Fig 2: Immunohistochemical staining of AR, IGF-1, and IGF-1R in Dunning G cells growing in rat tibia. Representative pictures showing immunostaining of the AR (a–c), IGF-1 (e–g), and IGF-1R (i–k) in Dunning G tumors located within the tibial bone marrow cavity of non-castrated (a, b, e, f, i, j) and castrated (c, g, k) rats. Negative controls stainings are represented by (d, h, l). Bar in (a, c-e, g-i, k-l) indicates 200 µm and bar in (b, f, j) 50 µm

Fig 3: IGF-1R and IGF-2R expression was predominant in resistant oculomotor neurons and extraocular muscles.Phosphorylated IGF-1R (pIGF-1R) protein was present in oculomotor motor neurons in wild-type and SOD1G93A mice (a,c,e), with signficantly lower levels in motor neurons in spinal cord in control (b) arrow heads, (e) t(113) = 8.69, n = 4 mice, P < 0.0001, Student’s t test, values represent means ± SEM) and SOD1G93A mice ((d) arrow heads, (e) t(141) = 4.30, P < 0.0001, n = 5 mice, Student’s t test, values represent means ± SEM). IGF-1R protein was strongly expressed and co-localized with endplates in extraocular muscles in wild-type and SOD1G93A mice (f,h), while it was barely detectable in lumbrical muscles (g,i). Western blot analysis confirmed that the pIGF-1R protein level was 4-fold higher in extraocular muscles than in lumbrical muscles ((j) t(8) = 8.20, n = 5 mice, P < 0.0001, Student’s t test, values represent means ± SEM). Phosphorylated IGF-2R (pIGF-2R) protein was present at comparable levels in oculomotor (k,m) and spinal (l,n) motor neurons in both wild-type ((o) t(92) = 0.6829, P = 0.4964, n = 3 mice, Student’s t test) and SOD1G93A mice ((o) t(102) = 1.880, P = 0.0630, n = 3 mice, Student’s t test). Peripherally, IGF-2R protein was barely detectable in extraocular muscles (p,r) and undetectable in lumbrical muscles (q,s) of wild-type and SOD1G93A mice using immunofluorescence. Western blot analysis showed that IGF-2R was indeed present in extraocular muscles at 3.8-fold higher levels than in lumbrical muscles ((t) t(8) = 2.86, P = 0.021, n = 5 mice/group, Student’s t test, values represent means ± SEM). Scale bars: (p) 50 µm (applicable to (a–d) and (m–o)), (q) 40 µm (applicable to (e–h) and (n–q)).

Fig 4: Immunohistochemical staining of IGF-1R in human prostate cancer bone metastases. A total IGF-1R score was calculated by multiplying tumor cell staining intensity (0–3) with the fraction of stained cells (0–4) for each metastasis. Sections show representative staining of different intensity, a no staining (0), b weak (1), c moderate (2), and d intense (3) staining (bar indicates 100 µm). e A total IGF-1R score was determined in bone metastases from 14 hormone-naïve and 47 castration-resistant prostate cancer patients. f Cancer-specific survival of castration-resistant patients in relation to the total IGF-1R score in their bone metastases, dichotomized as high (6–12, i.e. above median) or low (0–4, i.e. below median)

Fig 5: Detection of assembled receptor complex using proximity ligation assay. A, Staining of fixed UM-SCC47 cells pretreated for 6 hours with vehicle or 30 µmol/L hSSTNIGF1R, then stained for SDC1, IGF1R, or ITGAV compared with control rabbit (Rbt) or goat (Gt) IgG (bar = 50 µm). Data are representative of duplicate experiments. B, Model of PLA between ITGAV and IGF1R. C, PLA between ITGAV and IGF1R (ITGAV:IGF1R) in fixed UM-SCC47 cells pretreated for 6 hours with vehicle (left top) or 30 µmol/L hSSTNIGF1R (right top), compared with PLA between ITGAV and control goat IgG or IGF1R and control rabbit IgG in vehicle-treated cells (bottom left; bar = 50 µm). Individual cells are identified by DAPI-stained nuclei. PLA is quantified as dots per cell across 15 images over three experiments and graphed as a percentage of PLA observed in untreated cells for ease of comparison (± SD, Student t test; ***, P = 0.001). For reference, the average for the untreated cells was 72 dots per cell.