JCS03102 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 (Adobe PDF) -

  Table S1. Processing of MK2, IKK, JNK1, Akt, and ERK kinase activity data for Adv-infected HT-29 cells treated with 100 ng/ml TNF.

• Supplemental Table 2 (Adobe PDF) -

  Table S2. Processing of MK2, IKK, JNK1, Akt, and ERK kinase activity data for uninfected HT-29 cells treated with 100 ng/ml TNF.

• Supplemental Table 3 (Adobe PDF) -

  Table S3. Processing of MK2, IKK, JNK1, Akt, and ERK kinase activity data for Adv-infected HT-29 cells.

• Supplemental Table 4 (Adobe PDF) -

  Table S4. Processing of Akt fold activation from stimulated HT-29 cells.

• Supplemental Figure 1 -

  Fig. S1. Apoptosis is independent of the timing between Adv removal and TNF stimulation. Cells were infected at 6×10⁸ pfu/ml for 3 hours from 0-3, 3-6 and 9-12 hours. TNF was added at 24 hours (21, 18 and 12 hours post-infection), and apoptosis was measured at 48 hours by flow cytometry for cleaved caspase-cytokeratin. Measurements are plotted as the mean of three biological replicates ± s.e.m.

• Supplemental Figure 2 -

  Fig. S2. Adv sensitizes multiple human cell lines to TNF-induced apoptosis. (A-B) HT-29 (black), A549 (green), HeLa (red) and C3A (blue) cells were infected with increasing doses of Adv (expressed as multiplicities of infection, p.f.u./cell) and treated with (A) carrier or (B) 100 ng/ml TNF. HT-29 and A549 cells were collected 48 hours post TNF treatment. HeLa and C3A cells were collected 24 hours post TNF treatment. Cleaved caspase 3-cytokeratin measurements are plotted as the mean of three biological replicates ± s.e.

• Supplemental Figure 3 -

  Fig. S3. ERK and JNK are not differentially activated by TNF following Adv infection. Dynamic activation status and inhibitor response data for the (A) JNK1 pathway and (B) ERK pathway in uninfected cells treated with 100 ng/ml TNF (TNF; blue) and Adv-infected cells treated with 100 ng/ml TNF (Adv + TNF; red). Lysates were collected at 0, 5, 15, 30, 60 and 90 minutes and 2, 4, 8, 12, 16, 20 and 24 hours, and kinase activity was measured by a high throughput kinase activity assay and normalized to total protein content. Results are plotted as the mean relative activation of two biological replicates normalized to activity of the untreated control at 0 minutes (i.e. uninfected cells). The significance of the difference between each pair of curves was calculated by two-way ANOVA (JNK1: P=0.71; ERK: P=0.43). Apoptosis in the presence of kinase inhibition was measured by flow cytometry for cleaved caspase-cytokeratin. The following inhibitor concentrations were used: 10 μM SP600125 (SP) and 10 μM U0126 (U0). Cells were collected at 24 hours, rather than 48 hours, to minimize baseline apoptosis resulting from inhibition. Measurements are plotted as the mean percentage change in apoptosis resulting from inhibition (i.e., mean percentage apoptosis in the presence of inhibitor − mean percentage apoptosis without inhibitor) of three biological replicates ± s.e.m.

• Supplemental Figure 4 -

  Fig. S4. Adv infection alone upregulates Akt but not other TNF- and growth factor-activated pathways. (A-B) Dynamic activation status for (A) TNF-activated pathways and (B) growth factor-activated pathways in Adv-infected cells following mock stimulation (Adv; green). Activation status of Adv-infected cells treated with 100 ng/ml TNF (Adv + TNF; red) is plotted for reference (see also Fig. 2B-D). Lysates were collected at 0, 5, 15, 30 and 60 minutes and 2, 4, 8, 12, 16, 20 and 24 hours and kinase activity was measured by a high through kinase activity assay. Results are plotted as the mean relative activation of two biological replicates normalized to activity of the untreated control at 0 minutes (i.e. uninfected cells). The significance of the difference between each pair of curves was evaluated by two-way ANOVA (MK2: P<10⁻¹²; IKK: P<0.001; JNK1: P<10⁻⁶; Akt: P<0.01; ERK: P<10^-4). Note that Akt activity is plotted on a linear scale (as compared to a semi-log scale for the other kinases).

• Supplemental Figure 5 -

  Fig. S5. TNF-induced apoptosis is altered in the presence of IKK and Akt inhibition. Cells were treated with (A) 40 μM SN50 or SN50M (control peptide) or (B) 1 μM wortmannin 1 hour prior to TNF stimulation, collected after 24 hours, and measured by flow cytometry for cleaved caspase-cytokeratin. Measurements are plotted as the mean percentage change in apoptosis resulting from inhibition (i.e., mean percentage apoptosis in the presence of inhibitor − mean percentage apoptosis without inhibitor) of three biological replicates ± s.e.m. Changes are labeled as significant (*) if P<0.05.

• Supplemental Figure 6 -

  Fig. S6. Adv infection upregulates multiple Akt-pSubs before the addition of TNF. Cells were treated as described in Fig. 3, lysed at 12 hours and analyzed by western blot by probing with anti-Akt-pSub. 10% fetal bovine serum (FBS) stimulation for 15 minutes was used as the positive-control stimulus. 50 μg protein was loaded into each lane. The protein band at 42 kDa (Akt-pSub^42kDa) is shown in Fig. 4A.

• Supplemental Figure 7 -

  Fig. S7. Adv increases baseline Akt signaling. (A) IFNγ- and (B) Adv-sensitized HT-29 cells were treated with TNF (100 ng/ml and 50 ng/ml, respectively) in the presence and absence of 100 nM insulin. Cells were lysed at 0 minutes (before TNF addition) or at 12 hours and assayed for Akt kinase activity. Fold activation is indicated as described in Materials and methods. Measurements are plotted as the mean of six biological replicates (two independent experiments) ± s.e.m.