Actin is shown as a loading control
Actin is shown as a loading control. and ESCs in ES and Ndiff+2i media. (C) GLI transcription is not affected by deletion in ESCs. mRNA levels were measured by qPCR (= 3, biological replicates). (D) GLI transcription remains unchanged in ESCs treated with HH pathway targeting compounds. ESCs were treated for 48 hours with S (100 ng/ml), SAG (0.5 M), PMP (1 M), Cyclopamine-KAAD (1 M), and SANT-1 (10 M) as indicated, and mRNA levels were measured by qPCR. Samples do not show statistically significant differences (= 3, biological replicates). (E, F) Chemical inhibition of GLI activity does not affect ESCs survival. (E) Survival of ESCs after 48 hours with or without addition of the GLI inhibitor GANT61 (1M). (F) Effect of GANT61 treatment on expression. Expression of mRNA with or without GANT61 (1M) treatment in ESCs (= 3, biological replicates) (left). Induction of mRNA by SHH with or without GANT61 (1 M) treatment in NIH-3T3 (= 3, biological replicates) (right). (G, H) Frequency of ciliated ESCs. (G) Immunofluorescence showing ciliated cells in an ESC colony. ARL13B marker was used to stain cilia (indicated with an arrow). Scale bar = 10 m. (H) Plot of the percentage of ciliated cells over the total number of cells. Each dot represents the percentage of cells with cilia detected in an ESC colony (= 25). The data underlying all the graphs shown in the figure are included in the S1 Data file. ESC, embryonic stem cell; gRNA, guide RNA; HH, hedgehog; NHEJ, nonhomologous end joining; PMP, purmorphamine; qPCR, quantitative PCR; SAG, smoothened agonist; SHH, Sonic hedgehog; SMO, Smoothened; WT, wild-type.(PDF) pbio.3001596.s004.pdf (218K) GUID:?85183408-E788-40FD-A7EF-49F1A764F2A4 S2 Fig: High concentrations of SAG and PMP induce cell death by sequestering SMO protein. (A) transcriptional activity in NIH-3T3 cells treated with compounds shown in Fig 2B. mRNA was measured by RT-qPCR. (B) Effects of compounds indicated on survival of NIH-3T3 cells. NIH-3T3 cells were treated for 48 hours with S (50C500 ng/ml), SAG (1C5 M), PMP (2.5C10 M), Cyclopamine-KAAD (1C5 M) or SANT-1 (10C50 M). Survival rate is normalized to DMSO treated sample. (C) Dissociation-induced apoptosis is prompted by high PMP concentrations. Survival of ESCs treated for 48 hours with different PMP concentrations (2 nM20 M). Red dotted line highlights IC50 at 2.5 M. (D, E) High concentrations of PMP repress GLI activity and HH signaling. (D) RT-qPCR of mRNA in NIH-3T3 cells treated with increasing concentrations of PMP (2 nM to 40 M) and normalized to untreated samples. Dotted lines mark PMP EC50 (green, at 30 nM) and IC50 (red, at 4 M). (E) RT-qPCR of mRNA in MEFs treated with increasing concentrations of PMP (2 nM to 40 M) and normalized to untreated samples. Red dotted line highlight IC50. (F) SAG and PMP cytotoxic effects are reduced in ESCs. Survival of and control ESCs treated for 48 hours with SAG (2.5 to 5 M) and PMP (5 to 10 M). The effect Febantel on 2 independent clones is shown. Asterisks denote statistical significance for difference from the DMSO treated Febantel samples. Febantel (G) PMP induces blebbing and death after dissociation IL17RA of ESCs. On the left, ESC morphology 24 hours after dissociation and plating on Matrigel (upper panels, pretreated with PMP for 24 hours; lower panels, pretreated with PMP and with ROCKi). Cells showing membrane blebbing (arrow), and apoptotic bodies (asterisk) are indicated. Images at 1, 3, and 6 hours after plating are shown on the right. (H) Survival of ESCs treated for 48 hours with PMP with or without the addition of ROCKi. (I) Phosphorylated MYL2 (MYL2-P) distribution in WT and mutant ESCs treated with or without PMP for 24 hours. Bars represent 5 m. (J) Constitutive active as a new modulator of HH signaling. (A) Number of insertions in genes of the p24 family that were detected in selected and control samples. (B) Generation of and ESCs lines using.