Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis

Summary

The study demonstrates a quantitative PCR-based assay for primary cilia-mediated Sonic Hedgehog (SHH) signaling pathway activation using SHH agonist in a cellular model of ciliogenesis.

Abstract

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of motile or non-motile (primary) cilia that are microtubule-based cell protrusions. Primary cilia (PC) are present in most human epithelial and endothelial tissues and serve as a hub for physiological signal transduction, including Sonic Hedgehog (SHH) signaling. Various situations demand examining the function of PC, which may be achieved by measuring the canonical SHH signaling pathway that is almost exclusively mediated by PC. Here, a quantitative PCR (qPCR) based technique is developed to measure the transcriptional level of SHH pathway genes in quiescent hTERT-RPE1 (or RPE1) cells that mostly contain PC. Quiescence in RPE1 cells is achieved by serum starvation for 48 h, while activation of the SHH pathway is promoted by a specific agonist. This cell culture-based assay is easy to follow and sensitive. Successful demonstration of this assay in ciliated RPE1 cells indicates its immense potential as an in vitro assay to examine the proper function of PC upon genetic or epigenetic alteration of one or multiple genes, which may be associated with various ciliopathies.

Introduction

A cilium is a microtubule-based and membrane-ensheathed protrusion from the cell surface, which is assembled on a basal body¹. In vertebrates, two types of cilia, namely motile and non-motile, are observed. Motile cilia are located in specialized tissues and confer motility. Non-motile primary cilia (PC) are present in most vertebrate cells, and perform sensory functions and transduce critical signals from the extracellular milieu to the interior of the cells². Once assembled, ciliary elongation is helped by intraflagellar transport (IFT) machinery, which is a bidirectional transport system carrying cargo driven by kines....

Protocol

1. Cell culture

1. Passage a near-confluent T-25 cell culture flask of RPE1 cells at 1:5 dilution of the original culture into a fresh T-25 cell culture flask containing 5 mL of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycin mix (final concentration- 100 U/mL penicillin G and 100 µg/mL streptomycin). This medium is referred to as the complete medium. Grow cells at 37 °C in the presence of 5% CO₂.

Discussion

Examining the functionality of PC is a crucial step towards understanding the dynamics of ciliation and diseases or disorders associated with the dysfunction of PC. Apart from examining ciliation by immunostaining for ciliary markers or fluorophore-tagged ciliary structural components, the function of PC is commonly determined by assaying the canonical SHH pathway activation by measuring the ciliary translocation of Smo in tissues and cells using fluorescence microscopy^27,

Materials

Name	Company	Catalog Number	Comments
DMSO, sterile filtered	Sigma	D2650
Donkey, anti-Mouse IgG (H+L) Alexa Fluor 488	ThermoFisher Scientific	A21202
Donkey, anti-Rabbit IgG (H+L) Alexa Fluor 568	ThermoFisher Scientific	A10042
Dulbecco's Modified Eagle Medium (DMEM)	Gibco, ThermoFisher Scientific	41965039
Dulbecco's phosphate-buffered saline (DPBS)	Gibco, ThermoFisher Scientific	14190144
Fetal bovine serum (FBS)	Himedia	RM1112
Fluorescence Microscope fitted with 60X objective	Carl Zeiss		Axio observer colibri 5
hTERT-RPE1 cells	ATCC	CRL-4000
iScript cDNA Synthesis Kit	Bio-Rad Laboratories	1708891
Mouse, monoclonal anti-acetylated tubulin antibody	Milipore Sigma-Aldrich	T7451	Final dilution 1:1000
Penicillin-Streptomycin, 100X	Gibco, ThermoFisher Scientific	15140122
Quantitaive PCR machine, CFX 96	Bio-Rad Laboratories
Rabbit, polyclonal Arl13B antibody	ProteinTech	17711-1-AP	Final dilution 1:250
Rabbit, polyclonal Cep135 antibody	ProteinTech	24428-1-AP	Final dilution 1:500
SlowFade gold Antifade moutant	ThermoFisher Scientific	S36940
Smoothened Agonist (SAG)	Abmole	M4865	Stock solution: in DMSO, 5 mM
SsoAdvanced Universal SYBR Green Supermix	Bio-Rad Laboratories	1725271
TRIzol Reagent	Invitrogen	15596026
TrypLE	Gibco, ThermoFisher Scientific	12605028