Supplementary Materials and Methods
Quantitative PCR probes
The cDNA samples were analyzed with specific probes (Applied Biosystems) to detect the
expression of murine filaggrin (flg, Cat# Mm01716522_m1), involucrin (IVl, Mm00515219_s1),
loricrin (Lor, Cat#Mm01219285_m1), fatty acid synthase (fas, Mm00662319_m1), HMG-CoA
reductase (HMGCR, Mm01282501_m1), serine palmitoyl transferase (SPtlc1,
Cat#Mm00447343_m1), beta defensin 1 (Defb1, Mm_00432803_m1), beta defensin 3 (Defb3,
Mm01614469_m1), beta defensin 14 (Defb14, Mm00806979_m1), cathelicidin-related
antimicrobial peptide (Cramp, Cat# Mm00438285_m1), hepcidin-1 (Hamp,
Cat#Mm00519025_m1), hepcidin-2 (Hamp2, Cat# Mm00842044_g1), psoriasin (S100a7a,
Mm02018201_m1), kallikrein-5 (Klk5, Mm01203811_m1), and kallikrein-7 (Klk7,
Mm01203811_m1), beta-actin (ACTB, 4352341E).
Immunohistochemistry
Routine staining methods were performed on mouse skin to assess burn depth. Skin was
embedded in OCT compound, stored at -80ºC, and subsequently sectioned to 6-8μm using a
cryostat. Routine H&E staining was performed (reagents from Fisher). Sections were examined
with a standard microscope using a 10x and 20x objective; digital pictures were taken. In
addition, Gomori Trichrome collagen staining (ThermoScientific) was performed to further
evaluate the depth of involvement. Sections were similarly examined with a standard microscope
using a 4x and 10x objective; digital pictures were taken. All samples were analyzed in a blinded
manner, and all experiments were performed in duplicate.
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Supplementary Figure Legends
Figure S1. Burn injury suppresses AMP production in bladder epithelium. Following burn
injury, bladder tissue at (A) 24 hours and (B) 96 hours post-injury were analyzed for the relative
gene expression of several antimicrobial peptides and a protease, including beta-defensin-1
(DefB1), beta-defensin-3 (DefB3), beta-defensin-14 (DefB14), cathelicidin-related antimicrobial
peptide (Cramp), hepcidin-1 (HAMP1), hepcidin-2 (HAMP2), and psoriasin (s100A7) and
kallikrein-7 (Klk7). N=5-6 samples/group, experiments performed in duplicate. Data analyzed by
Unpaired t test (*p=0.058, #p<0.05) and Mann-Whitney test ($p<0.05). (C) Bladder tissue was
stained by IHF techniques for hepcidin (HAMP1), 20x magnification. N=3-4 samples/group,
experiments performed in duplicate.
Figure S2. Burn injury alters AMP production in lung epithelium. Following burn injury,
lung tissue at (A) 24 hours and (B) 96 hours post-injury were analyzed for the relative gene
expression of several antimicrobial peptides and a protease, including beta-defensin-1 (DefB1),
beta-defensin-14 (DefB14), cathelicidin-related antimicrobial peptide (Cramp), hepcidin-1
(HAMP1), hepcidin-2 (HAMP2), and psoriasin (s100A7) and kallikrein-7 (Klk7). N=6
samples/group, experiments performed in duplicate. Data analyzed by Unpaired t test (#p<0.05)
and Mann-Whitney test (*p<0.05). (C) Lung tissue was stained by IHF techniques for
cathelicidin-related antimicrobial peptide (CRAMP), 40x magnification. N=3-4 samples/group,
experiments performed in duplicate.
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Figure S3. Burn wounds at multiple timepoints were compared using routine H&E
staining. Mice were subjected to a sham (~22°C water) or scald burn (~92-94°C water) for 6, 8,
or 10 seconds. Skin was harvested at 24 hours post-burn. Routine H&E staining was performed;
10x and 20x magnification, as indicated. N=3 samples/group, experiments performed in
duplicate.
Figure S4. A full thickness burn injury was achieved following a 10 second scald burn, as
verified using routine trichrome collagen staining. Mice were subjected to a sham (~22°C
water) or scald burn (~92-94°C water) for 6, 8, or 10 seconds. Skin was harvested at 24 hours
post-burn. Routine trichrome collagen staining was performed; 4x and 10x magnification, as
indicated. N=3 samples/group, experiments performed in duplicate.
Figure S5. Denervation is consistently demonstrated with a 10 second scald burn. Mice
were subjected to a sham (~22°C water) or scald burn (~92-94°C water) for 6, 8, or 10 seconds.
Skin was harvested at 24 hours post-burn. Skin samples from the burn wound were evaluated
using IHF staining for neurofilament M; 20x magnification. N=3 samples/group, experiments
performed in duplicate.
Figure S6. Additional resuscitation does not alter the local or distal barrier responses to
burn injury and tape-stripping. Immediately after scald burn injury, all mice received
resuscitative fluids. The standard resuscitation included 1ml of fluid, while a subgroup received
an additional 0.5ml at 4 hours and 1ml at 24 hours following injury (administration indicated by
red vertical arrow on all graphs). We then measured (A) pH and (B) transepidermal water loss
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(TEWL) every 12 hours on the burn wounds for 72 hours post-burn. N=5 mice/group. Data
analyzed by two-way ANOVA, bonferroni post-test (all p>0.05). These same animals were
simultaneously subjected to tape-stripping on their ventral surface. Subsequent (C) pH and (D)
TEWL measurements were taken immediately following burn injury, immediately following
tape-stripping, and at several additional time-points thereafter (i.e. 4, 12, 24, 36 hours). N=5
mice/group. Data analyzed by two-way ANOVA, bonferroni post-test (all p>0.05).
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