Journal: Nature Communications
Article Title: Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine
Figure Lengend Snippet: EGFR and ErbB2 generate two distinct modes of ERK activity in intestinal organoids. a , b EKAREV-NLS organoids were treated with EGFRi (PD153035) (1 μM), and/or ErbB2i (CP-724714) (10 μM), at time point 0. a Time courses of average ERK activity under each condition (EGFRi: n = 70, ErbB2i: n = 62, EGFRi + ErbB2i: n = 57 cells). b Quantification of ERK activity before and after treatment with EGFRi or ErbB2i (EGFRi: n = 113, ErbB2i: n = 147 cells, pooled from two organoids). c Time course of average ERK activity in organoids that were cultured under EGF-starved condition (NR) for 24 h, treated with either EGFRi or ErbB2i for 60 min, and then stimulated with 50 ng ml −1 of EGF (EGFRi: n = 48, ErbB2i: n = 32 cells). d Quantification of ERK activity in organoids infected with a control lentivirus (control) or lentiviruses expressing either a dominant negative form of EGFR (dnEGFR) or that of ErbB2 (dnErbB2) (Control: n = 134, dnEGFR: n = 129, dnErbB2: n = 151 cells, pooled from five organoids cultured in the ENR medium). e , f Quantification of ERK activity pulses in EKAREV-NLS organoids cultured in the ENR medium and treated with EGFRi and/or ErbB2i (−/−: n = 71, EGFRi/−: n = 49, −/ErbB2i: n = 36, EGFRi/ ErbB2i: n = 53 cells). Organoids were treated with indicated inhibitors and imaged for 90 min. ERK activity data were smoothened by 6-min moving average, and fitted to flat lines or multi-peak functions. e The proportion of cells exhibiting the pulse-like ERK activation (ERK-pulse + ) under each condition. f Frequencies of ERK activity pulses under each condition. Duration ( g ) and frequencies ( h ) of ERK activity pulses in organoids cultured in the ENR medium and treated with 0, 10, 1, or 0.1 μM of a BRAF inhibitor (SB590885) for 90 min ( n = 55, 52, 29, and 65 cells, respectively). Scale bars, 50 µm. Red lines represent mean. Error bars represent s.e.m. Mann–Whitney U -test ( b ) and Steel–Dwass test ( d , f – h ) were used for comparison. * P
Article Snippet: Sections were subjected to immunofluorescence staining with the following primary antibodies: anti-EGFR (Medical & Biological Laboratories, clone 6F1), anti-ErbB2 (Cell Signaling Technology, clone 29D8), anti-phospho-ErbB2 (Cell Signaling Technology, clone 6B12), anit-Ki67 (Abcam, ab15580), and anti-E-cadherin (Cell Signaling Technology, clone 24E10).
Techniques: Activity Assay, Cell Culture, Infection, Expressing, Dominant Negative Mutation, Activation Assay, MANN-WHITNEY