Supplemental Figure Legends
Figure S1 (related to Figure 1)
(A) Body weight loss of WT and Birc3-/- mice during the AOM-DSS treatment. Each
symbol on the graph represents the mean ± SEM.
(B) Epifluorescence images of colon sections from WT or Birc3−/− mice on day 89 postAOM-DSS treatment stained with TUNEL and antibodies against active caspase-3 to
stain cell death, E-cadherin to mark IECs and Hoechst to label nuclei. Insets correspond
to boxed regions.
(C) Genotyping of caspase-11 using PCR and restriction enzyme digestion with DPNII.
Cleaved DNA fragments represent an intact caspase-11. WT and Ice-/- are positive
controls for intact and mutant caspase-11, respectively. 'C' is water control for PCR.
(D) Western blots depicting caspase-11 and actin levels in colon homogenates from WT
or Birc3-/- mice following AOM-DSS treatment (day 89). Each lane represents one
mouse.
(E) Western blots depicting IBα, NF-B2 and actin levels in colon homogenates from
WT or Birc3-/- mice following AOM-DSS treatment (day 89). Each lane represents one
mouse.
(F) Epifluorescence images of colon sections from WT or Birc3−/− mice on day 89 postAOM-DSS treatment stained with antibodies against phospho-p65 and Hoechst to label
nuclei. Insets correspond to boxed regions.
(G) The numbers of p-p65-positive cells per mm2 stratified according to nuclear or
cytoplasmic localization. 8 fields were analyzed per mouse per genotype (n=3
mice/genotype). The graph represents the mean ± SEM.
Figure S2 (related to Figure 2)
(A) Epifluorescence images of colon sections from WT or Birc3−/− mice on day 23 post3% DSS treatment stained for immune infiltrates using antibodies against F4/80, Gr-1,
CD19, CD3.
(B) Colon length of untreated WT and Birc3-/- mice is shown. Each symbol on the graph
represents 1 mouse; the error bars represent the mean. Statistical analysis was performed
using Student’s t-test (n=3 mice per genotype).
(C) Representative photographs of colon and cecum from untreated mice are depicted.
(D) Representative H&E staining of colon sections from untreated WT or Birc3-/- mice.
The scale bar represents 200 m. Insets correspond to boxed regions.
(E) Representative photographs of colon and cecum from WT or Birc3-/- mice on day 3 or
5 following 3% DSS treatment.
(F) Colon length of WT or Birc3-/- mice following DSS treatment. Each symbol on the
graph represents 1 mouse; (day 3: WT [n=6], Birc3-/- [n=6]; day 8: WT [n=5], Birc3-/[n=6]) the horizontal line represents the mean. Statistical analysis was performed using
Student’s t-test (day 3 p=0.0012; day 5 p=0.0416).
(G) Representative H&E staining of colon sections from WT or Birc3-/- mice on day 3 or
5 following DSS treatment. The scale bar represents 200 m. Insets correspond to boxed
regions.
(H) Kaplan Meier Survival curves of bone marrow chimeric mice following DSS
treatment (WT>WT [n=9], Birc3-/->WT [n=10], WT>Birc3-/- [n=12], Birc3-/->Birc3-/-
[n=11]). Statistical significance was calculated using Mantel-Cox test (WT>WT vs Birc3/>Birc3-/- p=0.0487, WT>WT vs. WT>Birc3-/- p=0.319)
Figure S3 (related to Figure 3)
(A) Epifluorescence images of colon sections from WT or Birc3−/− mice on days 0, 3 and
5 post-DSS treatment stained with antibodies against PCNA to mark dividing cells, Ecadherin to mark IECs and Hoechst to label nuclei.
(B) The numbers of PCNA-positive cells per crypt is shown (11-22 crypts were scored
per mouse; 3 mice per genotype per time point were quantified). Data represent the mean
± SEM. Statistical analysis was performed using Student’s t-test (p>0.05).
(C) ELISA for IL-18 performed on supernatants of organ culture. Each symbol on the
graph represents 1 mouse; the horizontal line represents the mean. The horizontal dotted
line represents the lower detection limit of the assay. Statistical analysis was performed
using Student’s t-test (n=5-6 mice post-DSS per time point per genotype, p>0.05).
(D)Western blots depicting caspase-1, caspase-11 and actin levels in colon homogenates
from WT or Birc3-/- mice following DSS treatment (days 0, 3 and 5). Each lane
represents one mouse.
(E) Western blots depicting NLRP3, NLRP6 and actin levels in colon homogenates from
WT or Birc3-/- mice following DSS treatment (day 8). Each lane represents one mouse.
(F) Western blots depicting IBα, Phospho-p38, Total p38, Phospho-Erk, Total Erk, NFB2 and actin levels in colon homogenates from WT or Birc3-/- mice following DSS
treatment (day 23). Each lane represents one mouse.
(G) Western blots depicting caspase-11 and actin levels in colon homogenates from WT
or Birc3-/- mice following DSS treatment (day 23). Each lane represents one mouse.
(H) Mice were injected with AOM (10 mg/kg) on day 0 and allowed to recover for 1
week. On day 7, 2% DSS was given in the drinking water (black boxe) for 5 days. During
DSS treatment, Birc3-/- mice were injected daily i.p. with IL-18bp (20 g/kg) or vehicle
control. Mice were sacrificed on day 5 post DSS treatment.
(I) Epifluorescence images of colon sections from Birc3-/- mice treated with IL-18bp or
PBS on 5 post-DSS treatment stained with antibodies against PCNA to mark dividing
cells, E-cadherin to mark IECs and Hoechst to label nuclei.
(J) The numbers of PCNA-positive cells per crypt is shown (11-28 crypts were scored per
mouse; 3-4 mice per genotype were quantified). Data represent the mean ± SEM.
Statistical analysis was performed using Student’s t-test (p<0.0001).
Figure S4 (related to Figure 4)
(A) Schematic representation of the experimental procedure for the DSS model. WT mice
were treated with 4% DSS for 5 days, followed by 3 days of clean water. Mice were
injected daily from days 0 to 4 with either rIL-18 (0.05 g) or Necrostatin-1 (5 mg/kg) or
vehicle control or on days 0, 2 and 4 with zVAD-FMK (3 mg/kg) or vehicle control (n=56 mice per treatment).
(B) Representative photographs of colon and cecum from WT mice injected with vehicle
control, recombinant IL-18, Nec-1 or zVAD-FMK on day 8 following DSS treatment
(C) Colon length of DSS-treated mice was measured on day 8. Each symbol on the graph
represents 1 mouse; the horizontal line represents the mean. Statistical analysis was
performed using Student’s t-test (p>0.05)
(D) Representative H&E staining of colon sections from Birc3-/- mice on day 8.
(E) Western blots depicting NF-B2, IBα, RIP3 and GAPDH levels in colon
homogenates from untreated (NT) Birc3-/- mice or DSS-treated Birc3-/- mice injected with
PBS or rIL-18. Each lane represents one mouse.
(F) mRNA derived from the colon of Birc3-/- mice injected with rIL-18 or PBS on day 8
post-DSS treatment was analyzed for expression of Il6 and Il11 by qPCR. Data represent
the mean ± SD. Statistical analysis was performed using Student’s t-test (n=6-7 mice per
time point).
Figure S5 (related to Figure 5)
(A) Western blots depicting Caspase-8 (low and high exposure), cFLIP, Rip3 and actin
levels in colon homogenates from WT or Birc3-/- mice following DSS treatment (day 23).
Each lane represents one mouse.
(B) Kaplan-Meier Survival curves of WT, Birc3-/-, Ripk3-/- or Ripk3-/-/Birc3-/- mice
following DSS treatment (black boxes represent weeks of 3% DSS treatment). Statistical
significance was calculated using Mantel-Cox test (n=4-6 mice per genotype, WT vs.
Ripk3-/- p=0.0147, WT vs. Ripk3-/-/Birc3-/- p=0.0389).
(C) The colon length of WT, Birc3-/-, Ripk3-/- or Ripk3-/-/Birc3-/- mice after DSS treatment
is shown. Each symbol on the graph represents 1 mouse; the horizontal line represents the
mean. Statistical analysis was performed using one-way ANOVA (p<0.05 for WT vs. all
genotypes).
(D) Representative H&E staining of colon sections from WT, Birc3-/-, Ripk3-/- or Ripk3-//Birc3-/- mice after DSS treatment is shown. The scale bar represents 200 m.
(E) Intestinal tissue damage and erosion were quantified as described in the Methods.
Data represent the mean ± SEM. Statistical analysis was performed using 2-way
ANOVA.
The data is representative of 2 experiments with similar results.