Article Figures & Data
Figures
- Figure 1.
Phosphorylation of EGFR and downstream proteins of SCC cells is similar in 3D cell cultures and SCC xenograft tumors. A, schematic and images of cells cultured in 2D or 3D and as tumor xenograft (in vivo). B, Western blot analysis on whole-cell lysates from 2D and 3D SCC cell cultures and homogenates of tumors grown on nude mice and detection of EGFR and associated signaling molecules. Representative images are shown. β-Actin served as loading control. C, fold change of protein phosphorylation shown in B upon normalization to total protein expression (mean ± SD; n = 2; t test; *, P < 0.05; **, P < 0.01).
- Figure 2.
Cytotoxicity and radiosensitizing potential of cetuximab in 2D and 3D SCC cell cultures. A, representative images of cell colonies. B, clonogenic survival of SCC cells treated with cetuximab (5 μg/mL). Results show mean ± SD (n = 3). C and D, colony formation of 2D and 3D SCC cell cultures treated with cetuximab for 24 hours before irradiation (0–6 Gy single X-ray doses). Results show mean ± SD (n = 3; t test; *, P < 0.05; **, P < 0.01).
- Figure 3.
Phosphoproteome array data of cetuximab-treated 3D FaDu cell cultures (EGFR and MAPK signaling). A, the Phospho Explorer Antibody Array (details in Materials and Methods) was used to screen total cell lysates from untreated and cetuximab-treated 3D FaDu cell cultures (n = 1). Data show fold change of indicated phosphoproteins upon cetuximab treatment after normalization to total protein expression. Gray-colored area indicates the defined induction/reduction boundaries (≤67%/≥150%). B, confirmatory Western blot analyses of 2D and 3D cell cultures and tumor xenografts after treatment with cetuximab. C, densitometric analysis of Western blot analyses shown in B. Phosphorylation was normalized to total protein expression (mean ± SD; n = 2; t test; *, P < 0.05; ** P, < 0.01). D, schematic of activated and deactivated proteins of the MAPK signaling pathway upon cetuximab treatment (according to www.phosphosite.org).
- Figure 4.
Phosphoproteome array data of cetuximab-treated 3D FaDu cell cultures (PI3K/Akt and JNK signaling). A, the Phospho Explorer Antibody Array (details in Materials and Methods) was used to screen total cell lysates from untreated and cetuximab-treated 3D FaDu cell cultures (n = 1). Data show fold change of indicated phosphoproteins upon cetuximab treatment after normalization to total protein expression. Gray-colored area indicates the defined induction/reduction boundaries (≤67%/≥150%). B, confirmatory Western blot analyses of 2D and 3D cell cultures and tumor xenografts after treatment with cetuximab. C, densitometric analysis of Western blot analyses shown in B. Phosphorylation was normalized to total protein expression (mean ± SD; n = 2; t test; *, P < 0.05; **, P < 0.01). D, schematic of activated or deactivated proteins of the PI3K/Akt and JNK signaling pathway upon cetuximab treatment (according to www.phosphosite.org).
- Figure 5.
Effects of combined cetuximab plus JNK2 knockdown on 3D SCC cell-culture radiosensitivity. A, immunofluorescence staining and confocal laser scanning microscopy of total and phosphorylated c-Jun S63 upon cetuximab. B, clonogenic radiation survival of 3D-grown MEK1 and JNK2 knockdown cell cultures additionally treated with cetuximab. Results show mean ± SD (n = 3; t test). C, colony formation of 4 to Gy irradiated 3D grown cell cultures pretreated with JNKi (10 μmol/L) for 30 minutes before 1-hour cetuximab treatment (mean ± SD; n = 3; t test). D, Western blot analysis and clonogenic survival of 3D-grown cells upon treatment with cetuximab under EGFR knockdown. Results show mean ± SD (n = 3; t test). Co siRNA, nonspecific siRNA control (*, P < 0.05; **, P < 0.01).
- Figure 6.
JIP-4 is recruited to EGFR upon treatment with cetuximab and facilitates JNK2 phosphorylation. A, immunoprecipitation (nonspecific IgG control and input lysates used for immunoprecipitation) in cetuximab-treated and untreated 3D lrECM FaDu cell cultures (see also Supplementary Table S1). B, Western blot analysis and clonogenic survival of cetuximab-treated, JIP-4 knockdown 3D cell cultures. Results show mean ± SD (n = 3; t test). C, immunofluorescence staining of EGFR and JIP-4 in 3D grown cells with and without cetuximab treatment. Colocalization was determined with LSM510 software. Results show mean ± SD (n = 3; t test). D, schematic of how cetuximab results in JNK2 activation via recruitment of JIP-4 to EGFR. Co siRNA, nonspecific siRNA control (*, P < 0.05; **, P < 0.01).
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1-8 - PDF file - 621K, Supplementary Figure S1. Treatment with Cetuximab inhibits EGF-mediated EGFR phosphorylation under 2D and 3D cell culture conditions in a cell line-dependent manner. Supplementary Figure S2. Induction of Cetuximab-mediated apoptosis in non-irradiated and irradiated 3D grown SCC cells. Supplementary Figure S3. EGFR inhibition with Cetuximab results in minor Caspase 3 cleavage. Supplementary Figure S4. Treatment with Cetuximab modulates tyrosine and serine protein phosphorylation. Supplementary Figure S5. JNK inhibition using siRNA or pharmacological inhibitor (SP600125) reduces cell survival of SCC cells. Supplementary Figure S6. JNK inhibition impairs Cetuximab-mediated c-Jun S63 phosphorylation. Supplementary Figure S7. JNK2 knockdown sensitizes UTSCC5 cells to Cetuximab-mediated radiosensitization. Supplementary Figure S8. EGFR and JIP-4 co-precipitate in untreated FaDu 3D cell cultures
- Supplementary Methods , Figure Legends 1-8, Tables 1-3 - PDF file - 109K, Supplementary Table S1. Plating efficiencies of SCC cells differ between 2D and 3D cell culture conditions cell line-dependently. Supplementary Table S2. JIP-4, Grb2 and STE20-like kinase bind to EGFR in untreated FaDu 3D cell cultures as determined by immunoprecipitation and subsequent mass spectrometry. Supplementary Table S3. Knockdown of JIP-4 and JNK2 in combination with Cetuximab synergistically impact on radiation survival of SCC cells
Volume 73, Issue 1
