Journal: Oncogene

Article Title: LRIG1 is a conserved EGFR regulator involved in melanoma development, survival and treatment resistance

doi: 10.1038/s41388-021-01808-3

Figure Lengend Snippet: A Normalized microarray data on LRIG1 expression from normal skin tissue ( N = 7), benign nevi ( N = 18), and primary melanoma ( N = 45). The boxplot shows Tukey whiskers. Statistical significance was determined with Dunnett’s multiple comparisons test. **** P < 0.0001. B Micrographs showing tissue sections from normal skin, nevus, primary melanoma, and metastatic melanoma stained with the Atlas anti-LRIG1 antibody. Scale bars are 100 μm.

Article Snippet: The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. . For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml.

Techniques: Microarray, Expressing, Staining

Journal: Oncogene

Article Title: LRIG1 is a conserved EGFR regulator involved in melanoma development, survival and treatment resistance

doi: 10.1038/s41388-021-01808-3

Figure Lengend Snippet: A Western blot showing LRIG1 expression in A375 parental cells and clones that were either overexpressing (oe) LRIG1 or sequentially silenced for LRIG1 using CRISPR/Cas9. Pc1-5 was sequenced and found to maintain one functional copy of LRIG1 (−/−/ + ). Pc1-5-4 was sequenced found to possess an insertion close to the splice acceptor site of LRIG1 exon 11 (−/−/−). Quantification of bands are indicated below the blot as the LRIG1/Actin fold change from A375. B A representative phospho-RTK array analysis on lysates from A375 cells and LRIG1 knockout A375 Pc1-5-4 cells. Duplicate spots for phospho-EGFR, phospho-RYK, and phospho-insulin receptor are indicated. C Quantified phospho-RTK array data. The graph shows all datapoints and distribution range from three independent experiments. Statistical significance between A375 and A375 Pc1-5-4 lysates was determined using paired, two-tailed Student’s t tests for each phospho-RTK. * P < 0.05. D Western blot showing EGFR expression in A375 cells and in LRIG1 knockdown and knockout subclones. Quantifications are indicated as the EGFR/Actin fold change from A375. E Micrographs showing EGFR-LRIG1 proximity ligation assay micrographs in A375 cells treated with shRNA control (sh-control) or shRNA against LRIG1 (sh-LRIG1). F Western blot analysis of phospho-ERK1/2 protein in A375 cells and LRIG1 knockout A375 Pc1-5-4 cells after stimulation with 10 ng/ml EGF for the indicated time. Quantifications are indicated as the p-Erk1/2/Actin fold change from untreated A375 cells.

Article Snippet: The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. . For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml.

Techniques: Western Blot, Expressing, Clone Assay, CRISPR, Functional Assay, Knock-Out, Two Tailed Test, Knockdown, Proximity Ligation Assay, shRNA, Control

Journal: Oncogene

Article Title: LRIG1 is a conserved EGFR regulator involved in melanoma development, survival and treatment resistance

doi: 10.1038/s41388-021-01808-3

Figure Lengend Snippet: Kaplan–Meier estimates based on RNAseq data, mutation status, and observed survival (OBS) data extracted from a TCGA data set on metastasized melanoma patients: A All 299 patients in the metastatic melanoma cohort with sequenced tissue from TCGA was divided into LRIG1 high/low groups based on the median. This cohort was further divided in two sub-cohorts at the median for EGFR tumor tissue expression. The resulting sub-cohorts of patients with ( B ) low EGFR expression or ( C ) high EGFR expression were then analyzed based on LRIG1 expression and OBS using Kaplan–Meier estimates. D – G Similarly, using the same dataset, we extracted patient samples with available mutational data ( N = 296). We then divided those into molecular subtypes as defined previously : D Patients with activating V600 or K601 mutations in BRAF . E Patients with activating G12, G13, or Q61 mutations in HRAS , KRAS , or NRAS . F Patients with inactivating mutations in NF1 . G Triple wild-type patients without any of the above mutations.

Article Snippet: The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. . For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml.

Techniques: Mutagenesis, Expressing

Journal: Oncogene

Article Title: LRIG1 is a conserved EGFR regulator involved in melanoma development, survival and treatment resistance

doi: 10.1038/s41388-021-01808-3

Figure Lengend Snippet: A Western blot analysis of LRIG1 protein expression in a panel of melanoma cell lines. The blot has been cropped vertically for clarity. Quantifications are indicated as the LRIG1/Actin fold change from A375. B Proliferation (WST intensity) after treatment with recombinant protein in four different cell lines. Normalization between experiments was done separately for each individual cell line by division with the mean WST intensity value of the two untreated (0 μg/mL) conditions. Concentrations of supplemented proteins are indicated for recombinant LRIG1 ectodomain (sLRIG1, blue bars) and His-MBP-Strep control protein (gray bars). Statistical significance vs. 0 µg/mL protein was tested using a two-way ANOVA with a Tukey’s multiple comparisons test.

Article Snippet: The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. . For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml.

Techniques: Western Blot, Expressing, Recombinant, Control

Journal: Oncogene

Article Title: LRIG1 is a conserved EGFR regulator involved in melanoma development, survival and treatment resistance

doi: 10.1038/s41388-021-01808-3

Figure Lengend Snippet: A , B Western blot showing protein expression in naïve A375 cells and two resistant lines with increasing levels of vemurafenib resistance probed for ( A ) LRIG1 and ( B ) EGFR. The blots have been cropped vertically for clarity. Quantifications are indicated as the LRIG1/Actin or EGFR/Actin fold change from A375 respectively. C Proliferation (normalized WST intensity) after treatment with recombinant protein in four different cell lines. Normalization and statistics were performed as in Fig. . Concentrations of supplemented proteins are indicated for recombinant LRIG1 ectodomain (sLRIG1, blue bars) and His-MBP-Strep control protein (gray bars). D Western blots on cell lysates form vemurafenib-resistant cells, stimulated as indicated and probed for phospho-AKT and phospho-Erk1/2. Quantifications are indicated as the phospho-AKT/Actin fold change from FBS-stimulated cells treated with control peptide or as the phospho-Erk1/2/Actin fold change from serum-starved cells treated with control peptide.

Article Snippet: The antigen peptides for all LRIG1 antibodies used are indicated in Supplementary Fig. . For immunohistochemistry: Anti-LRIG1 antibody (Atlas Antibodies AB, Bromma, Sweden, catalog no. HPA011846), 4.0 mg/ml.

Techniques: Western Blot, Expressing, Recombinant, Control