Fig 2: IGSF10-RET-GAS1 signaling mediates migration of GnRH neurons(A) IGSF10 knockdown activates RET and cdc42 in GN11 cells. GN11 cells were transfected with IGSF10 siRNA or control siRNA, and the levels of the indicated proteins were assessed by immunoblotting.(B) IGSF10 knockdown inhibits GN11 cell migration, which is rescued by wild-type IGSF10. GN11 cells were transfected with IGSF10 siRNA or control siRNA and treated with the indicated conditioned medium expressing wild-type, R156L, and E161K IGSF10 or empty vector. Cell migration was assessed using Transwell migration assays. Data are represented as mean ± SEM.(C) IGSF10 knockdown inhibits GN11 cell proliferation, which is rescued by wild-type IGSF10. GN11 cells were transfected with IGSF10 siRNA or control siRNA and treated with the indicated conditioned medium expressing wild-type, R156L, and E161K IGSF10 or empty vector. Cell proliferation was assessed using IncuCyte.(D) siRNA knockdown of IGSF10 and RET in GN11 cells. GN11 cells were transfected with IGSF10 siRNA and/or RET siRNA (filled up with control siRNA), and the levels of the indicated proteins were assessed by immunoblotting.(E) RET knockdown rescues inhibition of GN11 cell migration by IGSF10 knockdown. GN11 cells were transfected with IGSF10 siRNA and/or RET siRNA (filled up with control siRNA), and cell migration was assessed by Transwell assays. Data are represented as mean ± SEM.(F) RET knockdown rescues inhibition of GN11 cell proliferation by IGSF10 knockdown. GN11 cells were transfected with IGSF10 siRNA and/or RET siRNA (filled up with control siRNA), and cell proliferation was assessed using IncuCyte.(G) A RET kinase inhibitor rescues the inhibition of GN11 cell migration by IGSF10 knockdown. GN11 cells were transfected with IGSF10 siRNA or control siRNA and treated with and without 1 μM RET inhibitor 1. Cell migration was assessed by Transwell assays. Data are represented as mean ± SEM.(H) A RET kinase inhibitor rescues inhibition of GN11 cell proliferation by IGSF10 knockdown. GN11 cells were transfected with IGSF10 siRNA or control siRNA and treated with and without 1 nM RET inhibitor 1. Cell proliferation was assessed using IncuCyte.(I) A cdc42 inhibitor rescues inhibition of GN11 cell migration by IGSF10 knockdown. GN11 cells were transfected with IGSF10 siRNA or control siRNA and treated with and without 2.5 μM ML141. Cell migration was assessed by Transwell assays. Data are represented as mean ± SEM.(J) A cdc42 inhibitor rescues inhibition of GN11 cell proliferation by IGSF10 knockdown. GN11 cells were transfected with IGSF10 siRNA or control siRNA and treated with and without 2.5 μM ML141. Cell proliferation was assessed using IncuCyte.(K) GAS1 knockdown activates RET and cdc42 in GN11 cells. GN11 cells were transfected with GAS1 siRNA or control siRNA, and the levels of the indicated proteins were assessed by immunoblotting.(L) A RET kinase inhibitor rescues inhibition of GN11 cell migration by GAS1 knockdown. GN11 cells were transfected with GAS1 siRNA or control siRNA and treated with and without 1 μM RET inhibitor 1. Cell migration was assessed by Transwell assays. Data are represented as mean ± SEM.(M) A RET kinase inhibitor rescues inhibition of GN11 cell proliferation by GAS1 knockdown. GN11 cells were transfected with GAS1 siRNA or control siRNA and treated with and without 1 nM RET inhibitor 1. Cell proliferation was assessed using IncuCyte.(N) A cdc42 inhibitor rescues inhibition of GN11 cell migration by GAS1 knockdown. GN11 cells were transfected with GAS1 siRNA or control siRNA and treated with and without 2.5 μM ML141. Cell migration was assessed by Transwell assays. Data are represented as mean ± SEM.(O) A cdc42 inhibitor rescues inhibition of GN11 cell proliferation by GAS1 knockdown. GN11 cells were transfected with GAS1 siRNA or control siRNA and treated with and without 2.5 μM ML141. Cell proliferation was assessed using IncuCyte.(P) Migration assays for GnRH neurons in nasal explants. Left: Graph of cDNA products for IGSF10, normalized using β-tubulin, from randomer- and antisense-treated explants, diluted 1:8, showing a 71% decrease in IGSF10 in antisense-treated GnRH neurons. Middle: Photomicrographs of an explant treated with a randomer or IGSF10 antisense oligomer. These panels show low-power photomicrographs on which 100 μm concentric circles have been superimposed (white asterisk = tip of nasal cartilage). Scale bar: 500 μm. Boxed areas in the low magnification panels are shown at a higher magnification in the next panels. Scale bar: 100 μm. GnRH neurons are stained blue-black, and olfactory axons are stained brown. Right: histogram. A significant difference between randomer- and IGSF10 antisense-treated explants was found in the location of GnRH neurons (χ2 p ≤ 0.0001; χ2 = 51.3). Cells in the IGSF10 knockdowns (hatched bars) remained closer to the explant compared to those treated with randomers (black bars). χ2 analysis creates an unbiased group (expected) based on treated and control groups (represented by zero baseline in the graph). Plotted here is the % difference (based on cell number in each zone) that the randomer versus antisense treated groups were relative to the expected group at three different migration distances: 1–400 μm, 500–800 μm, and >900 μm from the main explant tissue.

Fig 3: GAS1 serves as a co-receptor for IGSF10-RET(A) GAS1 knockdown activates RET and cdc42 in Ewing sarcoma cells. A673 and ES-1 cells were transfected with GAS1 siRNA or control siRNA, and the levels of the indicated proteins were assessed by immunoblotting.(B) GAS1 knockdown inhibits Ewing sarcoma cell proliferation. A673, EW8, ES-1, and PDX1 Ewing sarcoma cells were transfected with GAS1 siRNA or control siRNA, and cell proliferation was assessed by IncuCyte (right). (Left) GAS1 knockdown was verified by immunoblotting.(C) GAS1 serves as the IGSF10 co-receptor in Ewing sarcoma. C-terminally FLAG-tagged IGSF10 was produced by transfection in 293T cells. A673 cells were transfected with GAS1 siRNA or control siRNA and incubated with FLAG-tagged IGSF10 for 0, 1, 4, or 8 h, as indicated. Cells were washed, and the whole-cell lysate was prepared to assess the binding of IGSF10-FLAG to the cells. IGSF10-FLAG bound to control siRNA-transfected cells, and the binding was abolished by GAS1 silencing. The silencing of GAS1 was verified by immunoblotting.(D) GAS1 binds to the N-terminal region of IGSF10. 293T cells were transfected with GAS1 and FLAG empty vector, IGSF10(1–281)-FLAG, or IGSF10(1–668)-FLAG, as indicated. The interaction between GAS1 and IGSF10 N-terminal region was assessed by anti-FLAG immunoprecipitation, followed by anti-GAS1 and anti-FLAG immunoblotting. Specific protein bands are indicated by asterisks.(E) IGSF10 mutations linked to delayed puberty reduce binding to GAS1. 293T cells were transfected with GAS1, RET, and FLAG empty vector, IGSF10(1–281)-FLAG, IGSF10(1–281) R156L-FLAG, or IGSF10(1–281) E161K-FLAG. The interaction between GAS1 and wild-type and mutant IGSF10(1–281) was assessed by anti-FLAG immunoprecipitation, followed by anti-GAS1 and anti-FLAG immunoblotting.(F) IGSF10 knockdown reduces RET-GAS1 co-immunoprecipitation and increases RET-GFRA2 co-immunoprecipitation in A673 cells. A673 cells were infected with lentiviruses expressing control shRNA, IGSF10 shRNA-1, or IGSF10 shRNA-2. Cell lysate was immunoprecipitated with anti-RET antibody or control IgG, followed by anti-IGSF10, anti-RET, anti-GAS1, and anti-GFRA2 immunoblotting. (Right) IGSF10 shRNA knockdown was verified by immunoblotting.(G) IGSF10 binds RET-GAS1, while GDNF binds RET-GFRA2. (Top) 293T cells were transfected with RET, GAS1, and/or GFRA2, as indicated. Cells were treated with C-terminally FLAG-tagged IGSF10 for 0, 1, or 2 h. Cells were washed, and whole-cell lysate was prepared to assess the binding of IGSF10-FLAG to the cells by anti-FLAG immunoblotting. (Bottom) 293T cells were transfected with RET, GAS1, and/or GFRA2, as indicated. Cells were treated with C-terminally FLAG-tagged GDNF for 0, 1, or 2 h. Cells were washed, and the whole-cell lysate was prepared to assess the binding of GDNF-FLAG to the cells by anti-FLAG immunoblotting.(H) ITC analysis of GAS1–IGSF10(1–281) interaction. The interaction of recombinant GAS1 (R&D, 2636-GS-050) and GST-IGSF10(1–281) was analyzed by ITC.(I) Multiple domains in GAS1 are involved in IGSF10 binding. 293T cells were transfected with C-terminally HA-tagged IGSF10(1–281) and FLAG empty vector, GAS1-FLAG, GAS1Δ(48–143)-FLAG, GAS1Δ(166–243)-FLAG, or GAS1Δ(254–345)-FLAG, as indicated. The interaction between IGSF10(1–281) and GAS1 deletion mutants was assessed by anti-FLAG immunoprecipitation, followed by anti-HA and anti-FLAG immunoblotting.