Antibodies, Cytokines, and Growth Factors
Supplementary Materials and Methods
Cell Culture
Nontransformed, conditionally immortalized YAMC, TNFR1−/−, and TNFR2−/− mouse colon epithelial cell lines were established by Dr Robert Whitehead in the Vanderbilt Digestive Diseases Research Center Novel Cell Line Core from H-2Kb-tsA58 (Immorto) mice. Cells were maintained under permissive conditions (RPMI 1640 with 5% fetal bovine serum, 5 U/mL mouse interferon gamma [Intergen, Norcross, GA], 100 U/mL penicillin and streptomycin, 5 g/mL insulin, 5 g/mL transferrin, and 5 ng/mL selenous acid [BD Biosciences, San Jose, CA] at 33C). In preparation for experiments, cells were transferred to nonpermissive conditions (RPMI 1640 containing 0.5% fetal bovine serum, streptomycin and penicillin without interferon gamma, insulin, transferrin, or selenous acid at 37C).
Antibodies, Cytokines, and Growth Factors
Antibodies were purchased as follows: polyclonal anti-ErbB4 (c-18) and anti-ErbB3 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-EGFR and anti-ErbB2 from Millipore; monoclonal anti-actin from Sigma Corp (St Louis, MO); anti–phospho-ErbB4 from Orbigen (San Diego, CA); anti-cleaved caspase-3, phospho-ErbB2, phospho-ErbB3, phospho-Akt, phopho-p38, IB-, and horseradish peroxidase–conjugated secondary antibodies from Cell Signaling (Danvers, MA); anti-active mitogen-activated protein kinase (ERK) from Promega (Madison, WI); and horseradish peroxidase polymer–conjugated anti-rabbit secondary antibody from DAKO USA (Carpinteria, CA).
Purified human EGF was a gift of Dr Stan Cohen (Vanderbilt University). Recombinant heregulin-1 (HRG) was purchased from R&D Systems (Minneapolis, MN). Murine TNF was purchased from Peprotech (Rocky Hill, NJ).
Transfections
Nontargeting control, ErbB4, and IB- siRNA pools were purchased from Dharmacon (Lafayette, CO). Cells were transfected with 100 nmol/L siRNA using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Experiments were performed 48 hours after transfection.
Cell Lysates, Immunoprecipitation, and Western Blot Analysis
Whole cell lysates were extracted in 50 mmol/L Tris, pH 7.4, containing 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1% Triton X-100, 0.2% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 0.1% protease and phosphatase inhibitor cocktails (Sigma), cleared by centrifugation, and boiled in Laemmli sample buffer. ErbB4 was immunoprecipitated by incubating 1 mg precleared lysate for 1 hour at 4°C with 2 g anti-ErbB4 and then for 1 hour with Protein A/G PLUS-Agarose beads (Santa Cruz). Beads were washed 4 times with lysis buffer and eluted by boiling in sample buffer.
Samples were separated on sodium dodecyl sulfate/polyacrylamide gels (6%–10% as appropriate) and blotted on nitrocellulose membranes using the manufacturer's instructions for each antibody. Loading was monitored by Western blot for actin and at least one additional uninvolved protein for whole cell lysates or by Western blot of input actin and recovered heavy chain immunoglobulin G for immunoprecipitations.
<CASE>RNA</CASE> Isolation and Analysis
Total RNA was purified with RNeasy columns (Qiagen, Valencia, CA), including on-column deoxyribonuclease treatment. ErbB4 mRNA was measured by RT-qPCR using an independent complementary DNA synthesis step with iScript (Bio-Rad, Hercules, CA), SYBR Green reaction mix (Sigma), and iCycler with IQ5 software (Bio-Rad). Relative mRNA levels were determined using the 2-CT method with 18S RNA as the reference. Primers were designed, verified, and optimized by the Molecular Cell Biology Resource Core Laboratory using Beacon Design software (Premier Biosoft International, Palo Alto, CA).
Primer sequences used were as follows: 5′-CGTCTGCCCTATCAACTTTCG (18s Fwd), 5′-CCTTCCTTGGATGTGGTAGCC (18s Rev), 5′-TCAACTGGAACACAACTTCAATGC (ErbB4 Fwd), 5′-GGCTCGTACACAAGAACTGGAATC (ErbB4 Rev), 5′-ccacccttgccatccaaactg (JM-a Fwd), 5′-cgtgttgtggtaaagtggaatgg (JM-a Rev), 5′-gcagatcaggatcgggagtg (JM-b Fwd), 5′-gcgtgttgtggtaaagtggaag (JM-b Rev), 5′-acatccagaacaagaattgactcc (CYT-1 Fwd), 5′-acacaaactgatttcccgacatg (CYT-1 Rev), 5′-catccagaacaagaattgactcc (CYT-2 Fwd), 5′-catgggcattccttgttgtg (CYT-2 Rev).
Supplementary Figure 1. Expression of ErbB4 isoforms in young adult mouse colon epithelial cells and mouse colon. (A) Total RNA prepared from cultured cells or 4-week-old mouse colon was subjected to standard end point (nonquantitative) reverse-transcription polymerase chain reaction using ErbB4 primers that detect either both the JM-a and JM-b isoforms or both the Cyt-1 and CYT-2 isoforms. −, no RT control with cultured cells (identical results with tissue). (B) Quantitative real-time polymerase chain reaction was performed using primer sets specific to each isoform to establish JM-a/b and CYT-1/2 expression ratios.
Supplementary Figure 2. Schematic and histology of acute DSS-induced colitis. Mice were given 3% DSS (wt/vol) in drinking water for 4 days as shown in the diagram to induce colonic ulcers and wound healing responses. H&E staining shows an ulcerated region (middle panel) and a hyperproliferative regrowth phase (bottom panel). During the injury/ulceration phase, significant regions of spared epithelium are also present (Figure 2).
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Supplementary Figure 3. TNF signaling is partially required for ErbB4 induction during murine DSS-induced colitis and recovery. (A) TNFR1/TNFR2 double-knockout mice were injected with TNF (104 U TNF, 24 hours). Fixed colon tissue was immunostained for ErbB4 expression and localization. (B) Mice were given DSS (3%) for 4 days, or 4 days followed by 3-day recovery. Bars = 100 μm.
Supplementary Figure 4. TNF does not stabilize the ErbB4 protein. ErbB4-overexpressing (JM-a, CYT-2 isoform) cells were preincubated with TNF for 8 hours and chased with 1 μg/mL TNF for indicated times. ErbB4 protein retention was assessed by Western blot analysis. Graph plots densitometry from 3 independent experiments. *P < .05 vs control.
Supplementary Figure 5. Either TNFR1 or TNFR2 is sufficient to mediate TNF-induced ErbB4 accumulation. YAMC, TNFR1−/−, or TNFR2−/− mouse colon epithelial cells were treated with 100 ng/mL TNF for 16 hours. ErbB4 was immunoprecipitated and immunocomplexes subjected to Western blot analysis for ErbB4. Actin blots were performed on input lysate. fl, full-length.
Supplementary Figure 6. ErbB4 siRNA effects on YAMC cells are rescued by ErbB4 re-expression. (A) YAMC cells were transfected with nontargeting or ErbB4-specific siRNA and treated with TNF for 16 hours. ErbB4 expression was determined by Western blot analysis of whole cell lysates. (B) YAMC cells expressing human ErbB4 construct were transfected with the siRNA pool used in our experiments, which primarily (3 of 4 sequences no sequence overlap) targets primarily mouse ErbB4. ErbB4 expression was determined by Western blot. (C) Cells expressing vector or human ErbB4 construct were transfected with anti-mouse ErbB4 siRNA and subjected to wound healing assays. (D) Cells expressing vector or human ErbB4 were transfected and subjected to TUNEL apoptosis assay after 6-hour TNF exposure. *P < .05 vs all other conditions (no other significant differences).
Supplementary Figure 7. ErbB4 expression results in increased Akt phosphorylation. YAMC cells expressing either vector or ErbB4 were treated with TNF for (A) 1–5 hours or (B) 24 hours, and whole cell lysates were subjected to Western blot analysis using antibody specific for phosphorylated Akt.