Journal: bioRxiv

Article Title: YAP1 status defines two intrinsic subtypes of LCNEC with distinct molecular features and therapeutic vulnerabilities

doi: 10.1101/2023.12.19.572449

Figure Lengend Snippet: YAP1 status does not predict sensitivity to cisplatin in LCNEC cell lines (A) or cell line/PDX xenograft models (B). C, Treatment of LCNEC cell lines with DMSO, cisplatin, MYF-01-37, XAV-939 and decitabine did not change YAP1, but verteporfin reduced YAP1 levels. D, YAP1 status does not predict response to verteporfin. E, Waterfall plot of drug sensitivity in LCNEC cell lines. A comparison of high YAP1 levels and IC50 values identified several drugs with similar targets, including MEK1/2, CDK4/6, and Src family kinase inhibitors. F, Treatment of YAP1-high LCNEC and SCLC (SW1271) cell lines with trametinib did not change YAP1 levels. G, Tumor growth curves from YAP1-high cell line xenografts H1915 (LCNEC) and SW1271 (SCLC) demonstrate a delay in tumor growth with trametinib treatment.

Article Snippet: One million cells each for H810, H1155, H1755, H1833, H2106, MKL1, HCC2374, HCC4017, HOP92, H1359, H2066, H1770, H2106, H1570, H661, HCC3051, H460, HCC4017, H1299, and H1915 in triplicate were surfaced stained with DLL3 (FAB4315P; R&D Systems), AXL (386202; Biolegends), CD56 (362534), EPCAM (324222) or IgG control and then fixed in 2% PFA.

Techniques: Comparison

Journal: Cancer reports (Hoboken, N.J.)

Article Title: The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling.

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: FIGURE 1 Generation of A431D cells that stably express CD148 forms (WT, Q276P/R326Q). (A) DNA sequence of mutated CD148 cDNA, confirming Q276P and R326Q mutations. (B) CD148 surface expression were examined in A431D-CD148 (WT, Q276P/R326Q) cells and the control A431D cells that was generated by the infection of empty retrovirus (Mock) by flow cytometry using a PE-conjugated anti-CD148 antibody. Representative results of four independent experiments are shown. Mean fluorescence intensity is also shown. The fluorescence intensity of CD148 WT cells is expressed as 1.0. A431D-CD148 WT and Q276P/R326Q cells express comparable levels of CD148. A431D-Mock cells show no CD148 expression. (C) Expression of CD148 in A431D-CD148 (WT, Q276P/R326Q) cells was also examined by Western blotting using anti-CD148 or anti-HA antibodies. Fifty micrograms of protein cell lysates were loaded into each well. The loading of proteins was assessed by anti-β actin Western blot. The ratios of CD148 or HA to β actin are also shown. The ratios in A431D-CD148 WT cells are expressed as 1.0. Representative results of four independent experiments are shown. (D) Immunolocalization of CD148 or HA epitope (both red) were examined in A431D-Mock and A431D-CD148 (WT, Q276P/R326Q) cells. The antibody against CD148 extracellular domain was used. Representative results of four independent experiments are shown. Scale bar = 25 μm. CD148 WT and Q276P/R326Q are similarly localized to the cell membrane (arrowheads). In anti-HA immunostaining, there is perinuclear CD148 presence that is generally observed in stable cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSAPBS, and 2 105 cells were incubated with 5 μl of PE-conjugated anti-human CD148 mouse monoclonal (clone 143-41, R&D Systems) or 10 μl of PE-conjugated anti-human EGFR (BD Biosciences) for 45 min at 4 C. Cells were washed with 0.5% BSA-PBS and analyzed using the three-laser BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Techniques: Stable Transfection, Sequencing, Expressing, Control, Generated, Infection, Flow Cytometry, Fluorescence, Western Blot, Membrane, Immunostaining

Journal: Cancer reports (Hoboken, N.J.)

Article Title: The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling.

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: FIGURE 3 Expression of epidermal growth factor receptor (EGFR) and EGF-induced ERK phosphorylation in A431D-CD148 (WT, Q276P/ R326Q) cells. (A) Cell surface expression of EGFR in A431D-CD148 (WT and Q276P/R326Q) and A431D-Mock (control) cells were examined by flow cytometry using a PE-conjugated anti-human EGFR antibody. HEK293 cells that do not express EGFR were used as a negative control. Mean fluorescence intensity is also shown (bottom right). The fluorescence intensity of A431D-Mock cells is expressed as 1.0. Representative results of three independent experiments are shown. All three A431D cells showed comparable level of EGFR expression. (B) EGF-induced ERK1/2 phosphorylation was examined in A431D-CD148 (WT and Q276P/R326Q) and A431D-Mock cells by ELISA as described in Section 2. Cells were treated with 10 ng/ml EGF for 5, 10, 15, and 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a control. The p-ERK/ERK ratios were normalized to serum-starved (30 min) A431D-Mock cells. Representative results of five independent experiments are shown. Data are means ± SEM of quadruplicate measurements. ***p < .001

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSAPBS, and 2 105 cells were incubated with 5 μl of PE-conjugated anti-human CD148 mouse monoclonal (clone 143-41, R&D Systems) or 10 μl of PE-conjugated anti-human EGFR (BD Biosciences) for 45 min at 4 C. Cells were washed with 0.5% BSA-PBS and analyzed using the three-laser BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Techniques: Expressing, Phospho-proteomics, Control, Flow Cytometry, Negative Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer reports (Hoboken, N.J.)

Article Title: The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling.

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: FIGURE 4 Immunoblotting to assess EGF-induced epidermal growth factor receptor (EGFR) and ERK1/2 phosphorylation in A431D-CD148 (WT, Q276P/R326Q) cells. (A) and (B) EGF-induced EGFR and ERK1/2 phosphorylation was examined in A431D-CD148 (WT, Q276P/R326Q) and A431D-Mock cells by Western blotting as described in Section 2. Cells were plated in 100 mm dishes, serum was reduced (0.5% FBS), then cells were treated with 10 ng/ml EGF for 5, 10, 15, 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a negative control. The p-EGFR/EGFR ratios were normalized to A431D-Mock cells treated with EGF for 5 min as EGFR phosphorylation was undetectable in serum starved (0.1% FBS) cells. The p-ERK/ERK ratios were normalized to serum-starved A431D-Mock cells. Representative results of five independent experiments are shown

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSAPBS, and 2 105 cells were incubated with 5 μl of PE-conjugated anti-human CD148 mouse monoclonal (clone 143-41, R&D Systems) or 10 μl of PE-conjugated anti-human EGFR (BD Biosciences) for 45 min at 4 C. Cells were washed with 0.5% BSA-PBS and analyzed using the three-laser BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Negative Control