Figures and data in PARP1 inhibitors trigger innate immunity via PARP1 trapping-induced DNA damage response

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4 figures, 1 table and 4 additional files

Figures

Figure 1 with 2 supplements

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PARPi induces the innate immune response via the cGAS-STING pathway.

(A) The level of trapped PARP1 in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ^***p < 0.001. (B) Staining of cytosolic dsDNA in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Left, representative image of PicoGreen (green) staining. DAPI (blue) was used to visualize the nucleus. Scale bars represent 10 μm. Right, the graph shows the quantification of the number of cells with cytosolic dsDNA. Values were presented as means ± SEM from three biological replicates (n = 3 fields,≥100 cells counted per condition). Significance was determined with unpaired Student’s t-test. ^****p < 0.0001. (C) The extent of DNA damage in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of γH2AX levels. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ^**p < 0.01. (D) The level of pS172 TBK1 in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of pS172 TBK1 levels. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ^**p < 0.01. (E) The level of pS396 IRF3 in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Left, representative image of pS396 IRF3 levels (green). DAPI (blue) was used to visualize the nucleus. Scale bars represent 20 μm. Right, the graph shows the quantification of the number of cells stained positive for pS396 IRF3 in nucleus. Values were presented as means ± SEM from three biological replicates (n = 3 fields,≥100 cells counted per condition). Significance was determined with unpaired Student’s t-test. ^***p < 0.001. (F) RT-qPCR of type I interferons levels in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Values of *Inf-α* and *Inf-β* were presented as means ± SEM from three biological replicates. Significance was determined with unpaired Student’s t-test. ^*p < 0.05, ^**p < 0.01. (G) Knock-down of cGAS. HeLa cells expressing the control shRNA (shScramble) or shcGAS (shcGAS #1 or #2) were probed using the indicated antibodies. Right, the graph shows the ratio of cGAS depletion. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^***p < 0.001. (H) Depletion of cGAS abolishes PARPi-induced activation of innate immune signaling. HeLa cells expressing shRNA against control (shScramble) or cGAS (shcGAS #1 or #2) were treated with or without Talazoparib (10 µM for 72 hr). The cells were lysed and were immunoblotted using the indicated antibodies. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^****p < 0.0001, n.s., not significant. (I) RT-qPCR analyses of type I interferons. HeLa cells expressing shRNA against control (shScramble) or cGAS (shcGAS #1 or #2) were treated with or without Talazoparib (10 µM for 72 hr). Values of *Inf-α* and *Inf-β* mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with unpaired Student’s t-test. ^****p < 0.0001, n.s., not significant.

Figure 1—figure supplement 1

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Proteomic profiling of PARPi-induced innate immune response (Part 1).

(A) RT-qPCR analyses of cGAS-STING target gene expression in HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Values of cytokines and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with unpaired Student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. (B) The levels of trapped PARP1 in HeLa cells expressing shRNA against control (shScramble) or cGAS (shcGAS #1 or #2) that were treated with or without Talazoparib (10 µM for 72 hr). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the levels of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^***p < 0.001, n.s., not significant. (C) The extent of DNA damage in HeLa cells expressing shRNA against control (shScramble) or cGAS (shcGAS #1 or #2) that were treated with or without Talazoparib (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of γH2AX levels. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^***p < 0.001, n.s., not significant. (D) RT-qPCR analyses of cGAS-STING target gene expression in HeLa cells expressing shRNA against control (shScramble) or cGAS (shcGAS #1 or #2) that were treated with or without Talazoparib (10 µM for 72 hr). Values of cytokines and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ^***p < 0.001, ^****p < 0.0001, n.s., not significant.

Figure 1—figure supplement 2

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Proteomic profiling of PARPi-induced innate immune response (Part 2).

(E) The levels of trapped PARP1 in MHH-ES-1 cells treated with or without Talazoparib (1 µM for 24 hr). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ^**p < 0.01. (F) The extent of DNA damage in MHH-ES-1 cells treated with or without Talazoparib (1 µM for 24 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of γH2AX levels. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ^**p < 0.01. (G) The level of pS172 TBK1 in MHH-ES-1 cells treated with or without Talazoparib (1 µM for 24 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of pS172 TBK1 levels. Values were presented as means ± SD from three biological replicates. Significance was determined with unpaired Student’s t-test. ^**p < 0.01. (H) Reproducibility of the TMT experiments. The signal-to-noise (SN) values of the corresponding TMT channels for each protein was extracted from the two biological replicate experiments. (I) Quantification of protein expression in MHH-ES-1 cells treated with Talazoparib 1 µM for 24 hr (Supplementary file 1). Top, the graph shows the log₂ value of total protein expression in Talazoparib-treated vs. DMSO control. Bottom, the heatmap shows quantification reproducibility of the up- and down-regulated protein. Red: up-regulated proteins; Green: down-regulated proteins. (J) GO analysis of the up-regulated proteins as shown in (I). The list shows the top 10 enriched biological processes of the up-regulated proteins. (K) RT-qPCR analyses of the cGAS-STING target gene expression in MHH-ES-1 cells treated with or without Talazoparib (1 µM for 24 hr). Values of type I interferons, cytokines, and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with unpaired Student’s t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

Figure 2 with 1 supplement

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The PARP1 protein is required for PARPi-induced innate immune signaling.

(A) PARPi-induced PARP1 trapping in wild-type (WT) and PARP1 knockout (KO) HeLa cells. Cell were also treated with or without Talazoparib (10 µM for 72 hr). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^****p < 0.001, n.s., not significant. (B) DDR in WT and PARP1 KO HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of γH2AX levels. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^***p < 0.001, n.s., not significant. (C) The level of pS172 TBK1 in WT and PARP1 KO HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of pS172 TBK1 levels. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^****p < 0.0001, n.s., not significant. (D) Staining of pS396 IRF3 levels in WT and PARP1 KO HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Left, a representative image of pS396 IRF3 levels (green). DAPI (blue) was used to visualize the nucleus. Right, the graph shows the quantification of the number of cells stained positive for pS396 IRF3 in the nucleus. Values were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ^****p < 0.0001, n.s., not significant. (E) RT-qPCR analyses of type I interferons in WT and PARP1 KO HeLa cells treated with or without Talazoparib (10 µM for 72 hr). Values of *Inf-α* and *Inf-β* mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ^*p < 0.05, ^**p < 0.01, n.s., not significant.

Figure 2—figure supplement 1

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The PARP1 protein is required for PARPi-induced innate immune response.

(A) The level of PARP1 in WT and PARP1 KO HeLa cells. Whole cell lysates were probed using the indicated antibodies. GAPDH used as the loading control. (B) RT-qPCR analyses of cGAS-STING target gene expression in WT and PARP1 KO HeLa cells that were treated with or without Talazoparib (10 µM for 72 hr). Values of cytokines and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ^**p < 0.01, ^***p < 0.001, ^****p < 0.001, n.s., not significant.

Figure 3 with 1 supplement

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PARP1 trapping is the major contributor of PARPi-induced innate immune signaling.

(A) The level of trapped PARP1 in HeLa cells treated with or without the indicated PARPi (10 µM for 72 hr). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^*p < 0.05, ^***p < 0.001. (B) The extent of DNA damage in HeLa cells treated with or without the indicated PARPi (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of γH2AX levels. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^**p < 0.01, ^***p < 0.001. (C) The level of pS172 TBK1 in HeLa cells treated with or without the indicated PARPi (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of pS172 TBK1 levels. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^*p < 0.05, ^***p < 0.001. (D) Heatmap of PARP1 trapping, DNA damage, and pS172 TBK1 levels for each PARPi. The normalized levels of PARP1 trapping (A), γH2AX (B), and pS172 TBK1 (C) are shown. (E) PARPi does not induce the trapping of a PARP1 mutant with defective DNA binding. Top, HeLa PARP1 KO cells expressing WT PARP1 or R138C mutant PARP1 (R138C) were treated with or without Talazoparib (10 µM for 72 hr). Chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the levels of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^**p < 0.01, ^***p < 0.001. (F) The extent of DNA damage in HeLa PARP1 KO cells expressing WT PARP1 or R138C PARP1 that were treated with or without Talazoparib (10 µM for 72 hr). Left, whole cell lysates were probed using the indicated antibodies. Right, the graph shows the quantification of γH2AX and pS172 TBK1 levels. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ^**p < 0.01, ^***p < 0.001. (G) RT-qPCR analyses of type I interferons in HeLa PARP1 KO cells expressing WT or R138C PARP1 that were treated with or without Talazoparib (10 µM for 72 hr). Values of *Inf-α* and *Inf-β* mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ^**p < 0.01, ^***p < 0.001.

Figure 3—figure supplement 1

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PARP1 trapping is required for PARPi-induced cytotoxicity and innate immune response.

(A) The levels of PAR signals in HeLa cells treated with the indicated PARPi (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of PAR levels. GAPDH was used as the loading control. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^***p < 0.001. (B) RT-qPCR analyses of cGAS-STING target gene expression in HeLa cells treated with Talazoparib or Rucaparib (10 µM for 72 hr). Values of type I interferons, cytokines, and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. (C) RT-qPCR analyses of cGAS-STING target gene expression in HeLa PARP1 KO cells expressing PARP1 WT or R138C that were treated with or without Talazoparib (10 µM for 72 hr). Values of cytokines and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ^**p < 0.01, ^***p < 0.001. (D) Cell death analyses. MHH-ES-1 cells were treated with the PARP inhibitors as indicated (1 µM for 24 hr). Cell viability was measured by CellTiter-Glo assays. Cell survival is presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ^***p < 0.001, ^****p < 0.0001.

Figure 4 with 1 supplement

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PARP1 degraders abolish PARP1-trapping induced innate immune signaling.

(A) The level of PARP1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 µM for 72 hr). Whole cell lysates were probed using the indicated antibodies. GAPDH was used as the loading control. (B) The level of trapped PARP1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 µM for 72 hr). Top, chromatin-bound fractions were isolated and were probed using the indicated antibodies. Histone H3 was used as the loading control. Bottom, the graph shows the quantification of the level of PARP1 trapping. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^***p < 0.001. (C) The extent of DNA damage in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of γH2AX levels. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^***p < 0.001. (D) The level of pS172 TBK1 in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 µM for 72 hr). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of pS172 TBK1 levels. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ^**p < 0.01. (E) RT-qPCR analyses of the cGAS-STING target gene expression in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (10 µM for 72 hr). Values of type I interferons, cytokines, and ISGs mRNA levels were presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. (F) The model of the activation of innate immune response via PARPi-induced PARP1 trapping.

Figure 4—figure supplement 1

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PARP1 degraders prevent PARP1 trapping-induced cytotoxicity.

(A) Cell death analyses. MHH-ES-1 cells were treated with Rucaparib or iRucaparib-AP6 as indicated (1 µM for 24 hr). Cell viability was measured by CellTiter-Glo assays. Cell survival is presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ^***p < 0.001, ^****p < 0.0001.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Antibody	Anti-PAR (Mouse monoclonal)	Trevigen	Cat# 4335-MC-100, RRID:AB_2572318	IB (1:3000)
Antibody	Anti-PARP1 (Rabbit polyclonal)	Cell Signaling Technology	Cat# 9542, RRID:AB_2160739	IB (1:1000)
Antibody	Anti-γH2AX (Rabbit monoclonal)	Cell Signaling Technology	Cat# 9718, RRID:AB_2118009	IB (1:3000)
Antibody	Anti-Histone H3 (Rabbit monoclonal)	Cell Signaling Technology	Cat# 4499, RRID:AB_10544537	IB (1:3000)
Antibody	Anti-GAPDH (Mouse monoclonal)	Santa Cruz Biotechnology	Cat# sc-32233, RRID:AB_627679	IB (1:5000)
Antibody	Anti-Phospho-TBK1/NAK (pS172 TBK1) (Rabbit monoclonal)	Cell Signaling Technology	Cat# 5483, RRID:AB_10693472	IB (1:1000)
Antibody	Anti-TBK1/NAK (Rabbit monoclonal)	Cell Signaling Technology	Cat# 3504, RRID:AB_2255663	IB (1:1000)
Antibody	Anti-Flag (Rabbit polyclonal)	MilliporeSigma	Cat# F7425, RRID:AB_439687	IB (1:1000)
Antibody	Anti-cGAS (Rabbit monoclonal)	Cell Signaling Technology	Cat# 15102, RRID:AB_2732795	IB (1:1000)
Antibody	Anti-Phospho-IRF-3 (pS396 IRF3) (Rabbit monoclonal)	Cell Signaling Technology	Cat# 4947, RRID:AB_823547	IF (1:1000)
Antibody	Alexa Fluor 488-conjugated goat anti-rabbit IgG (Goat polyclonal)	Thermo Fisher Scientific	Cat# A32731, RRID:AB_2633280	IF (1:1000)
Antibody	Goat Anti-Mouse IgG Antibody, HRP conjugate, Species Adsorbed (Goat polyclonal)	MilliporeSigma	Cat# AP181P, RRID:AB_11214094	IB (1:3000)
Antibody	ECL Rabbit IgG, HRP-linked fragment (Donkey polyclonal)	GE Healthcare life sciences	Cat# NA9340, RRID:AB_772191	IB (1:3000)
Chemical compound, drug	Talazoparib	Selleck	Cat# S7048	PARP1 inhibitor
Chemical compound, drug	Niraparib	Selleck	Cat# S2741	PARP1 inhibitor
Chemical compound, drug	Rucaparib	Selleck	Cat# S1098	PARP1 inhibitor
Chemical compound, drug	Olaparib	Selleck	Cat# S1060	PARP1 inhibitor
Chemical compound, drug	Veliparib	Selleck	Cat# S1004	PARP1 inhibitor
Chemical compound, drug	iRucaparib-AP6	Our laboratory	N/A	PARP1 degrader
Chemical compound, drug	Polybrene (Hexadimethrine bromide)	MilliporeSigma	Cat# H9268; CAS 28728-55-4
Chemical compound, drug	Puromycin	MilliporeSigma	Cat# P7255; CAS 58-58-2
Cell line (human)	HeLa	ATCC	Cat# CCL-2, RRID:CVCL_0030
Cell line (human)	HeLa PARP1 KO	In this study	N/A	PARP1 deficient HeLa
Cell line (human)	MHH-ES-1	DSMZ	Cat# ACC 167, RRID:CVCL_1411
Cell line (human)	HeLa	ATCC	Cat# CCL-2
Recombinant DNA reagent	pCMV-hPARP1-3xFlag-WT	Addgene	Cat# 11157	In pCMV; tagged with 3XFlag on its N-terminus
Recombinant DNA reagent	pCMV-hPARP1-3xFlag-R138C	In this study	Modified by R138C mutation	In pCMV; tagged with 3XFlag on its N-terminus
Strain, strain background (Escherichia coli)	DH5alpha	Thermo Fisher Scientific	Cat# 18258012	Competent cells
Strain, strain background (Escherichia coli)	Stbl3 Competent E. coli	Thermo Fisher Scientific	Cat# C737303	Competent cells
Commercial assay or kit	Quant-iT PicoGreen dsDNA Reagent	Thermo Fisher Scientific	Cat# P7581
Commercial assay or kit	Subcellular Protein Fractionation Kit for Cultured Cells	Thermo Fisher Scientific	Cat# 78840
Commercial assay or kit	RNeasy Mini Kit	QIAGEN	Cat# 74104
Commercial assay or kit	QIAprep Spin Miniprep Kit	QIAGEN	Cat# 27106
Commercial assay or kit	e-Myco PLUS Mycoplasma PCR Detection Kit	BOCA SCIENTIFIC	Cat# 25237
Commercial assay or kit	TMT6plex Mass Tag Labeling Kits	Thermo Fisher Scientific	Cat# 90110
Commercial assay or kit	CellTiter-Glo Luminescent Cell Viability Assay	Promega	Cat# G7571
Software, algorithm	ImageJ 1.49 v	NIH	https://imagej.net/ImageJ2
Software, algorithm	DAVID Bioinformatics Resources v6.8	DAVID	https://david.ncifcrf.gov/
Software, algorithm	PRISM v8.2.0	GraphPad	https://www.graphpad.com/scientific-software/prism/
Software, algorithm	human protein sequences (Uniprot)	UniProt	https://www.uniprot.org/UniProt database (2019_07,560,537 sequences;taxonomy, Homo sapiens, 20,431 )
Software, algorithm	human IPI protein database v3.60	EMBL-EBI	ftp://ftp.ebi.ac.uk/pub/databases/IPI
Software, algorithm	The Sequest algorithm v28	Cell. 2010 Dec 3;143(7):1174–89	N/A
Others	DAPI	MilliporeSigma	Cat# D9542
Others	Dimethyl sulfoxide (DMSO)	Thermo Fisher Scientific	Cat# BP231-1; CAS 67-68-5
Others	Lipofectamine 2000	Thermo Fisher Scientific	Cat# 11668500
Others	Dulbecco’s Modified Eagle’s Medium (DMEM)	MilliporeSigma	Cat# D5796
Others	RPMI1640	MilliporeSigma	Cat# R8758
Others	Fetal Bovine Serum (FBS)	MilliporeSigma	Cat# 12303C
Others	Lysyl Endopeptidase (Lys-C)	Wako	Cat# 129–02541; CAS 123175-82-6
Others	Gen5	BioTek	N/A
Others	BCA reagents	Thermo Fisher Scientific	Cat# 23224/23228
Others	0.45 μm filter	Thermo Fisher Scientific	Cat# 05-713-387
Others	Synergy HT Multi-Detection Microplate Reader.	BioTek	N/A
Others	SuperScript III Reverse Transcriptase	Thermo Fisher Scientific	Cat# 18080044
Others	CFX384 Touch Real-Time PCR Detection System	Bio-Rad	Cat# 1855484
Others	Applied Biosystems Power SYBR Green PCR Master Mix	Thermo Fisher Scientific	Cat# 43-676-59
Others	Oasis HLB solid-phase extraction (SPE) cartridges	Waters	Cat# 186000383
Others	3M Empore C18 Extraction Disk	Thermo Fisher Scientific	Cat# 14-386-2
Others	ZORBAX 300Extend-C18 HPLC column	Agilent	Cat# 761775–902
Others	Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer	Thermo Fisher Scientific	Cat# IQLAAEGAAPFALGMAZR
Others	PicoFrit nanospray columns	New Objective	PF360-75-15-N-5