Figure 7Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h and followed by an additional hour of treatment in the presence of 100 mM 5-ethynyl uridine (EU). After fixation cells were permeabilized and EU was stained with an AlexaFluor-488-coupled azide. Representative images are shown for each treatment (A). The EU intensity in the nucleus of each condition was ranked between the untreated control (Ctr, 0 % transcription inhibition) and the control that was not incubated with EU (corresponding to 100 % transcription inhibition) (B). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were treated for 2.5 h and before fixation and permeabilization. Then, cells were stained with a rabbit anti-fibrillarin antibody followed by a staining with an anti-rabbit AlexaFluor-647- or AlexaFluor-546-coupled secondary antibody as well as with a mouse anti-nucleolin antibody followed by a staining with an anti-mouse AlexaFluor-488-coupled secondary antibody. Then images were acquired and colocalization between both signals was assessed (C). The surface overlap coefficient (SOC) was calculated and ranked between the untreated control (Ctr) and the positive control (DACT) Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown Human osteosarcoma U2OS cells were pre-treated with dactinomycin (DACT), bortezomib (BTZ), daunorubicin (DAUN), docetaxel (DOC), doxorubicin (DOXO), epirubicin (EPI), mitoxantrone (MTX), paclitaxel (PACL), vinblastine (VB) and vincristine (VC) at 0.5, 1 and 5 µM ; with cisplatin (CDDP) at 75, 150 and 300 µM, with oxaliplatin (OXA) at 250, 500 and 1000 µM and with crizotinib (CRIZ) at 10, 20 and 40 µM for 1.5 to 2.5 h Cells were pre-treated overnight with the aforementioned compounds in complete medium followed by washout and treatment pursued in methionine-free medium for 30 min. Afterwards, the treatments were continued in methionine-free medium supplemented with 25 µM L-azidohomoalanine (AHA) for additional 1.5 h. AHA incorporation was detected after fixation, permeabilization and blocking by the addition of an AlexaFluor-488-coupled azide. Then images were acquired (E) and AHA intensity in the cells was ranked between the untreated control (Ctr, 0 % translation inhibition) and the untreated control without AHA (corresponding to 100 % translation inhibition) (F). Representative images of DACT 1 µM, BTZ 1 µM, CDDP 150 µM, CRIZ 20 µM, DAUN 0.5 µM, DOC 1 µM, DOXO 1 µM, EPI 1 µM, MTX 1 µM, OXA 500 µM, PACL 1 µM, VB 1 µM, VC 1 µM are shown Correlation between the transcription measured by EU incorporation and measured by fibrillarin and nucleolin colocalization is depicted with Pearson correlation coefficient (R) and p-value (p) (G). The same parameters are shown for the correlation between transcription measured by EU incorporation and translation measured with AHA incorporation