Journal: The Journal of Cell Biology

Article Title: Range of SHH signaling in adrenal gland is limited by membrane contact to cells with primary cilia

doi: 10.1083/jcb.201910087

Figure Lengend Snippet: Human adrenocortical carcinoma cells do not exhibit autocrine, canonical SHH signaling. (A) Semiquantitative RT-PCR showing the expression of SHH pathway components in mouse microdissected adrenal cortex, using Gapdh as a reference. (B) Semiquantitative RT-PCR showing the expression of SHH pathway components in NCI-H295R cells, using GAPDH as a reference. (C) Western blots of NCI-H295R cell and adrenal gland lysates, probed for SHH, PTCH1, SMO, GLI1, GLI2, GLI3, and ACTIN as a loading control. (D) Proliferation of NCI-H295R cells cultured with or without lipoproteins with the respective treatments, quantified by measuring BrdU incorporation. Data are presented as mean ± SD, n = 4 replicates, pooled from two experiments. (E and F) GLI1 and PTCH1 expression in NCI-H295R cells grown for 48 h with or without lipoproteins, treated with SHH pathway inhibitors 10 µg/ml 5E1 and 10 µM cyclopamine or appropriate controls (E), or with SHH pathway activators 200 nM SAG or 10 ng HEK-ShhNc (F). (G) Gli1 and Ptch1 expression in mouse fibroblasts NIH3T3 is used as a positive control for canonical SHH signaling. B-actin is used as a reference. Data are normalized to the respective No treatment values. (E–G) Data are presented as mean ± SD, n = 6–12 replicates, pooled from two to four experiments. ****, P < 0.001.

Article Snippet: Proliferation of NCI-H295R was measured using the BrdU Cell Proliferation ELISA Kit (Abcam), according to the manufacturer’s protocol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Cell Culture, BrdU Incorporation Assay, Positive Control