Motor neurons were derived from iPSC lines following a monolayer-based differentiation protocol81 (link). iPSCs were plated on Geltrex and cultured in mTeSR1 medium until confluent. Then, we initiated the differentiation induction with neuron differentiation media (DMEM/F12:Neurobasal 1:1 (ThermoFisher, 11330057 and 21103049) supplemented with non-essential amino acids, glutamax (ThermoFisher, 35050038), B27 (ThermoFisher, 12587010), and N2 (ThermoFisher, 17502048). From day 0 to day 6, neuron differentiation media was supplemented with 1 μM retinoic acid (Sigma-Aldrich, R2625), 1 μM smoothened agonist (Sigma-Aldrich, 566661), 0.1 μM LDN-193189 (Miltenyi Biotech, 130-103-925), and 10 μM SB-431542 (Miltenyi Biotech, 130-105-336). From day 7 to day 14, neuron differentiation media was supplemented with 1 μM retinoic acid, 1 μM smoothened agonist, 4 μM SU-5402 (Sigma-Aldrich, SML0443), and 5 μM DAPT (Sigma-Aldrich, D5942). Motor neurons were split and plated on poly-L-ornithine (Sigma-Aldrich, P3655) and laminin-coated (ThermoFisher, 23017015) plates containing neurobasal media supplemented with non-essential amino acids, glutamax, N2, B27, and neurotrophic factors 10 ng ml−1 BDNF (Biozol, 450-02) and 10 ng ml−1 GDNF (Biozol, 450-10).
Smoothened agonist
Manufactured by Merck Group
Sourced in United Kingdom
Smoothened agonist is a type of lab equipment used in research. It functions as an activator for the Smoothened protein, which is a key component of the Hedgehog signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (3 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (2 mentions)
Recombinant human fgf basic, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Selleck Chemicals (1 mentions)
Pd173074, by Selleck Chemicals (1 mentions)
Sb431542, by Selleck Chemicals (1 mentions)
Ldn193189, by Selleck Chemicals (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Pd0325901, by Selleck Chemicals (1 mentions)
Sch772984, by Selleck Chemicals (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Ultra low attachment 6 well plate, by Corning (1 mentions)
Anti arl13b, by Proteintech (1 mentions)
Irdye 800cw secondary antibody, by LI COR (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Irdye 680lt secondary antibody, by LI COR (1 mentions)
Anti acetylated tubulin, by Merck Group (1 mentions)
Anti pi 4 5 p2, by Echelon Biosciences (1 mentions)
Anti pi4p, by Echelon Biosciences (1 mentions)
Anti gli3, by Abcam (1 mentions)
7 protocols using smoothened agonist
1
Monolayer-Based Motor Neuron Differentiation
2
GNP Isolation and Gene Expression Analysis
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GNP isolation was performed as previously described (Kawauchi et al. 2012 (link)). For in vitro studies, five million P7 GNPs were plated per well in a six-well plate and cultured for 48 h in the presence or absence of 200 nM smoothened agonist (Merck). RNA was extracted and cDNA was generated with SuperScript II kit (Invitrogen), according to the manufacturer's instructions. RNA and cDNA from tumor samples was generated similarly. Relative gene expression was compared with Gapdh in all experiments except Figures 3 P and 4 B, which was compared with the geometric mean of Hrpt1, Rpl27, and Rer1, and Supplemental Figure S1D , which was compared with 18s rRNA. All primer sequences are listed in Supplemental Table S5 .
3
Medulloblastoma Histopathology and Gene Expression
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For histopathology, samples of mouse medulloblastomas (3 tumors per genotype) were formalin-fixed, paraffinembedded and sectioned at 5 μm. The sections were stained with hematoxylin and eosin (H&E), and histological classifications were performed blinded to genotype.
Immunostaining was performed as previously described All slides were counterstained with DAPI (300 nM). For quantification, we counted the number of average positive cells in four 100 μm x 100 μm regions in 3 independent cerebellum.
Quantitative PCR (qPCR)
GNP isolation was performed as previously described
Materials and Methods for Kutscher, Okonechnikov, Batora et al. (Kawauchi et al. 2012) . For in vitro studies, 5 million cells were plated per well in a 6-well plate and cultured for 48h in the presence or absence of 200 nM smoothened agonist (Merck). RNA was extracted and cDNA was generated with Superscript II kit (Invitrogen), according to manufacturer's instructions. RNA and cDNA from tumor samples was generated similarly. Relative gene expression was compared to Gapdh in all experiments except Fig. 3P and 4B, which was compared to the geometric mean of Hrpt1, Rpl27, and Rer1 (Thomas et al. 2014) . All primer sequences are listed in Supplemental Table S5.
Immunostaining was performed as previously described All slides were counterstained with DAPI (300 nM). For quantification, we counted the number of average positive cells in four 100 μm x 100 μm regions in 3 independent cerebellum.
Quantitative PCR (qPCR)
GNP isolation was performed as previously described
Materials and Methods for Kutscher, Okonechnikov, Batora et al. (Kawauchi et al. 2012) . For in vitro studies, 5 million cells were plated per well in a 6-well plate and cultured for 48h in the presence or absence of 200 nM smoothened agonist (Merck). RNA was extracted and cDNA was generated with Superscript II kit (Invitrogen), according to manufacturer's instructions. RNA and cDNA from tumor samples was generated similarly. Relative gene expression was compared to Gapdh in all experiments except Fig. 3P and 4B, which was compared to the geometric mean of Hrpt1, Rpl27, and Rer1 (Thomas et al. 2014) . All primer sequences are listed in Supplemental Table S5.
4
Directed Differentiation of Human Fetal Embryos
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Human fetal embryo of 6.3 weeks of gestation were obtained from pregnant women referred to the Department of Gynecology and Obstetrics at the Antoine Béclère hospital (Clamart, France) for legally induced abortions in the first trimester of pregnancy as previously described (Lambrot et al., 2006) . All women provided written informed consent for scientific use of the fetal tissues.
(Life technologies), penicillin/streptomycin 1%, β-Mercaptoéthanol 0.1% (Life Technologies), with Y-27632 (10µM, Stemgent or Stem Cell Technologies), CHIR-99021 (3 µM or 4 µM Selleckchem) SB431542 (20 µM, Selleckchem) and LDN 193189 (0.1 µM, Selleckchem). 2x10 5 cells ml -1 were seeded in ultra-low attachment 6 well plates (Corning) to form embryoid bodies (EBs). All conditions of differentiation received the same medium at day 2 and at day 3 but SB431542 was removed at day 3. Then, the differentiation proceeded according to the schemas presented above the figures. SAG (Smoothened Agonist, Merck millipore), FGF2 (Recombinant Human FGF basic, Peprotech), RA (Sigma-Aldrich), GDF11 (Recombinant Human/Murine/Rat GDF11, Peprotech), PD0325901 (Selleckchem), PD173074, SCH772984 (Selleckchem), DAPT (Stemgent) were added at indicated time points. For concentrations see Table S2. Media were changed every other day unless specified.
(Life technologies), penicillin/streptomycin 1%, β-Mercaptoéthanol 0.1% (Life Technologies), with Y-27632 (10µM, Stemgent or Stem Cell Technologies), CHIR-99021 (3 µM or 4 µM Selleckchem) SB431542 (20 µM, Selleckchem) and LDN 193189 (0.1 µM, Selleckchem). 2x10 5 cells ml -1 were seeded in ultra-low attachment 6 well plates (Corning) to form embryoid bodies (EBs). All conditions of differentiation received the same medium at day 2 and at day 3 but SB431542 was removed at day 3. Then, the differentiation proceeded according to the schemas presented above the figures. SAG (Smoothened Agonist, Merck millipore), FGF2 (Recombinant Human FGF basic, Peprotech), RA (Sigma-Aldrich), GDF11 (Recombinant Human/Murine/Rat GDF11, Peprotech), PD0325901 (Selleckchem), PD173074, SCH772984 (Selleckchem), DAPT (Stemgent) were added at indicated time points. For concentrations see Table S2. Media were changed every other day unless specified.
5
Antibody Validation for Ciliary Proteins
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The antibodies used in this study included anti-acetylated tubulin (Sigma-Aldrich, T7451 and Cell Signaling Technology, 5335); anti-ARL13B (Proteintech, 17711-1-AP); anti-gamma tubulin (Abcam, ab179503); anti-INPP5E (Proteintech, 17797-1-AP); anti-OSBPL2 (Proteintech, 14751-1-AP and Abclonal, A14199); anti-FLAG (Sigma-Aldrich, F1804); anti-HA (Cell Signaling Technology, 3724); anti-GAPDH (Cell Signaling Technology, 5174); anti–PI(4,5)P2 (Echelon, Z-P045); anti–PI4P (Echelon, Z-P004); anti-SMO (Santa Cruz, sc-166685); anti-Gli3 (Abcam, ab6050 and Proteintech, 19949-1-AP); anti-Gli1 (Proteintech, 66905-1-Ig); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Invitrogen, A31570); Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Invitrogen, A10040); Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A21202); Donkey F(ab′)2 Anti-Rabbit IgG H&L, Alexa Fluor 647 (Abcam, ab181347); IRDye 800CW Secondary Antibody (LI-COR, 925-32211); and IRDye 680LT Secondary Antibody (LI-COR, 925-68020). The regents used in this study included Smoothened Agonist (Sigma-Aldrich, 566661); Digitonin (MCE, HY-N4000); FLAG Immunoprecipitation Kit (Millipore, FLAGIPT1); DAPI (Sigma-Aldrich, F6057); and isopropyl β-D-thiogalactoside (IPTG, Sigma-Aldrich, I6758).
6
Modulation of Hedgehog Signaling in Embryos and Cells
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Co-injected embryos with 3.8 ng of control, ATG, or E14 MO with either 1 ng control or p53ATG MO (Chen et al., 2005 (link)) were treated with final concentrations of 0.15%v/v DMSO, 20 µM smoothened agonist (Sigma Aldrich), or 20 µM smoothened antagonist-1 (SANT-1) (Sigma, Dorset, UK, S4572) from 4 hpf until 48 hpf. Embryos were dechorionated and put in fresh treatment solution at 24 hpf. Images were taken with a fluorescence stereomicroscope (Leica Microsystems, Mannheim, Germany).
For cell experiments, hTERT-RPE1 cells were subjected to two rounds of transfection. Six hours after the second round, cells were serum starved in un-supplemented DMEM F12 HAM overnight, then treated with either 500 ng/ml recombinant Sonic Hedgehog (Shh) (C42II, N-terminus, R&D systems, Abingdon, UK, 1845-SH-025), 500 ng/ml SANT-1 (Sigma, Dorset, UK, S4572) or an equivalent volume of drug vehicle control for 24 h before methanol fixation and immunolabelling.
For cell experiments, hTERT-RPE1 cells were subjected to two rounds of transfection. Six hours after the second round, cells were serum starved in un-supplemented DMEM F12 HAM overnight, then treated with either 500 ng/ml recombinant Sonic Hedgehog (Shh) (C42II, N-terminus, R&D systems, Abingdon, UK, 1845-SH-025), 500 ng/ml SANT-1 (Sigma, Dorset, UK, S4572) or an equivalent volume of drug vehicle control for 24 h before methanol fixation and immunolabelling.
7
Leydig Cell Differentiation Protocol
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For LCs differentiation, a previously method reported by the group was applied.[17 ] In brief, the SLCs were induced in DIM containing M199 Medium (Gibco), 4% FBS, 1% ITS (Gibco), 1% GlutaMAX (Gibco), 10 ng mL−1 platelet‐derived growth factor‐AA (PDGF‐AA, PeproTech), 0.25 µM smoothened agonist (Sigma), 10 ng mL−1 LH (Sigma), 50 µg mL−1 insulin‐like growth factor 1 (IGF‐1, PeproTech). The cell supernatants were collected for evaluation of testosterone 2 weeks after induction. The differentiation was confirmed by immunostaining and quantitative RT‐PCR for LCs lineage‐specific markers (Primers and antibodies were listed in Table S2 and S5 , Supporting Information, respectively).
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