REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Calnexin	StressGen Biotechnologies	SPA-860, RRID: AB_312057
DHRSX	Sigma	HPA003035, RRID: AB_1080631
FLAG M2	Sigma	F3165, RRID: AB_259529
Penta-His	Qiagen	34660, RRID: AB_2619735
β-tubulin	Thermo Fisher Scientific	MA516308, RRID: AB_2537819
LAMP2 antibody	Santa-Cruz Biotech	SC-18822, RRID: AB_626858
Goat-anti-Mouse antibody	DAKO	P0447, RRID: AB_2617137
Goat-anti-Rabbit antibody	DAKO	P0448, RRID:AB_2617138
Alexa 488-labeled goat anti-rabbit IgG antibody	Thermo Fisher Scientific	A-11034, RRID: AB_2576217
Alexa 568-labeled goat anti-mouse IgG antibody	Thermo Fisher Scientific	A-11019, RRID: AB_143162
Alexa 488-labeled goat anti-mouse IgG antibody	Thermo Fisher Scientific	A-11001, RRID: AB_2534069
Alexa 568-labeled goat anti-rabbit IgG antibody	Thermo Fisher Scientific	A-11011, RRID: AB_143157
Chemicals, reagents, and recombinant proteins
Bovine Serum Albumin (BSA) Fraction V	Sigma	10735086001
Digitonin	Invitrogen (Thermo Fisher Scientific)	BN2006
DBPS	Cytiva	SH30264.01
Fetal Bovine Serum	Dutcher	S1810
Fetal Bovine Serum	BioWest	S1400
TurboFectin 8.0	Origene	TF81005
Lipofectamine3000	Thermo Fisher Scientific	15292465
Opti-MEM™	Gibco	11058021
DMEM/F-12	Gibco	21041–025
DMEM/F-12, HEPES	Gibco	31330038
IMDM	Gibco	12440–053
DMEM	Gibco	41966052
Cytiva HyClone™ Fetal Clone III Serum	Cytiva	12319862
0.5 mm glass beads	Sigma Aldrich	Z250465
DAPI Solution (1 mg/mL)	Thermo Fisher Scientific	62248
Saponin	Sigma-Aldrich	84510
Triton™ X-100	Sigma-Aldrich	T8787
beta-mercaptoethanol	Sigma-Aldrich	M3148
Pageruler Plus Prestained Protein Ladder	Thermo Fisher Scientific	11832124
DMSO	Sigma-Aldrich	317275
Para-formaldehyde	Thermo Fisher Scientific	28908
Mowiol® 4-88	Sigma-Aldrich	81381
RIPA buffer	Custom	N/A
SuperSignal™ West Pico PLUS Chemiluminescent Substrate	Thermo Fisher Scientific	34579
Puromycin Dihydrochloride	Gibco	A1113802
Difco™ Casamino Acids, Vitamin Assay	Gibco	16219861
Bacto™ Yeast Extract	Gibco	212750
Bacto™ Peptone	Gibco	16299741
Geneticin™ Selective Antibiotic	Gibco	10131035
Hygromycin B	Gibco	10687010
GeneRuler	Thermo Fisher Scientific	SM1331
PBS	Thermo Fisher Scientific	10010023
TBS 10X	Euromedex	ET220
Versene	Gibco	15040033
Methanol	Thermo Fisher Scientific	10606652
Methanol (LC-MS grade)	Biosolve	136878
Chloroform	VWR	22711290
Chloroform (LC-MS grade)	Biosolve	34806
trimethylsilyl diazomethane (TMSD)	Sigma-Aldrich	362832
NAD+ free acid grade II	ROCHE	10127990001
NADH disodium salt	ROCHE	10128023001
NADP+ sodium salt hydrate	Sigma-Aldrich	N0505-1G
NADPH tetrasodium salt	ROCHE	10102824001
Dolichol	Aventi polar lipids	9002000
Polyprenol	Aventi polar lipids	9002100
Dolichal	This study	N/A
Polyprenal	Aventi polar lipids	9002200
Phosphatidylcholine	Sigma-Aldrich	P-0378
Phosphatidylethanolamine	Sigma-Aldrich	P-0503
Glucose	Sigma-Aldrich	G7021
Magnesium Sulfate	Thermo Fisher Scientific	A14491.0I
Asahipak NH2P-50 4E column	Shodex	N/A
7-AAD	Thermo Fisher Scientific	A1310
Carbenicillin	VWR	J67159.AD
Dithiothreitol	Thermo Fisher Scientific	R0861
Gelred nucleic acid stain	Sigma-Aldrich	SCT123
T7 DNA Ligase	New England Biolabs	M0318S
CutSmart® Buffer	New England Biolabs	B7204
Agilent 6546 ion funnel mass spectrometer	Agilent	N/A
Agilent 1290 HPLC System	Agilent	N/A
Accucore C30 150 × 2.1 mm column	Thermo Fisher Scientific	27826–152130
Isopropopanol	Biosolve	162678
Acetonitrile	Biosolve	12078
Ammonium formate	Biosolve	19878
Formic acid	Biosolve	232478
iBlot® 2 NC mini Stacks	Invitrogen	IB23002
ECL WB substrate	Thermo Fisher Scientific	PIER32106
First-strand cDNA Synthesis Kit	Sigma Aldrich	GE27-9261-01
DNAse I	Sigma Aldrich	10104159001
Trypsin	Gibco	25300096
IPTG	Thermo Fisher Scientific	15529019
Bolt® 4–12% Bis-Tris Plus Gels, 12-well	Thermo Fisher Scientific	15324604
Bolt Transfer Buffer (20X)-1 L	Thermo Fisher Scientific	15256066
20X Bolt® MES SDS Running Buffer (500 mL)	Thermo Fisher Scientific	13266499
LDS Sample buffer	Thermo Fisher Scientific	11549166
cOmplete(TM), Mini, EDTA-free Protease	Thermo Fisher Scientific	11836170001
Trypsin	Sigma Aldrich	T8003-500MG
Trypsin-EDTA 1X in PBS w/o Calcium w/o Magnesium	Dominique Dutscher	L0940-100
Tween 20	Thermo Fisher Scientific	P1379
N-Glycosidase F	Roche	11365193001
6X DNA Loading Dye	Thermo Fisher Scientific	R0611
Cell Disruption Media	Scientific Industries, Inc.	SI-BG05
LipidSpot™ 610	Biotium	70069
Polybrene	Merck life science N.V (ex Sigma Aldrich)	TR-1003
Phusion™ DNA polymerase	Thermo Fisher Scientific	F630S
2-Chloroacetamide (CAM)	Sigma-Aldrich	C0267
Trizma® base	Merck Life Science BV (ex Sigma Aldrich)	T1503-100g
S-Trap™ micro units	Protifi LLC	N/A
Axygen™ MaxyClear Snaplock Microtubes, 1.5 mL (MCT-150-L-C)	Thermo Fisher Scientific	11311984
CaCl2	Sigma-Aldrich	793639
Water ULC/MS - CC/SFC	Biosolve	232141
Sequencing Grade Modified Trypsin (1x100μg)	Promega	V5117
Lys-C endopeptidase	Sopachem NV	125–02543
Phosphoric acid	Merck Life Science BV (ex Sigma Aldrich)	695017-100ML
Sodium dodecyl sulfate SDS	Merck Life Science BV (ex Sigma Aldrich)	62862-1KG
Critical commercial assays
Micro BCA™ Protein Assay Kit	Thermo Fisher Scientific	Cat# 23235
DNeasy Blood & Tissue Kit	Qiagen	69504
Ni-NTA Fast Start Kit	Qiagen	30600
Ni-NTA spin kit	Qiagen	31314
RNeasy Mini KIT	Qiagen	74104
NucleoSpin Plasmid Mini kit	Macherey-Nagel	740588.5
Plasmid mini kit	Qiagen	12123
Yeast Transformation Kit	Sigma Aldrich	YEAST1-1KT
NEBridge® Golden Gate Assembly Kit	New England Biolabs	R3733S
Experimental models: Cell lines
HAP1 Parental cells	Horizon Discovery	C631
HAP1 DHRSX KO cells	Horizon Discovery	HZGHC007899c011
HAP1 SRD5A3 KO Cells	This paper	N/A
HAP1 MPDU1 KO cells	Horizon Discovery	HZGHC004060c012
HEK293T	Gift from Reid Gilmore, Uni. Of Mass.	This paper
EBV-immortalized lymphoblasts (Controls and affected DHRSX and SRD5A3-deficient individuals)	This paper	N/A
Dermal fibroblasts (Controls and affected DHRSX and SRD5A3-deficient individuals)	This paper	N/A
Experimental models: Organisms/strains
Yeast BY4741	Euroscarf	Y00000
Yeast BY4741 dfg10 KO	This paper	N/A
BL21(DE3) E. Coli	Thermo Fisher Scientific	10749734
One Shot® TOP10 E. coli	Thermo Fisher Scientific	C404003
Oligonucleotides
DHRSX_gDNA_PCR_F TCTCCACTGTCTTCCCCAGT	This paper	N/A
DHRSX_gDNA_PCR_R CGCACAGACAGAGGGAGATG	This paper	N/A
SRD5A3_gDNA1_PCR_F GCCTTTGATGTCCCCAAGAGATATT	This paper	N/A
SRD5A3_gDNA1_PCR_R CATGGCACTTATACTGATGGGC	This paper	N/A
SRD5A3_gDNA2_PCR_F TCAGAATTCTCGGGGCGGC	This paper	N/A
SRD5A3_gDNA2_PCR_R AGTGAATGACCACTCCTGCTTTA	This paper	N/A
Ori-Amp_CW_F GAGGTACCGGTCTCTGGG GCGGTAATACGGTTATCCACAG	This paper	N/A
Ori-PmeI_CCW_F GAGGTACCGGTCTCAGTT TAAACTCATATATACTTTAGATTGATTTAAAAC	This paper	N/A
AmpR_CW_F GAGGTACCGGTCTCTAAACTT GGTCTGACAGTTACCAATGCTTAATC	This paper	N/A
Ori-Amp_CCW_F GAGGTACCGGTCTCATC ACTGGCACTTTTCGGGGAAATGTGC	This paper	N/A
DFG10-L_CW_F GAGGTACCGGTCTCTGT GAGTTTAAACTTTTTGCCCAAATAAATCATAATC	This paper	N/A
DFG10-L_CCW_F GAGGTACCGGTCTCTC GCAGCAATTGTTCTTCATCAAAGTACAT	This paper	N/A
pUG74_NatK7b_CW-F GAGGTACCGGTC TCGTGCGGTACGCTGCAGGTCGACAACC	This paper	N/A
pUG74_NatK7_CCW_F GAGGTACCGGTCT CTACGACACTAGTGGATCTGATATCACC	This paper	N/A
DFG10-R_CW_F GAGGTACCGGTCTCTTCG TTAATGTGCACACTAGGGCATTCATATAC	This paper	N/A
DFG10-R_CCW_F GAGGTACCGGTCTCT CCCCGTTTAAACAATGCCTAAAATATT AAATAAAAAATAGG	This paper	N/A
AmpR-Mut_CCW_F GAGGTACCGGTCTC GTCACGCGGTATCATTGCAGCACTG	This paper	N/A
pEF-1_CCW_F GAGGTACCGGTCTCTCG GTTGCTAGTGAACACAGTTGTGTCAG	This paper	N/A
DHRSX_CW_F GAGGTACCGGTCTCTACCG ATGTCGCCATTGTCTGCGGC	This paper	N/A
DHRSX-mut_CCW_F GAGGTACCGGTCTCTCT TCGTCTCTTTCTCGTTGTATAGGTAAC	This paper	N/A
DHRSX-mut_CW_F GAGGTACCGGTCTCTG AAGTCCCTCCACGTCACCTACAAC	This paper	N/A
DHRSX-stop_CCW_F GAGGTACCGGTCTCT GTTGTCACAGGGTCACATCAAGGAC	This paper	N/A
hBGt-R-CCW-F GAGGTACCGGTCTCTCA ACATCGATTAGACTAGTCTAGAAATTCACC	This paper	N/A
AmpR_Mut_CW_F GAGGTACCGGTCTCC GTGACCCACGCTCACCGGCTCCAG	This paper	N/A
pEF-2_CCW_F GAGGTACCGGTCTCTCGGTT CACGACACCTGAAATGGAAG	This paper	N/A
DHRSX-Ctag_CCW_F GAGGTACCGGT CTCTCCAGCAGGGTCACATCAAGGACCC	This paper	N/A
C3xFlag_CW_F GAGGTACCGGTCTCAC TGGGTCACCATCACCATCACCATGAC TACAAAGACCATGACGGTG	This paper	N/A
C3xFlag_CCW_F GAGGTACCGGTCTCG GTTGTCAGGCCGCAAGCTTGTCATCGT	This paper	N/A
hBGt-2-R-CW-F GAGGTACCGGTCTCT CAACCGACTGTGCCTTCTAGTTGCC	This paper	N/A
CeGFP_CW_F GAGGTACCGGTCTCA CTGGGTGTGAGCAAGGGCGAGGAGCTG	This paper	N/A
CeGFP_CCW_F GAGGTACCGGTCTCGG TTGTTACTTGTACAGCTCGTCCATGCCG	This paper	N/A
SRD5A3_CW_F GAGGTACCGGTCTCTA CCGATGGCTCCCTGGGCGGAGG	This paper	N/A
SRD5A3-Ctag_CCW_F GAGGTACCGGT CTCTCCAGAAACAAAAATGGTA GGAAAGCTTTCCTATG	This paper	N/A
Cen4Ars1_CW_F GAGGTACCGGTCTCTG TGACCGCATATATCGCTGGGCCATTC	This paper	N/A
Cen4Ars1_CCW_F GAGGTACCGGTCTCA CGCAGTTATTACTGAGTAGTATTTATT TAAGTATTGTTTG	This paper	N/A
pRS_DrugK7_CW_F GAGGTACCGGTCTCGTG CGGCATCAGAGCAGATTGTACTGAGAGTG	This paper	N/A
pRS_DrugK7_CCW_F GAGGTACCGGTCTC TACGATGCGGTATTTTCTCCTTACGCATCTGTG	This paper	N/A
DFG10-L2_CW_F GAGGTACCGGTCTCTTCGTG TTTAAACTTTTTGCCCAAATAAATCATAATC	This paper	N/A
DFG10-A_CW_F ACCAGTAGAATGCTGTCCTGC	This paper	N/A
DFG10-B_CW_F CTATGAAAAGTGAGCAAATGCCTAC	This paper	N/A
DFG10-C_CW_F CATGTTATTCTGGCCAATCTG	This paper	N/A
DFG10-D_CW_F GTTAGGCATTTGTCTCTGAAG	This paper	N/A
kanB CTGCAGCGAGGAGCCGTAAT	This paper	N/A
kanC TGATTTTGATGACGAGCGTAAT	This paper	N/A
SRD5A3sense_BglII_HIS TTATATAGATCTTGCCACCATGCATCATCAC	This paper	N/A
SRD5A3_rev_bsrGI ATACAATGTACACTAGAAGGCACAGTCGAGGC	This paper	N/A
SRD5A3_sense_BglII TTATATAGATCTCCACCATGGCTCCCTGGGCGGAG	This paper	N/A
hDHRSX_rev_bsrGI ATACCATGTACATCACAGGGTCACATCAAGGAC	This paper	N/A
hDHRSX_ sense_BglII TTATATAGATCTCCACCATGTCGCCATTGTCTGCGG	This paper	N/A
Deposited data
Proteomics data	This paper	ProteomeXchange: PXD049174
Recombinant DNA
pET15b DHRSX-N-His NM_145177.3	This paper	N/A
pcDNA3.1(+) SRD5A3-N-His NM_024592.5	This paper	N/A
pcDNA3.1(+) DFG10-N-His (Human optimized cDNA)	This paper	N/A
pSpCas9(BB)-2A-GFP (PX458) gRNA_1 sequence CACGACGTGAATCATGACAT	This paper	N/A
pSpCas9(BB)-2A-GFP (PX458) gRNA_2 sequence CTAGCCACCAAGTTAAGTTG	This paper	N/A
pDel-DFG10	This paper	N/A
pEF-DHRSX-3xFlag	This paper	N/A
pYCH-DFG10	This paper	N/A
psPAX2		Addgene #12260
pMD2.G		Addgene #12259
pUB83 SRD5A3	This paper	N/A
pUB83 SRD5A3-N-His	This paper	N/A
Software and algorithms
ImageJ	NIH	https://www.imagej.net/ij/
Fiji	NIH	https://doi.org/10.1038/nmeth.2019
Adobe Illustrator	Adobe	https://www.adobe.com
LAURA 6	LabLogic, UK	https://lablogic.com/life-sciences/software/laura
Alissa Interpret	Agilent	https://www.agilent.com/en/product/next-generation-sequencing/clinical-informatics-platform/alissa-interpret-930086
Interactive Genomics Viewer	Broad Institute	https://software.broadinstitute.org/software/igv/
Ugene	Unipro	https://www.ugene.net/
Graphpad Prism	Dotmatics	https://www.graphpad.com
Mass Hunter Quantitative Analysis	Agilent	https://www.agilent.com/en/product/software-informatics/mass-spectrometry-software/data-analysis/quantitative-analysis
Mass Hunter Qualitative Analysis	Agilent	https://www.agilent.com/en/product/software-informatics/mass-spectrometry-software/data-analysis/quantitative-analysis
Proteome Discoverer	Thermo Fisher Scientific	https://www.thermofisher.com/be/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis
Biorender	Science Suite Inc.	https://www.biorender.com/