Effect of JQ1 on the hypoxia status and blood vessels in TH-MYCN tumor-bearing mice. (A) Experimental design of JQ1 treatment of TH-MYCN tumor-bearing mice showing the schedule and dosing. Following the development of homozygote TH-MYCN tumors, the tumor volume and R2* values were assessed by an MRI in all mice. Mice were randomly assigned to a vehicle-treated group (n = 6) or JQ1-treated group (n = 9). Mice were treated twice with either the vehicle or JQ1 (50 mg/kg) by IP on days 1 and 2. On day 3 post-treatment, an MRI was performed to determine the tumor volume and R2* values. Tumors were harvested for subsequent experiments. (B) The volume of TH-MYCN tumors treated with either the vehicle (Veh) or JQ1 as described in (A) on day 3. Each dot represents one tumor. Results are shown as mean ± SEM (error bars). Statistically significant differences were calculated compared to the Veh-treated tumors using an unpaired two-tailed Student’s t-test (ns: not significant). R2* values in TH-MYCN tumors before and after treatment on day 3 with the vehicle (C) and JQ1 (D) according to the experimental design in (A). The R2* values in TH-MYCN tumors after the treatment with either the vehicle (Veh) or JQ1 are reported in (E). The R2* ratio of after to before treatment with the vehicle (Veh) or JQ1 is reported in (F). Each dot represents one tumor. Results are shown as mean ± SEM (error bars). Statistically significant differences are calculated using a Mann–Whitney test for (C) and (D) or an unpaired two-tailed Student’s t-test for (E) and (F) (* p < 0.05, ** p < 0.01; and *** p < 0.001; ns: non-significant). (G) Western-blot showing the protein expression of CAIX in three different (1, 2 and 3) TH-MYCN tumors treated with either the vehicle or JQ1 according to the experimental schedule described in (A). Actin was used as a loading control. (H) Staining of vehicle- or JQ1-treated TH-MYCN tumors described in (A) with H&E (upper panels), CD31 (middle panels), or aSMA lower panels. Enlarged images of the zones delineated with black boxes (in CD31 and aSMA stained tumors) are shown (Scale bars 100 or 50 µm).