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Myd88

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Myd88 is a protein that plays a key role in the innate immune response. It functions as an adapter protein, facilitating the activation of signaling pathways in response to the detection of pathogens or other stimuli. Myd88 is involved in the activation of transcription factors, such as NF-κB, which regulate the expression of genes involved in the inflammatory response.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (2 mentions) Superscript 3, by Thermo Fisher Scientific (2 mentions) Confocal dishes, by MatTek (2 mentions) Lamp1, by Thermo Fisher Scientific (2 mentions) Antibodies to mouse tlr7, by Thermo Fisher Scientific (2 mentions) Alexfluor550, by Thermo Fisher Scientific (2 mentions) Confocal microscope, by Nikon (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Rq manager software v2, by Thermo Fisher Scientific (1 mentions) Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) 7900ht real time pcr system, by Thermo Fisher Scientific (1 mentions) 18s rrna, by Thermo Fisher Scientific (1 mentions) Phospho sapk jnk, by Cell Signaling Technology (1 mentions) Dulbecco s modified eagle s medium dmem, by Solarbio (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Sapk jnk, by Cell Signaling Technology (1 mentions) Primescripttm rt reagent kit with gdna eraser kit, by Takara Bio (1 mentions) Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)

10 protocols using myd88

1

Quantitative RT-PCR for PML-RARA and MyD88

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The RQ-PCR method using Power SYBR Green for ORFs, PML-RARA and MyD88 (Applied Biosystems) and primer sequences used are detailed in Supplementary Table S2. The human PML-RARA clone [47 (link)] and the mouse MyD88 cloned by our laboratory were used to generate the standard curves. The reference gene used was mouse Abl, which was cloned by our laboratory. Target genes were ran for each sample in triplicate. In order to quantify the samples, a standard curve was software generated according to the Ct obtained with the diluted plasmids. The copy number of the gene of interest for each sample was determined as a function of the detected fluorescence extrapolated from the standard curve. The pVAX14+ATRA-treated group was stratified according to survival below or above the median of individual protocols.
Le Pogam C., Patel S., Gorombei P., Guerenne L., Krief P., Omidvar N., Tekin N., Bernasconi E., Sicre F., Schlageter M.H., Chopin M., Noguera M.E., West R., Abu A., Mathews V., Pla M., Fenaux P., Chomienne C, & Padua R.A. (2015). DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies. Oncotarget, 6(32), 32494-32508.
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2

Myeloid Cell Isolation and Gene Expression

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Myeloid cells were isolated from the spleens of SPF and CoH mice by plastic adherence (Inaba et al., 2009 ; Steinman et al., 1979 ). Spleens (5 SPF and 5 CoH) were prepared into a single cell suspension in 2 mL HBSS, washed, and resuspended in 10 mL of complete RPMI. Each cell suspension was added to two 10 cm culture dishes and incubated at37°Cfor90 min. Plates were washed twice with 5 mL PBS to remove nonadherent cells. Total RNA was isolated from the remaining adherent myeloid cells using TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 mg was reverse-transcribed using Superscript III (Invitrogen). Resulting cDNA was used as a template for qPCR using TaqMan primer/probe sets for Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, Tlr9, Cd14, Ly96, Myd88, Ticam2, Traf6, Iraki, and 18 s rRNA (Applied Biosystems).
Huggins M.A., Sjaastad F.V., Pierson M., Kucaba T.A., Swanson W., Staley C., Weingarden A.R., Jensen I.J., Danahy D.B., Badovinac V.P., Jameson S.C., Vezys V., Masopust D., Khoruts A., Griffith T.S, & Hamilton S.E. (2019). Microbial Exposure Enhances Immunity to Pathogens Recognized by TLR2 but Increases Susceptibility to Cytokine Storm through TLR4 Sensitization. Cell reports, 28(7), 1729-1743.e5.
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3

Quantifying Pattern Recognition Receptor mRNA

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cDNA was generated from 1 μg of isolated RNA from ectocervical, endocervical, and vaginal cells using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific). qPCR was performed on the 7900HT Real-Time PCR System (Applied Biosystems) using the TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturers’ protocols. The standard curve method was used for relative expression quantification using the RQ manager software v2.4 (Applied Biosystems). The relative abundance of the target of interest was divided by the relative abundance of 18S in each sample to generate a standardized abundance for the target transcript of interest. All mRNA primers were purchased from Applied Biosystems: TLR2, MYD88, NOD1, NOD2, and 18S.
Anton L., Ferguson B., Friedman E.S., Gerson K.D., Brown A.G, & Elovitz M.A. (2022). Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses. Microbiome, 10, 119.
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4

Isolation and Gene Expression Analysis of Myeloid Cells

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Splenocytes were cultured overnight in complete RPMI at 37°C 5% CO2 to permit the isolation of myeloid cells by plastic adherence (10 , 11 (link)). Non-plastic adherent cells were washed away with 2 washes of 5 mL PBS. Total RNA was isolated from the adherent myeloid cells using TRIzol reagent (Invitrogen, Carlsbad, CA), and 1 μg RNA was reverse transcribed into cDNA using random hexamers and Superscript III (Invitrogen, Carlsbad, CA). The resulting cDNA was used as template for qPCR using TaqMan primer/probe sets for Tlr1, Tlr2, Tlr3, Tlr4, Tlr6, Cd14, Myd88, and 18s rRNA (Applied Biosystems).
Zani S., Mellon M.T., Collier J.L, & Zehr J.P. (2000). Expression of nifH Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR. Applied and Environmental Microbiology, 66(7), 3119-3124.
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5

Intracellular Localization of TLR7 Signaling Components in BMDCs

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BMDCs were seeded in confocal dishes (Mattek) at a final concentration of 1×105 cells/mL in RPMI-complete media. The following day, cells were treated with 10 μg/mL GRD for 5 minutes and 15 minutes at 37C. Cells were carefully washed with PBS and fixed with 2% PFA for 10 minutes at room temperature in the dark. Fixed cells were permeabilized with SAP buffer (2% saponin in PBS) for 1 hour at 37C. Following permeabilization, BMDCs were intracellularly stained with antibodies to mouse TLR7, MyD88, EEA1, or LAMP1 (Thermo Scientific), followed by staining with secondary antibodies fluorescently labeled with AlexaFluor488 or AlexFluor550 (Invitrogen). Cells were washed 3 times with PBS. Finally, DNA was stained with 1:1000 dilution of DAPI (Invitrogen) for 10 minutes at RT. Cells were visualized using a Nikon confocal microscope and analysis performed using ZEN software. Data analysis and processing of images was conducted using ImageJ software (NIH).
Ramirez-Ortiz Z.G., Prasad A., Griffith J.W., Pendergraft WF I.I.I., Cowley G.S., Root D.E., Tai M., Luster A.D., Khoury J.E., Hacohen N, & Means T.K. (2015). TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity. Nature immunology, 16(5), 495-504.
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6

Exploring anti-inflammatory mechanisms in vitro

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Dulbecco’s Modified Eagle’s Medium (DMEM) and neutral red were purchased from Solarbio (Beijing, China). Fetal Bovine Serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Nitric oxide kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α and IL-6 ELISA kit were obtained from Beijing 4A Biotech Co, Ltd (Beijing, China). Primer iNOS, TNF-α, IL-6, TLR4 and MyD88 were purchased from Thermo Fisher scientific (Shanghai, China). Reactive Oxygen Species Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). PrimeScriptTMRT reagent kit with gDNA Eraser kit and TB Green TM Ex TaqTM II (Tli RNadeH Plus) and Bulk kit were purchased from TaKaRa. Antibody NF-κB p65, phospho-NF-κB p65, p38 MAPK, phospho-p38 MAPK, SAPK/JNK, phospho-SAPK/JNK, p44/p22 MAPK and phospho-p44/p22 MAPK were purchased from Cell Signaling (Beverly, MA, USA). NF-κB inhibitor (PDTC), ERK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580) Lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO, USA)
Wang H., Xu X., Yin Z., Wang M., Wang B., Ma C., Wang J, & Kang W. (2021). Activation of RAW264.7 cells by PCp-I, a polysaccharide from Psoralea corylifolia L, through NF-κB/MAPK signalling pathway. International Journal of Immunopathology and Pharmacology, 35, 20587384211010058.
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7

Intracellular Localization of TLR7 Signaling Components in BMDCs

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BMDCs were seeded in confocal dishes (Mattek) at a final concentration of 1×105 cells/mL in RPMI-complete media. The following day, cells were treated with 10 μg/mL GRD for 5 minutes and 15 minutes at 37C. Cells were carefully washed with PBS and fixed with 2% PFA for 10 minutes at room temperature in the dark. Fixed cells were permeabilized with SAP buffer (2% saponin in PBS) for 1 hour at 37C. Following permeabilization, BMDCs were intracellularly stained with antibodies to mouse TLR7, MyD88, EEA1, or LAMP1 (Thermo Scientific), followed by staining with secondary antibodies fluorescently labeled with AlexaFluor488 or AlexFluor550 (Invitrogen). Cells were washed 3 times with PBS. Finally, DNA was stained with 1:1000 dilution of DAPI (Invitrogen) for 10 minutes at RT. Cells were visualized using a Nikon confocal microscope and analysis performed using ZEN software. Data analysis and processing of images was conducted using ImageJ software (NIH).
Ramirez-Ortiz Z.G., Prasad A., Griffith J.W., Pendergraft WF I.I.I., Cowley G.S., Root D.E., Tai M., Luster A.D., Khoury J.E., Hacohen N, & Means T.K. (2015). TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity. Nature immunology, 16(5), 495-504.
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8

Western Blot Analysis of NF-κB Signaling Pathway

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Proteins (50 μg) from all sample groups were processed as previously described [40 (link)]. Sheets were incubated 12 h at 4°C in with primary antibodies to NFκB (1 : 500, Santa Cruz Biotechnology), MyD88 (1 : 1000, ThermoFisher Scientific), p300 (1 : 750, EpiGentek), DNMT1 (1 : 750, OriGene), and β-actin (1 : 1000, Santa Cruz Biotechnology) [41 (link), 42 (link)]. Then, sheets were maintained at room temperature for 30 min with peroxidase-conjugated secondary antibody diluted 1 : 1000 in 1x TBS, 5% milk, and 0.05% Tween-20 [43 (link)]. The ECL method was used for band visualization, and the protein level were measured by means of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) [44 (link)].
Marconi G.D., Fonticoli L., Guarnieri S., Cavalcanti M.F., Franchi S., Gatta V., Trubiani O., Pizzicannella J, & Diomede F. (2021). Ascorbic Acid: A New Player of Epigenetic Regulation in LPS-gingivalis Treated Human Periodontal Ligament Stem Cells. Oxidative Medicine and Cellular Longevity, 2021, 6679708.
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9

miRNA, siRNA, and Luciferase Transfection

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Indicated concentration of miRNAs, siRNAs and luciferase plasmids were transfected with either Lipofectamine RNAiMAX (Invitrogen) or Attractene transfectant (Qiagen) (see supplement). The synthetic miRNAs (mimic) of has-miR-1248 and All Star negative control miRNA (miR-N.C.) were from Qiagen. The siRNAs of IFNAR1, IFNAR2, AGO2, DAK, HMGB1, MAVS, MYD88, LGP2, RIG-1, ITPR3 were obtained from Ambion. Firefly and renilla luciferase activities were determined by Dual-Luciferase Reagent Assay System (Promega) with Fluostar Omega microplate reader (BMG Labtech). Firefly luciferase activity was normalized with renilla luciferase activity and expressed as fold change over control (see supplement).
Jang S.I., Tandon M., Teos L., Zheng C., Warner B.M, & Alevizos I. (2019). Dual function of miR-1248 links interferon induction and calcium signaling defects in Sjögren's syndrome. EBioMedicine, 48, 526-538.
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10

Investigating siRNA-mediated Knockdown in PAECs

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ON TARGETplus SMARTpool siRNA (Horizon), Silencer Select siRNA (Thermo Fisher Scientific), and Gene Silencer (Santa Cruz Biotechnology) (see list below) were transfected into PAECs by the 4D Nucleofector Core unit using p5 Primary Cell Solution according to manufacturer’s recommendations (Lonza). The knockdown efficiency was determined by qPCR and immunoblotting. PAECs transfected with siRNA were incubated for 48 hours, and then treated with 10 μg/mL of HERV-K dUTPase for 72 hours.
siRNAs used for transfection: TLR4 (Ambion, catalog s14194); TLR2 (Ambion, catalog s162); TLR6 (Ambion, catalog s20215); MYD88 (Ambion, catalog s9134); NRP1 (Dharmacon, catalog L019484); ENG (Dharmacon, catalog L011026); MCAM (Ambion, catalog s8572); RELA (Ambion, catalog s11915); SMAD3 (Ambion, catalog s535079); STAT1 (Ambion, catalog s279); ATF2 (Santa Cruz, catalog sc29205); Negative Control No.1 (Ambion, catalog 4390844); Nontargeting Control (Dharmacon, catalog D001810); and Control siRNA-A (Santa Cruz, catalog Sc37007).
Otsuki S., Saito T., Taylor S., Li D., Moonen J.R., Marciano D.P., Harper R.L., Cao A., Wang L., Ariza M.E, & Rabinovitch M. (2021). Monocyte-released HERV-K dUTPase engages TLR4 and MCAM causing endothelial mesenchymal transition. JCI Insight, 6(15), e146416.
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