Supplentmental Materials

Supplentmental materials

1. Gastrointestinal transit (GI transit)

Gastrointestinal transit was measured 24 hours after the operationusing standard methods as described previously.Briefly, mice transduced withHuR-RNAi lentivirus or recevied surgery were gavaged with 250 smg/ 10 mL/kg of fluorescein-labeled dextran of 70 000 MW (FD70; Sigma-Aldrich, St. Louis, MO, USA). After 30 min, mice were euthanized by decapitation and the The transit of fluorescein-labeled dextran along the gastrointestinal tract was immediately excised. Fluorescence was visualized and quantified using the G-box system (Syngene, Cambridge,UK) 24 and the geometric center was calculated using the formula:GC =Σ(% FD70 per segment 9 segment number)/100.

2.Immunohistochemistry

Paraffin-embedded specimenswere deparaffinized in xylene, subjected to heat-mediated antigen-retrieval in10mM sodium citrate (pH 6.0), permeabilized in 0.2% Triton X-100 (Sigma),treated with mouse on mouse blocking reagent (Vector Laboratories) and blockedin 5% donkey sera. The mast cells, resident macrophages, neutrophils, and monocyte-derived macrophageswas detected using a mouse monoclonal, and Anti-mast cell chymase (ab186417)Anti-CD163 antibody (ab199402) (Abcam, U.K.) MPO (22225-1-AP) and CD68 (25747-1-AP) rabbit polyclonal antibodies were purchased from Wuhan Proteintech (1:50). And an HRP-conjugated donkey anti-rabbitsecondary (1:250, Jackson Immunochemicals), amplified with AB reagent(Vectastain) and detected using DAB reagent (Thermo Scientific). Images wereacquired using a Zeiss Axioplan 2 microscope.

Thisinvestigationwasconductedonatotalof60

paraffin-embeddedNPCsamplesand10normalnasophar-

yngealmucosasamples.Allsamplesweretakenatthetime

ofdiagno sisforNPCbeforethetreatment.Paraffin-

embeddedtissueswerecutatathicknessof4μm.Tissue

sectionsweredeparaffinizedinxylene,rehydratedina

gradedseriesofethanolsolution,andwashedwith

phosphate-bufferedsaline(PBS)(pH7.4).Thedeparaffi-

nizedsectionswerethentreatedwith3%H

2

O

2

in

methanolfor30minutestoinactivateendogenous

peroxidases.Beforeincubationwithprimaryantibodies,

thesectionsweresubmittedtotreatmentinamicrowave

oven,usingthree5-minutecyclesat700Win10mmol/L

citricacid(pH6.0).Toreducenonspecificbinding,

sectionswereblockedwith3%bovineserumalbuminin

Thisinvestigationwasconductedonatotalof60

paraffin-embeddedNPCsamplesand10normalnasophar-

yngealmucosasamples.Allsamplesweretakenatthetime

ofdiagno sisforNPCbeforethetreatment.Paraffin-

embeddedtissueswerecutatathicknessof4μm.Tissue

sectionsweredeparaffinizedinxylene,rehydratedina

gradedseriesofethanolsolution,andwashedwith

phosphate-bufferedsaline(PBS)(pH7.4).Thedeparaffi-

nizedsectionswerethentreatedwith3%H

2

O

2

in

methanolfor30minutestoinactivateendogenous

peroxidases.Beforeincubationwithprimaryantibodies,

thesectionsweresubmittedtotreatmentinamicrowave

oven,usingthree5-minutecyclesat700Win10mmol/L

citricacid(pH6.0).Toreducenonspecificbinding,

sectionswereblockedwith3%bovineserumalbuminin

Thisinvestigationwasconductedonatotalof60

paraffin-embeddedNPCsamplesand10normalnasophar-

yngealmucosasamples.Allsamplesweretakenatthetime

ofdiagno sisforNPCbeforethetreatment.Paraffin-

embeddedtissueswerecutatathicknessof4μm.Tissue

sectionsweredeparaffinizedinxylene,rehydratedina

gradedseriesofethanolsolution,andwashedwith

phosphate-bufferedsaline(PBS)(pH7.4).Thedeparaffi-

nizedsectionswerethentreatedwith3%H

2

O

2

in

methanolfor30minutestoinactivateendogenous

peroxidases.Beforeincubationwithprimaryantibodies,

thesectionsweresubmittedtotreatmentinamicrowave

oven,usingthree5-minutecyclesat700Win10mmol/L

citricacid(pH6.0).Toreducenonspecificbinding,

sectionswereblockedwith3%bovineserumalbuminin

Thisinvestigationwasconductedonatotalof60

paraffin-embeddedNPCsamplesand10normalnasophar-

yngealmucosasamples.Allsamplesweretakenatthetime

ofdiagno sisforNPCbeforethetreatment.Paraffin-

embeddedtissueswerecutatathicknessof4μm.Tissue

sectionsweredeparaffinizedinxylene,rehydratedina

gradedseriesofethanolsolution,andwashedwith

phosphate-bufferedsaline(PBS)(pH7.4).Thedeparaffi-

nizedsectionswerethentreatedwith3%H

2

O

2

in

methanolfor30minutestoinactivateendogenous

peroxidases.Beforeincubationwithprimaryantibodies,

thesectionsweresubmittedtotreatmentinamicrowave

oven,usingthree5-minutecyclesat700Win10mmol/L

citricacid(pH6.0).Toreducenonspecificbinding,

sectionswereblockedwith3%bovineserumalbuminin

Table 1. Interference RNA sequences

Table 2. qRT-PCR primers