Table 1.

Reagents and resources

Reagent or ResourceSourceIdentifier
Antibodies 
Smoothened Santa Cruz Biotechnology sc-166685, RRID:AB2239686 
Acetylated α-Tubulin Cell Signaling 5335S, RRID:AB_0544694 
ARL13B Antibodies Incorporated 75-287, RRID:AB_2341543 
ARL13B BiCell Scientific ARL2L1 
EP4 Santa Cruz Biotechnology sc-55596, RRID:AB_2174775 
AC3 (IF) Proteintech 19492-1-AP RRID:AB_10638445 
AC3 (WB) Abcam 352098 
AC5 (WB) Life Technologies BS-3922R RRID:AB_10857032 
AC5/6 (IF) FabGennix PAC-501AP 
AC6 (WB) Proteintech 14616-1-AP-20UL
RRID:AB_2242343 
ANKMY2 (WB) Sigma-Aldrich HPA067100
RRID:AB_2685777 
PAX6 Developmental Studies Hybridoma Bank PAX6-s
RRID:AB_528427 
OLIG2 Millipore AB9610, RRID:AB_570666 
GPR161 Saikat Mukhopadhyay Pal et al. (2016)  
ABCC4 John Schuetz Wijaya et al. (2020)  
Kinesin Abcam ab167429
RRID:AB_2715530 
AlexaFluor 488 Life Technologies A11029 (mouse), RRID:AB_2534088; A11034 (rabbit), RRID:AB_2576217 
AlexaFluor 555 Life Technologies A21424 (mouse), RRID:AB_141780; A21429 (rabbit), RRID:AB_2535850 
AlexaFluor 647 Life Technologies A21236 (mouse), RRID:AB_2535805; A21245 (rabbit), RRID:AB_2535813 
Chemicals, peptides, and recombinant proteins 
Arachidonic acid Cayman Chemical Company 90010-50 
Giripladib WuXi Custom Synthesis Duvernay et al. (2015)  
L161,982 Tocris Chemicals 2,514 
Dibutyryl-cAMP (dBcAMP) MedChem Express HY-B0764 
Sonidegib (LDE225) MedChem Express HY-16582 
Prostaglandin E2 (PGE2) Sigma-Aldrich Chemicals P5640 
Celecoxib (Coxib) Cayman Chemical Company 10008672 
Forskolin (FSK) Sigma-Aldrich Chemicals F6886-10MG 
SAG Selleck Chemical Company S7779-2MG 
Lipofectamine 3000 Thermo Fisher Scientific L3000008 
Lipofectamine RNAiMAX Thermo Fisher Scientific 13778150 
IBMX Sigma-Aldrich I5879-1G 
DMSO Sigma-Aldrich D2650 
Hygromycin Life Technologies 10687010 
Commercial assay kits 
Prostaglandin E2 parameter assay kit R&D Systems KGE004B 
BCA protein assay kit Thermo Fisher Scientific Pierce 23227 
Flp-In complete system Thermo Fisher Scientific K601001 
RNeasy plus mini kit Qiagen 74136 
High-capacity cDNA reverse transcription kit Applied Biosystems 4368813 
Direct cAMP ELISA kit Enzo Life Sciences ADI900066 
Ratio-metric cilia-targeted cADDis cAMP Montana Molecular D0211G 
PowerUp SYBR green master mix Applied Biosystems A25742 
PowerPlex fusion system Promega DC2402 
MycoAlert plus Lonza LT07-710 
Oligonucleotides 
Ptch1 Forward: 5′-TCA​CCT​CCA​TCA​GCA​ATG​TC-3′ Thermo Fisher Scientific N/A 
Ptch1 Reverse: 5′-GAA​TAC​CAC​CAC​CAC​AGC​AG-3′ Thermo Fisher Scientific N/A 
Gli1 Forward: 5′-CCA​AGC​CAA​CTT​TAT​GTC​AGG​G-3′ Thermo Fisher Scientific N/A 
Gli1 Reverse: 5′-AGC​CCG​CTT​CTT​TGT​TAA​TTT​GA-3′ Thermo Fisher Scientific N/A 
Adcy3 Forward: 5′-GCT​TTC​TTT​GTC​TTC​TCC​TTC-3′ Thermo Fisher Scientific N/A 
Adcy3 Reverse: 5′-ATG​ATA​GCA​CAC​AGG​TAG​AG-3′ Thermo Fisher Scientific N/A 
Adcy5 Forward: 5′-GTC​TCA​GAA​AGT​GGC​AAT​TC-3′ Thermo Fisher Scientific N/A 
Adcy5 Reverse: 5′-CAT​TCA​GGT​AGT​TGA​GTG​TG-3′ Thermo Fisher Scientific N/A 
Anmky2 Forward: 5′-GCT​GCC​AAA​ACA​TAA​GAG​ACA​G-3′ Thermo Fisher Scientific N/A 
Anmky2 Reverse: 5′-CTT​CTT​TCT​CCC​TTC​CAC​AG-3′ Thermo Fisher Scientific N/A 
Ppia Forward: 5′-AGC​ACT​GGA​GAG​AAA​GGA​TT-3′ Thermo Fisher Scientific N/A 
Ppia Reverse: 5′-ATT​ATG​GCG​TGT​AAA​GTC​ACC​A-3′ Thermo Fisher Scientific N/A 
Btf3 Forward: 5′-GAA​CAA​CAT​CTC​TGG​TAT​TGA​AGA-3′ Thermo Fisher Scientific N/A 
Btf3 Reverse: 5′-AAT​GGT​GAA​GGT​GTT​TGC​TG-3′ Thermo Fisher Scientific N/A 
NGS primer Adcy3 forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TGG​TGT​ACT​GGG​CTC​CTC​AGG​GCT​AG-3′ Integrated DNA Technologies N/A 
NGS primer Adcy3 reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TAC​ACA​CCA​TCG​CTC​CTG​AGT​CTC-3′ Integrated DNA Technologies N/A 
Adcy5 primer forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TTC​TGG​CAG​GCA​GCT​TAC​TCC​AAG​GA-3′ Integrated DNA Technologies N/A 
NGS Adcy5 primer reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TGA​AAG​CCA​GGA​GCC​CCG​GAG​CAG​AG-3′ Integrated DNA Technologies N/A 
NGS primer Adcy6 forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TTG​ATC​TAG​GCA​GGC​TGG​GAT​CTG​AA-3′ Integrated DNA Technologies N/A 
NGS primer Adcy6 reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TAC​CTA​CAA​GGC​ACT​GGG​GGT​GAA​GGC-3′ Integrated DNA Technologies N/A 
NGS primer Ankmy2 forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TTG​TGT​GTA​TGT​GTC​ATG​AAG​ATG​G-3′ Integrated DNA Technologies N/A 
NGS primer Ankmy2 reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TCT​GCC​CGG​CTG​TAA​TAG​CTT-3′ Integrated DNA Technologies N/A 
sgRNA spacer Adcy3: 5′-CUA​CCU​GUG​UGC​UAU​CAU​CG-3′ Integrated DNA Technologies N/A 
sgRNA spacer Adcy5: 5′-AUU​GUU​ACU​ACU​GCG​UCU​CG-3′ Integrated DNA Technologies N/A 
sgRNA spacer Adcy6: 5′-CCA​ACA​GCU​CGG​UGC​UAA​CG-3′ Integrated DNA Technologies N/A 
sgRNA spacer Ankmy2: 5′-CUC​AUG​UUU​GCU​GCG​CUU​UC-3′ Integrated DNA Technologies N/A 
Adcy3 siRNA Thermo Fisher Scientific Assay ID: S98223 
Adcy5 siRNA Thermo Fisher Scientific Assay ID: S104765 
Anmky2 siRNA Thermo Fisher Scientific Assay ID: MSS235827 
Experimental models: Cell lines 
IMCD3 ATCC CRL-2123 
IMCD3 Flp-In cells Maxence Nachury Lab Ye et al. (2013)  
IMCD3 Adcy3−/− cells This paper N/A 
IMCD3 Adcy5−/− cells This paper N/A 
IMCD3 Adcy6−/− cells This paper N/A 
IMCD3 Ankmy2−/− cells This paper N/A 
IMCD3 EF1α-cyto-bPAC-GFP This paper N/A 
IMCD3 EF1α-Arl13b-Cilia-bPAC-GFP This paper N/A 
NIH-3T3 ATCC CRL-1658 
Abcc4−/− NIH-3T3 John Schuetz lab Wijaya et al. (2020)  
Ep4−/− mouse embryonic fibroblast This paper N/A 
Wild type mouse embryonic fibroblast This paper N/A 
Experimental models: Mouse model 
Ep4−/− mice Elaine Shelton Segi et al. (1998)  
Recombinant DNA 
pgLAP5-EF1α dTATA-bPAC-GFP Gift from Reiter lab Truong et al. (2021)  
pgLAP5-EF1α dTATA-Arl13b-bPAC-GFP Gift from Reiter lab Truong et al. (2021)  
pEF1α-mIFT88-EGFP Addgene 149697 
pCDNA-DEST47-Arl13b-GFP Addgene 40872 
pCDNA3-EGFP Addgene 13031 
PTGER4-Tango Addgene 66486 
5HT6-mCherry-G-GECO1.0 Addgene 47500 
pKK44-ABCC4 Gift from Schuetz lab Wijaya et al. (2020)  
pCDNA-h-mmPtch1-B-HA-GFP Addgene 120903 
Software and algorithms 
GraphPad prism v. 10 GraphPad Software http://www.graphpad.com/ 
ImageJ/Fiji Schindelin et al. (2012)  https://imagej.net/software/fiji/ 
LAS X Leica https://www.leica-microsystems.com/products/microscope-software/details/product/leica-las-x-ls/ 
JACoP Open Source https://imagej.nih.gov/ij/plugins/track/jacop2.html 
KymographDirect v. 2.0 Open Source https://sites.google.com/site/kymographanalysis/ 
NIS-elements viewer v. 4.50.00 Nikon https://www.microscope.healthcare.nikon.com/products/software/nis-elements/viewer 
Inkscape Open Source https://inkscape.org/ 
StarDist Stevens et al. (2022)  https://github.com/stardist/stardist 
CRIS.py (v2.0) Connelly and Pruett-Miller (2019)  https://github.com/patrickc01/CRIS.py 
Reagent or ResourceSourceIdentifier
Antibodies 
Smoothened Santa Cruz Biotechnology sc-166685, RRID:AB2239686 
Acetylated α-Tubulin Cell Signaling 5335S, RRID:AB_0544694 
ARL13B Antibodies Incorporated 75-287, RRID:AB_2341543 
ARL13B BiCell Scientific ARL2L1 
EP4 Santa Cruz Biotechnology sc-55596, RRID:AB_2174775 
AC3 (IF) Proteintech 19492-1-AP RRID:AB_10638445 
AC3 (WB) Abcam 352098 
AC5 (WB) Life Technologies BS-3922R RRID:AB_10857032 
AC5/6 (IF) FabGennix PAC-501AP 
AC6 (WB) Proteintech 14616-1-AP-20UL
RRID:AB_2242343 
ANKMY2 (WB) Sigma-Aldrich HPA067100
RRID:AB_2685777 
PAX6 Developmental Studies Hybridoma Bank PAX6-s
RRID:AB_528427 
OLIG2 Millipore AB9610, RRID:AB_570666 
GPR161 Saikat Mukhopadhyay Pal et al. (2016)  
ABCC4 John Schuetz Wijaya et al. (2020)  
Kinesin Abcam ab167429
RRID:AB_2715530 
AlexaFluor 488 Life Technologies A11029 (mouse), RRID:AB_2534088; A11034 (rabbit), RRID:AB_2576217 
AlexaFluor 555 Life Technologies A21424 (mouse), RRID:AB_141780; A21429 (rabbit), RRID:AB_2535850 
AlexaFluor 647 Life Technologies A21236 (mouse), RRID:AB_2535805; A21245 (rabbit), RRID:AB_2535813 
Chemicals, peptides, and recombinant proteins 
Arachidonic acid Cayman Chemical Company 90010-50 
Giripladib WuXi Custom Synthesis Duvernay et al. (2015)  
L161,982 Tocris Chemicals 2,514 
Dibutyryl-cAMP (dBcAMP) MedChem Express HY-B0764 
Sonidegib (LDE225) MedChem Express HY-16582 
Prostaglandin E2 (PGE2) Sigma-Aldrich Chemicals P5640 
Celecoxib (Coxib) Cayman Chemical Company 10008672 
Forskolin (FSK) Sigma-Aldrich Chemicals F6886-10MG 
SAG Selleck Chemical Company S7779-2MG 
Lipofectamine 3000 Thermo Fisher Scientific L3000008 
Lipofectamine RNAiMAX Thermo Fisher Scientific 13778150 
IBMX Sigma-Aldrich I5879-1G 
DMSO Sigma-Aldrich D2650 
Hygromycin Life Technologies 10687010 
Commercial assay kits 
Prostaglandin E2 parameter assay kit R&D Systems KGE004B 
BCA protein assay kit Thermo Fisher Scientific Pierce 23227 
Flp-In complete system Thermo Fisher Scientific K601001 
RNeasy plus mini kit Qiagen 74136 
High-capacity cDNA reverse transcription kit Applied Biosystems 4368813 
Direct cAMP ELISA kit Enzo Life Sciences ADI900066 
Ratio-metric cilia-targeted cADDis cAMP Montana Molecular D0211G 
PowerUp SYBR green master mix Applied Biosystems A25742 
PowerPlex fusion system Promega DC2402 
MycoAlert plus Lonza LT07-710 
Oligonucleotides 
Ptch1 Forward: 5′-TCA​CCT​CCA​TCA​GCA​ATG​TC-3′ Thermo Fisher Scientific N/A 
Ptch1 Reverse: 5′-GAA​TAC​CAC​CAC​CAC​AGC​AG-3′ Thermo Fisher Scientific N/A 
Gli1 Forward: 5′-CCA​AGC​CAA​CTT​TAT​GTC​AGG​G-3′ Thermo Fisher Scientific N/A 
Gli1 Reverse: 5′-AGC​CCG​CTT​CTT​TGT​TAA​TTT​GA-3′ Thermo Fisher Scientific N/A 
Adcy3 Forward: 5′-GCT​TTC​TTT​GTC​TTC​TCC​TTC-3′ Thermo Fisher Scientific N/A 
Adcy3 Reverse: 5′-ATG​ATA​GCA​CAC​AGG​TAG​AG-3′ Thermo Fisher Scientific N/A 
Adcy5 Forward: 5′-GTC​TCA​GAA​AGT​GGC​AAT​TC-3′ Thermo Fisher Scientific N/A 
Adcy5 Reverse: 5′-CAT​TCA​GGT​AGT​TGA​GTG​TG-3′ Thermo Fisher Scientific N/A 
Anmky2 Forward: 5′-GCT​GCC​AAA​ACA​TAA​GAG​ACA​G-3′ Thermo Fisher Scientific N/A 
Anmky2 Reverse: 5′-CTT​CTT​TCT​CCC​TTC​CAC​AG-3′ Thermo Fisher Scientific N/A 
Ppia Forward: 5′-AGC​ACT​GGA​GAG​AAA​GGA​TT-3′ Thermo Fisher Scientific N/A 
Ppia Reverse: 5′-ATT​ATG​GCG​TGT​AAA​GTC​ACC​A-3′ Thermo Fisher Scientific N/A 
Btf3 Forward: 5′-GAA​CAA​CAT​CTC​TGG​TAT​TGA​AGA-3′ Thermo Fisher Scientific N/A 
Btf3 Reverse: 5′-AAT​GGT​GAA​GGT​GTT​TGC​TG-3′ Thermo Fisher Scientific N/A 
NGS primer Adcy3 forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TGG​TGT​ACT​GGG​CTC​CTC​AGG​GCT​AG-3′ Integrated DNA Technologies N/A 
NGS primer Adcy3 reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TAC​ACA​CCA​TCG​CTC​CTG​AGT​CTC-3′ Integrated DNA Technologies N/A 
Adcy5 primer forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TTC​TGG​CAG​GCA​GCT​TAC​TCC​AAG​GA-3′ Integrated DNA Technologies N/A 
NGS Adcy5 primer reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TGA​AAG​CCA​GGA​GCC​CCG​GAG​CAG​AG-3′ Integrated DNA Technologies N/A 
NGS primer Adcy6 forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TTG​ATC​TAG​GCA​GGC​TGG​GAT​CTG​AA-3′ Integrated DNA Technologies N/A 
NGS primer Adcy6 reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TAC​CTA​CAA​GGC​ACT​GGG​GGT​GAA​GGC-3′ Integrated DNA Technologies N/A 
NGS primer Ankmy2 forward: 5′-CTA​CAC​GAC​GCT​CTT​CCG​ATC​TTG​TGT​GTA​TGT​GTC​ATG​AAG​ATG​G-3′ Integrated DNA Technologies N/A 
NGS primer Ankmy2 reverse: 5′-CAG​ACG​TGT​GCT​CTT​CCG​ATC​TCT​GCC​CGG​CTG​TAA​TAG​CTT-3′ Integrated DNA Technologies N/A 
sgRNA spacer Adcy3: 5′-CUA​CCU​GUG​UGC​UAU​CAU​CG-3′ Integrated DNA Technologies N/A 
sgRNA spacer Adcy5: 5′-AUU​GUU​ACU​ACU​GCG​UCU​CG-3′ Integrated DNA Technologies N/A 
sgRNA spacer Adcy6: 5′-CCA​ACA​GCU​CGG​UGC​UAA​CG-3′ Integrated DNA Technologies N/A 
sgRNA spacer Ankmy2: 5′-CUC​AUG​UUU​GCU​GCG​CUU​UC-3′ Integrated DNA Technologies N/A 
Adcy3 siRNA Thermo Fisher Scientific Assay ID: S98223 
Adcy5 siRNA Thermo Fisher Scientific Assay ID: S104765 
Anmky2 siRNA Thermo Fisher Scientific Assay ID: MSS235827 
Experimental models: Cell lines 
IMCD3 ATCC CRL-2123 
IMCD3 Flp-In cells Maxence Nachury Lab Ye et al. (2013)  
IMCD3 Adcy3−/− cells This paper N/A 
IMCD3 Adcy5−/− cells This paper N/A 
IMCD3 Adcy6−/− cells This paper N/A 
IMCD3 Ankmy2−/− cells This paper N/A 
IMCD3 EF1α-cyto-bPAC-GFP This paper N/A 
IMCD3 EF1α-Arl13b-Cilia-bPAC-GFP This paper N/A 
NIH-3T3 ATCC CRL-1658 
Abcc4−/− NIH-3T3 John Schuetz lab Wijaya et al. (2020)  
Ep4−/− mouse embryonic fibroblast This paper N/A 
Wild type mouse embryonic fibroblast This paper N/A 
Experimental models: Mouse model 
Ep4−/− mice Elaine Shelton Segi et al. (1998)  
Recombinant DNA 
pgLAP5-EF1α dTATA-bPAC-GFP Gift from Reiter lab Truong et al. (2021)  
pgLAP5-EF1α dTATA-Arl13b-bPAC-GFP Gift from Reiter lab Truong et al. (2021)  
pEF1α-mIFT88-EGFP Addgene 149697 
pCDNA-DEST47-Arl13b-GFP Addgene 40872 
pCDNA3-EGFP Addgene 13031 
PTGER4-Tango Addgene 66486 
5HT6-mCherry-G-GECO1.0 Addgene 47500 
pKK44-ABCC4 Gift from Schuetz lab Wijaya et al. (2020)  
pCDNA-h-mmPtch1-B-HA-GFP Addgene 120903 
Software and algorithms 
GraphPad prism v. 10 GraphPad Software http://www.graphpad.com/ 
ImageJ/Fiji Schindelin et al. (2012)  https://imagej.net/software/fiji/ 
LAS X Leica https://www.leica-microsystems.com/products/microscope-software/details/product/leica-las-x-ls/ 
JACoP Open Source https://imagej.nih.gov/ij/plugins/track/jacop2.html 
KymographDirect v. 2.0 Open Source https://sites.google.com/site/kymographanalysis/ 
NIS-elements viewer v. 4.50.00 Nikon https://www.microscope.healthcare.nikon.com/products/software/nis-elements/viewer 
Inkscape Open Source https://inkscape.org/ 
StarDist Stevens et al. (2022)  https://github.com/stardist/stardist 
CRIS.py (v2.0) Connelly and Pruett-Miller (2019)  https://github.com/patrickc01/CRIS.py 
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