Fig 2: Effect of the lack of LCN2 on muscle phenotype. WT and Lcn2 ‐/− mice were subjected to (a) grip force analysis at the ages indicated in the abscissa. Results are expressed as grip force (g)/body weight(g). After sacrifice, sera were harvested and analysed for (b) myoglobin, (c) CK, (d) IL6 and (b, d, e) DKK1 by ELISA or (c) by the Reflotron assay. Curve fitting test. *p < .05 versus WT. CK, creatine kinase; ELISA, enzyme‐linked immunosorbent assay; IL6, interleukin‐6; WT, wild‐type

Fig 3: Validation of the digital image analysis algorithm to quantify DKK1 RNAscope signal. (a) Six gastric/gastroesophageal junction tumor biopsies with a range of DKK1 expression were stained for DKK1 by RNAscope. Images were scored in duplicate using an image analysis solution from Flagship Biosciences (left graph) and an H-score (range 0–300) was calculated as described in the methods. The same images were scored manually in duplicate with a one week washout period by two separate pathologists (right panel). Mean and standard deviation are shown. (b) Representative images from 3 tumor resections with no, low, and high DKK1 signal and the image analysis markup are shown. Markup up colors correspond to the following; blue indicates tumor cells with no DKK1 signal, yellow represents tumors cells with low signal (1–3 dots), orange selected tumors cells have medium signal (4–9 dots), and red identifies tumors cells with high signal (10 + dots). Stromal cells which are not highlighted by the algorithm are not scored. Image analysis (IA). Scale bar: 10 μm. (c) Images from replicate DKK1 staining conducted on 3 different days were analyzed and H-scores were quantified. All replicates were required to be in the same bin category or within an H-score of ± 20 if discordant binning occurred. Bins were defined as, negative signal (H-score = 0), low signal (H-score < 34), and high signal (H-score ≥ 35). Asterisk indicates a resection that did not meet the precision criteria.

Fig 4: Specificity and accuracy of the DKK1 RNAscope assay. (a) Cell lines were identified using RNA-Seq data from the Cancer Cell Line Encyclopedia database that expressed very high levels of the indicated Dickkopf family member and low levels of DKK1. Red boxes denote the expression level of the Dickkopf family member that is highly expressed in the indicated cell line. Reads per kilobase per million mapped reads (RPKM). (b) The specificity FFPE cell pellet array with the indicated cell lines was stained for DKK1, quantified using QuPath morphometric software and an H-score (range 0–300) was calculated as described in the methods. Scale bar: 50 μm. (c) FFPE cell pellet arrays with different cancer cell lines were stained for DKK1, quantified using QuPath morphometric software and an H-score (range 0–300) was calculated as described in the methods. DKK1 RNA-Seq data was obtained from the Cancer Cell Line Encyclopedia database. Reads per kilobase per million mapped reads (RPKM). (d) The indicated cells were stained for DKK1 RNA by RNAscope (top row), DKK1 protein by IHC (middle row) or an isotype control antibody (bottom row). Scale bar: 50 μm.

Fig 5: The DKK1 RNAscope assay in tumor resections. Representative images from 3 tumor resections with no (top row), moderate (middle row) and high (bottom row) DKK1 signal are shown with the PPIB control for RNA integrity and the dapB background control. DKK1 H-scores (range 0–300) were semi-quantified manually as described in the methods. Black arrows denotes non tumoral cells without DKK1 signal. Blue arrows denote tumor cells with low levels of DKK1 signal (one dot per cell). Scale bar: 50 μm.