PPT1 regulation of HSP90α depalmitoylation participates in the pathogenesis of hyperandrogenism

. 2023 Feb 3;26(3):106131. doi: 10.1016/j.isci.2023.106131

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Rabbit anti-HSP90α polyclonal antibody	Proteintech	Cat# 13171-1-AP, RRID: AB_2120924
Rabbit anti-PPT1 polyclonal antibody	Proteintech	Cat# 10887-1-AP, RRID: AB_2168906
Rabbit anti-LAMP1 monoclonal antibody	Abcam	Cat#ab208943, RRID: AB_2923327
Mouse anti-DYDDDDK Tag monoclonal antibody	Proteintech	Cat# 66008-3-Ig, RRID: AB_2749837
Mouse anti-GAPDH monoclonal antibody	Proteintech	Cat# 60004-1-Ig, RRID: AB_2107436
Mouse anti-beta actin monoclonal antibody	Proteintech	Cat#66009-1-Ig, RRID: AB_2687938
Rabbit anti-HA tag polyclonal antibody	Proteintech	Cat#51064-2-AP, RRID: AB_11042321
Mouse anti-Vinculin monoclonal antibody	Proteintech	Cat#66305-1-lg, RRID: AB_2810300
Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP	Thermo Fisher Scientific	Cat#31430, RRID: AB_228307
Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP	Thermo Fisher Scientific	Cat#31460, RRID: AB_228341
HRP-labeled Streptavidin	Beyotime	Cat#A0303, RRID: AB_2933973

Bacterial and virus strains

Escherichia coli DH5a	TIANGEN	Cat#CB101
Escherichia coli TOP10	TIANGEN	Cat#CB104

Biological samples

paraffin-embedded specimens from patients	Jinling Hospital of Nanjing Medical University; Women’s Hospital of Nanjing Medical University	N/A

Chemicals, peptides, and recombinant proteins

Dehydroepiandrosterone	Sigma-Aldrich	Cat#D4000
Dipyridamole	Sigma-Aldrich	Cat#9766
Corn oil	Sigma-Aldrich	Cat#C8267
Penicillin/streptomycin	Gibco	Cat#15140-122
Normocin	InvivoGen	Cat#ant-nr-1
PMSG	Ningbo 2^nd Hormone Factory	N/A
hCG	Ningbo 2^nd Hormone Factory	N/A
Hyaluronidase	Sigma-Aldrich	Cat#H3884
Protease inhibitor cocktail	Roche	Cat#4693116001
Phosphatase inhibitor cocktails	Roche	Cat#04906837001
NEM	Sigma-Aldrich	Cat#E3876
Biotin-BMCC	Thermo Fisher Scientific	Cat#21900
HPDP-Biotin	Thermo Fisher Scientific	Cat#21341
DHT	Sigma-Aldrich	Cat#D-073
Trypsin, sequencing grade	Promega	Cat#V5280
Tris, mass spectrometry grade	Sigma-Aldrich	Cat#T1503
SDS, mass spectrometry grade	Bio-Rad	Cat#1610302
DTT, mass spectrometry grade	Bio-Rad	Cat#1610610
Iodoacetamide, mass spectrometry grade	Bio-Rad	Cat#1632109
Formic acid, mass spectrometry grade	Thermo Fisher Scientific	Cat#85178
Acetonirile, mass spectrometry grade	Sigma-Aldrich	Cat#34851

Critical commercial assays

Mouse oestradiol ELISA kit	Mlbio	Cat#ml001962
Mouse testosterone ELISA kit	Mlbio	Cat#ml001948
Mouse LH ELISA kit	Cloud-clone	Cat#CEA441Mu
Mouse FSH ELISA kit	Cloud-clone	Cat#CEA830Mu
Mouse estrone ELISA kit	Mlbio	Cat#ml266215
Mouse androgen ELISA kit	Mlbio	Cat#ml001954
BCA Protein Assay Kit	Thermo Fisher Scientific	Cat#23225
Muta-Direct Kit	SBS Genetech	Cat#SDM-15
Luciferase assay system	Beyotime	Cat#RG027
Total RNA Purification Kit	EZBioscience	Cat#B0004-plus
HiScript III RT SuperMix	Vazyme	Cat#R312-01
SYBR qPCR Master Mix	Vazyme	Cat#Q111-02
Lysosome isolation Kit for mammalian Cells/Tissues	Invent	Cat#LY-034

Experimental models: Cell lines

LNCaP clone FGC	ATCC	Cat# CRL-1740
HEK293T	ATCC	Cat#CRL-3216
KGN	BIUEFBIO	Cat#BFN60805961
GRM02	BIUEFBIO	Cat#BFN60810498

Experimental models: Organisms/strains

Mouse: ICR strain	Jiangsu ALF Biotechnology Company	N/A

Oligonucleotides

qPCR primers for mouse Cyp19a1: forward: 5′-AACCCCATGCAGTATAATGTCAC-3′ reverse: 5′-AGGACCTGGTATTGAAGACGAG-3′	Harvard Primer Bank	156139071c1
qPCR primers for mouse Gapdh: forward: 5′- AGGTCGGTGTGAACGGATTTG-3′ reverse: 5′-TGTAGACCATGTAGTTGAGGTCA-3′	Harvard Primer Bank	126012538c1
qPCR primers for human CYP19A1: forward: 5′-TCCTATCAGGACGGAAGGTCC-3′ reverse: 5′-GTTCCTTGACCTCAGAGGGG-3′	This paper	N/A
qPCR primers for mouse GAPDH: forward: 5′-GGGAAGCTTGTCATCAATGGAA-3′ reverse: 5′-AGAGATGATGACCCTTTTGGCTC-3′	This paper	N/A

Recombinant DNA

pcDNA3.1-3×Flag-mHsp90αα1-WT	This paper	N/A
pcDNA3.1-3×Flag-mHsp90αα1-C598S	This paper	N/A
pcDNA3.1-3×Flag-mHsp90αα1-C599S	This paper	N/A
pcDNA3.1-3×Flag-mHsp90αα1-C598,599S	This paper	N/A
pcDNA3.1-3×Flag-hHsp90αα1-WT	This paper	N/A
pcDNA3.1-3×Flag-hHsp90αα1-C597,598S	This paper	N/A
pcDNA3.1-3×HA-mLYPLA1	This paper	N/A
pcDNA3.1-3×HA-mLYPLA2	This paper	N/A
pcDNA3.1-3×HA-mABHD17A	This paper	N/A
pcDNA3.1-3×HA-mABHD17B	This paper	N/A
pcDNA3.1-3×HA-mABHD17C	This paper	N/A
pcDNA3.1-3×HA-mPPT1	This paper	N/A
pARE2-TATA-Luc	Moilanen et al., 1998⁵⁰	N/A

Software and algorithms

ImageCal version1.10	Tanon	http://www.biotanon.com
Prism 8.0	GraphPad	http://www.graphpad.com
SPSS version 21.0	IBM	https://www.ibm.com/cn-zh/analytics/spss-statistics-software
ImageJ	NIH	https://imagej.nih.gov/ij
The mass spectrometry proteomics data	Proteome Xchange Consortium (http://proteomecentral.proteomexchange.org)	The dataset identifier: PXD028835

Other

FuturePAGE™ 4–20% 12 wells	ACE Biotechnology	Cat#ET12420GEL
FuturePAGE™ 4–20% 15 wells	ACE Biotechnology	Cat#ET15420GEL
WB & IP lysis buffer	Beyotime	Cat# P0013
RIPA lysis buffer	Sigma-Aldrich	Cat#R0278
Protein A/G beads	Thermo Fisher Scientific	Cat#88847
Anti-Flag beads	Sigma-Aldrich	Cat#M8823
Streptavidin agarose resin	Thermo Fisher Scientific	Cat#20347
DMEM	Gibco	Cat#11995020
RPMI-1640	Gibco	Cat#61870036
Opti-MEM	Gibco	Cat#31980070
DMEM-F12	BasalMedia	Cat#L310KJ
FBS	Gibco	Cat#A3161002C
Lipofectamine 3000	Invitrogen	Cat#L3000015
PBS	GENOM	Cat#GNM10010
M2 medium	Sigma-Aldrich	Cat#M7167
2 × SDS loading buffer	FDbio	Cat#FD003
5 × SDS loading buffer	FDbio	Cat#FD002
PVDF membrane	Bio-Rad	Cat#1620177
Western antibody diluent	NCMbiotech	Cat#WB500D