Figure Legend
Figure 1: LPA attenuates radiation-induced disruption of
tight junctions in the intestinal epithelial monolayers .
Caco-2 and m-ICC12 cell monolayers on Transwell inserts
were irradiated (2-15 Gy) with or without LPA (10 µM) administered 30
min prior to irradiation. One hour after irradiation, the tight junction
integrity was examined. A & B : Caco-2 cell monolayers exposed
to radiation with or without LPA were fixed and stained for occludin
(green) and ZO-1 (red). Confocal images (A) and fluorescence density at
the junctions (B) are presented. Values are Mean ± SEM (n = 4). The
corresponding p-values (on top of the bars) indicate the significant
differences between groups. NS indicates that the p-value is greater
than 0.05. C : Caco-2 cell monolayers exposed to radiation (10
Gy) with or without LPA were stained for F-actin (green) and
cofilinpS3 (red). D : Caco-2 cell monolayers
were pretreated with toxin-B (TB) 30 min prior to LPA treatment.
Monolayers were irradiated (10 Gy) 30 min after LPA treatment and
stained for occludin (green), ZO-1 (red), and nucleus (blue).E : Confocal images for ZO-1 in m-ICC12 cell
monolayers that were treated with LPA or vehicle prior to irradiation.
Figure 2: Effect of LPA2 receptor deficiency on the
radiation-induced disruption of tight junction and adherens junction .
Wild type (WT) and LPA2 receptor knockout
(Lpar2-/- ) mice were subjected to TBI (9.5 Gy).
Two hours after irradiation, colonic sections were stained for tight
junction and adherens junction proteins. A : Merged fluorescence
images for occludin (green), ZO-1 (red), and nucleus (blue). B :
Fluorescence images for E-Cadherin (green), β-Catenin (red), and nucleus
(blue). Representative images from 4 mice per each irradiated group of
mice and 2 mice for control groups are presented.
Figure 3: RP1 blocks TBI-induced disruption of tight
junction and adherens junction, barrier dysfunction, and endotoxemia .
Wild type mice were injected with RP1 (0.5 mg/kg) or vehicle (Veh) 30
min prior to TBI (9.5 Gy) (IR and IR+RP1); the control group was
sham-treated. At 2 hours post-irradiation, colonic sections were
co-stained for occludin, ZO-1, and nucleus (A) or E-cadherin, β-catenin,
and nucleus (B). Confocal images were captured, and fluorescence
densities at the junctions were measured. Fluorescence densities for
ZO-1 (C) and E-cadherin (D) are presented. At 2 and 4 hours
post-irradiation, mucosal permeability in vivo in the colon (E)
and ileum (F) and the plasma LPS levels (G) were measured. Values are
Mean ± SEM (n = 4). The corresponding p-values (on top of the bars)
indicate the significant differences between groups. NS indicates that
the p-value is greater than 0.05. Experiment was repeated with similar
results.
Figure 4: RP1 mitigates TBI-induced disruption of AJC,
mucosal barrier dysfunction, and endotoxemia .
Adult wild type mice were subjected to TBI (9.5 Gy). At 24 hours after
irradiation, mice were injected with RP1 (0.5 mg/kg daily) or vehicle
(Veh); the control group was sham-treated. At 24 and 48 hours after RP1
treatment, junctional integrity (A-F), gut permeability in vivo(G & H), and endotoxemia (I) were evaluated. Sections of the colon were
co-stained for occludin, ZO-1, and nucleus (A), E-cadherin, β-catenin,
and nucleus (B) or claudin-3, Claudin-2, and nucleus (C). Confocal
fluorescence images were captured, and fluorescence densities at the
junctions were measured. Fluorescence densities for ZO-1 (D) and
β-catenin (E) and Claudin-3 (F) are presented. Mucosal permeabilityin vivo in the colon (G) and ileum (H) and the plasma LPS levels
(I) were measured. Values are Mean ± SEM (n = 4). The corresponding
p-values (on top of the bars) indicate the significant differences
between groups. NS indicates that the p-value is greater than 0.05.
Experiment was repeated with similar results. Similar results were also
produced in a similar experiment analyzed at 7 days post-irradiation.
Figure 5: RP1 alleviates TBI-induced downregulation of
antioxidant gene expression .
Wild type (WT) mice were subjected to TBI (9.5 Gy). At 24 hours after
irradiation, mice were injected with RP1 (0.5 mg/kg daily) or vehicle
(Veh); the control group was sham-treated. At 24 hours after RP1
treatment, RNA extracted from colonic mucosa was analyzed for mRNA forGpx1 (A), SOD1 (B), CAT (C), Prdx1 (D), andNrf2 (E) by RT-qPCR. Values are Mean ± SEM (n = 4). The
corresponding p-values (on top of the bars) indicate the significant
differences between groups. NS indicates that the p-value is greater
than 0.05.
Figure 6: RP1 blocks PBI-induced disruption of AJC,
barrier dysfunction, and endotoxemia .
Adult wild type mice were injected with RP1 (3 mg/kg) or vehicle (Veh)
daily starting 1 day after partial body irradiation (PBI; 15.6 Gy); the
control group was sham-treated. At varying times after irradiation,
colonic sections were co-stained for occludin, ZO-1, and nucleus (A) or
E-cadherin, β-catenin, and nucleus (B). Confocal fluorescence images
were captured, and the fluorescence densities of ZO-1 (C) and E-cadherin
(D) at the junctions were measured. Values are Mean ± SEM (n = 4). The
corresponding p-values (on top of the bars) indicate the significant
differences between groups. NS indicates that the p-value is greater
than 0.05. Similar results were produced in similar experiment when
examined at 72 and 96 hours post-irradiation.
Figure 7: RP1 mitigates PBI-induced oxidative stress .
Adult mice were injected with RP1 (3 mg/kg) or vehicle (Veh) daily
starting one day after partial body irradiation (PBI-BM5; 15.6 Gy); the
control group was sham-treated. At 48 hours after irradiation (or 24
hours after RP1), colonic sections were stained for reduced-protein
thiols, oxidized-protein thiols, and NRF2. Antioxidant gene expression
was analyzed by RT-qPCR. A & B : Confocal images for protein
thiols were captured (A), and the fluorescence densities were measured
(B). C & D : Colonic sections were co-stained for F-actin
(green), NRF2 (red) and nucleus (blue) (C). The protein extracts were
immunoblotted for NRF2, and the band densities were measured.E-J : RNA isolated from colonic mucosa was analyzed for mRNA forNrf2 (E), SOD1 (F), Gpx1 (G), CAT (H),Trx1 (I), and Prdx1 (J) by RT-qPCR. Values are Mean ± SEM
(n = 4). The corresponding p-values (on top of the bars) indicate the
significant differences between groups. NS indicates that the p-value is
greater than 0.05. Similar results were produced in similar experiment
when examined at 72 and 96 hours post-irradiation.
Figure 8: RP1 mitigates PBI-induced F-actin remodeling
and its association with apical junctional proteins .
Adult mice were injected with RP1 (3 mg/kg) or vehicle (Veh) daily
starting one day after partial body irradiation (PBI-BM5; 15.6 Gy); the
control group was sham-treated. A-E: At 48 hours after
irradiation (or 24 hours after RP1), detergent-insoluble fractions of
colonic mucosa were immunoblotted for tight junction and adherens
junction proteins as well as NRF2 and β-actin (A). Band densities for
E-cadherin (B), β-catenin (C), Cldn-3 (D), ZO-1 (E), and β-actin (F)
were measured. G: Cryosections of the colon were fixed and
stained for F-actin (red) and nucleus (blue). H & I: Colonic
sections were co-stained for cofilinpS3 (red), F-actin
(green) and nucleus (blue) (H). Mucosal extracts were immunoblotted for
cofilinpS3 and β-actin (I). Band densities were
measured, and the cofilinpS3 band densities presented
by values normalized to corresponding actin band densities (I). In all
graphs, values are Mean ± SEM (n = 4). The corresponding p-values (on
top of the bars) indicate the significant differences between groups. NS
indicates that the p-value is greater than 0.05.