Sc 20687

Cells treated with DMSO, Niebla sp. (1), or tumidulin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [24 (link)]. Antibodies against ALDH1, CD44, CD133, Lgr5, Msi1, and α-tubulin were used as previously described [17 (link)]. Antibodies against Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), and β-Actin (sc-47778; SANTA CRUZ) were detected with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) using an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA) and luminescence imaging on an Image Quant LAS 4000 mini. Bands were measured using Multi-Gauge 3.0 software, and the relative density was calculated based on the density of the β-actin bands in each sample. Values are expressed as arbitrary densitometric units corresponding to signal intensity.

CSC221, cells treated for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer^{24 (link)}. Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), Msi-1 (ab52865, Abcam), Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), Bmi-1 (ab38295; Abcam) were used to detection. α-tubulin (2125, Cell Signaling Technology) and β-Actin (sc-47778; SANTA CRUZ) antibody was used as an internal control. The bands were cut according to the protein size region of interest before intubating with antibodies and then imaged with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Uncropped images of the blot images are presented as additional data (Supple Figs. 10, 11). Bands relative density was calculated based on the density of α-tubulin and actin bands in each sample. Values were demonstrated as arbitrary densitometric units corresponding to signal intensity.

To stain SHH signaling proteins, D2 embryos were fixed in formalin solution overnight and washed three times in DPBS/PVA. Fixed embryos were incubated at RT for 1 h in DPBS containing 1% Triton X-100. Subsequently, embryos were washed three times in DPBS/PVA and stored in DPBS/PVA containing 1 mg/mL BSA (blocking solution) at RT for 1 h. The embryos were incubated with primary antibodies for SHH (1:100; sc-365112; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), SMO (1:100; sc-13943; Santa Cruz), PTCH1 (1:200; sc-9016; Santa Cruz), or GLI1 (1:200; sc-20687; Santa Cruz) at 4 °C overnight. After washing three times in DPBS/PVA, embryos were placed in blocking solution for 1 h at RT. Next, the embryos were reacted at RT for 1 h with the Alexa-Fluor-488-labeled goat anti-mouse IgG secondary antibody (200:1). The embryos were washed three times in DPBS/PVA and mounted on clean slide glasses with DAPI. The fluorescence intensities of SHH signaling proteins were observed using a fluorescence microscope (DMi8; Leica). The fluorescence signal intensities were analyzed using ImageJ software after normalization through subtraction of the background intensity to that of control embryos.