control and auxF596X clones. Means ± SDs are shown. n = 15. ****p < 0.0001.Scale bars, 20 µm (O, 10 µm).

Fig 3: BRN2 expression increases resistance to BRAF inhibition.a MM370, MM455, or MM603 cells transduced to express BRN2 (circles) or lacZ (squares) were seeded into 96-well plates, treated with doxycycline (open symbols) or vehicle (closed symbols) the following day before treating with the indicated concentration of PLX-4032 for an additional 5 days. Data were normalized to survival of doxycycline-exposed cells to account for the slower proliferation. Sulforhodamine B assay was used to measure cell survival following exposure to drug. Values indicate mean ± SD; n = 4 independent experiment with at least triplicate readings. b IC50 values of melanoma cell lines with differing expression of BRN2. Left graph shows individual cell line IC50 values. Values indicate mean ± SEM; n = at least two independent experiments with at least triplicate reading. Right graph shows grouping means for low, intermediate and high levels of BRN2 in cell lines as determined by western blot analysis. Values indicate mean ± SD. *p < 0.05; ****p < 0.0001; unpaired t test.

Fig 4: Generation of inducible BRN2-expressing human melanoma cell lines.a Production of cells with doxycycline-inducible induction of BRN2 or lacZ in melanoma cell lines. Western blot analysis was performed on MM370, MM455, and MM603 cells, 48 h after induction of expression by exposure to 50 ng/ml doxycycline. * long exposure for MITF in MM455 is also shown. b MM370, MM455, or MM603 cells transduced to express BRN2 (circles) or lacZ (squares) were seeded into 96-well plates and treated with doxycycline (open symbols) or vehicle (closed symbols) for 7 days. Sulforhodamine B assay was used to measure cellular proliferation rate. Values indicate mean ± SD; n = 3 independent experiments with at least triplicate readings.

Fig 5: Inhibition of c-MET reverses anoikis resistance induced by BRN2 expression.a–c Cellular viability counts following 7 days growth under ultra-low-adherence conditions either with or without induction of expression of BRN2 or lacZ as a control with or without inhibition of c-MET with 0.25 µm crizotinib (CRIZ). a MM370 cells, b MM455 cells, c MM603 cells. d, e MM370 cellular viability counts following 7 days under the above conditions with or without inhibition of c-MET with d 0.25 µm foretinib (FORE) or e 1.25 nm capmatinib (CAP). a–c Values indicate mean ± SEM, n = at least duplicate counts from three independent experiments. d, e Values indicate mean ± SEM, n = triplicate counts from two independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001; unpaired t test.