Journal: Cell Reports Medicine

Article Title: EGFR-directed antibodies promote HER2 ADC internalization and efficacy

doi: 10.1016/j.xcrm.2024.101792

Figure Lengend Snippet: High EGFR expression suppresses T-DXd internalization (A) Mass spectrometry-based quantification of expressed RTKs in 18 HER2-expressing CRC patient samples. Scale of expression is in amol/μg. (B) Schematic illustrating the pHrodo-conjugated antibody internalization assay. (C) Relative T-DXd or cetuximab internalization in DiFi cells treated with siRNA against non-targeting control (siNT) or against EGFR (siEGFR). Imaging was started 72 h after siRNA treatment, and images were acquired every 1 h for 48 h. Data are shown as mean ± SEM of three replicates and are representative of n = 3 independent experiments. The groups were compared using two-way ANOVA test. (D) Western blot analysis of DiFi cells treated with siNT and siEGFR for 72 h. Vinculin was used as a loading control. Data are representative of n = 3 independent experiments. (E) Flow cytometry histograms displaying membrane levels of HER2 and EGFR in DiFi cells treated with siNT or siEGFR for 72 h. Data are representative of n = 3 independent experiments. (F) Relative T-DXd or cetuximab internalization in NCI-H508 cells overexpressing doxycycline-inducible EV or EGFR. Imaging was started 48 h after 1 μg/mL doxycycline treatment, and images were acquired every 1 h for 48 h. Data are shown as mean ± SEM of three replicates and are representative of n = 3 independent experiments. The groups were compared using two-way ANOVA test. (G) Western blot analysis of NCI-H508 cells stably transduced with doxycycline-inducible EV or EGFR vectors. The cells were treated with or without 1 μg/mL doxycycline for 48 h before harvesting. Vinculin was used as a loading control. Data are representative of n = 3 independent experiments. (H) Flow cytometry histograms displaying membrane levels of HER2 and EGFR in NCI-H508 cells overexpressing doxycycline-inducible EV control or EGFR for 48 h. Data are representative of n = 3 independent experiments. (I) Western blot analysis of the indicated CRC cell lines to assay endogenous expression of EGFR, HER2, HER3, and HER4. Vinculin was used as a loading control. (J) Flow cytometry histograms displaying membrane levels of HER2 and EGFR in RKO, HCA46, NCI-H508, HT-55, SW48, and DiFi cells. Data are representative of n = 3 independent experiments. (K) Trafficking index of T-DXd or cetuximab in RKO, HCA46, NCI-H508, HT-55, SW48, and DiFi cells. The pHrodo-conjugated antibody-treated cells were imaged every 1 h for 48 h. Data are shown as mean ± SEM of three replicates and are representative of n = 3 independent experiments. See also Tables S1 and , Figure S1 , and .

Article Snippet: Membranes were probed using the following antibodies at 1:1000 dilution – EGFR (Cell Signaling Technology D38B1), HER2 (Cell Signaling Technology 29D8), HER3 (Cell Signaling Technology D22C5), HER4 (Cell Signaling Technology 111B2), Vinculin (Cell Signaling Technology E1E9V), p-HER2 Y1221/1222 (Cell Signaling Technology 6B12), p -EGFR Y1068 (Cell Signaling Technology D7A5), p -ERK T202/Y204 (Cell Signaling Technology D13.14.4E), ERK (Cell Signaling Technology 9102), p -AKT S473 (Cell Signaling Technology D9E), AKT (Cell Signaling Technology 11E7), p-S6 S235/236 (Cell Signaling Technology 2211), p-S6 S240/244 (Cell Signaling Technology 2215), S6 (Cell Signaling Technology 5G10), and p-4EBP S65 (Cell Signaling Technology D9G1Q).

Techniques: Expressing, Mass Spectrometry, Control, Imaging, Western Blot, Flow Cytometry, Membrane, Stable Transfection, Transduction

Journal: Cell Reports Medicine

Article Title: EGFR-directed antibodies promote HER2 ADC internalization and efficacy

doi: 10.1016/j.xcrm.2024.101792

Figure Lengend Snippet:

Article Snippet: Membranes were probed using the following antibodies at 1:1000 dilution – EGFR (Cell Signaling Technology D38B1), HER2 (Cell Signaling Technology 29D8), HER3 (Cell Signaling Technology D22C5), HER4 (Cell Signaling Technology 111B2), Vinculin (Cell Signaling Technology E1E9V), p-HER2 Y1221/1222 (Cell Signaling Technology 6B12), p -EGFR Y1068 (Cell Signaling Technology D7A5), p -ERK T202/Y204 (Cell Signaling Technology D13.14.4E), ERK (Cell Signaling Technology 9102), p -AKT S473 (Cell Signaling Technology D9E), AKT (Cell Signaling Technology 11E7), p-S6 S235/236 (Cell Signaling Technology 2211), p-S6 S240/244 (Cell Signaling Technology 2215), S6 (Cell Signaling Technology 5G10), and p-4EBP S65 (Cell Signaling Technology D9G1Q).

Techniques: Amplification, Mutagenesis, Recombinant, Transfection, Lysis, Staining, Labeling, Bicinchoninic Acid Protein Assay, In Situ, Antibody Labeling, Mass Spectrometry, Software

Journal: Cancers

Article Title: Landscape of Epidermal Growth Factor Receptor Heterodimers in Brain Metastases

doi: 10.3390/cancers14030533

Figure Lengend Snippet: Primary antibodies, pairing, and optimised conditions.

Article Snippet: (1)HER3-(2)HER2 , CST (12708) , D22C5 , Rabbit , 1/100 , Abcam (ab16901) , 3B5 , Mouse , 1/2000 , O/N, 4 °C.

Techniques: In Vitro, In Situ