P rb1

Primary antibodies against CRM1, GAPDH, actin, RanBP1, and p53 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against cyclin D1, Cdc25B, p27, p21, Foxo1, p-Foxo1, Akt, p-Akt, p-Rb1, and histone H3 were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).

Cells were labeled with CD31-FITC antibody (0.5 μg/sample) to validate EC isolation (BD-Pharmigen, 558738) and incubated on ice for 30 min. Cells were fixed in ice-cold 1% PFA for 10 min. For EdU cell cycle phase determination, we used the Click-iT EdU Pacific Blue flow cytometry assay kit, per manufacturer recommendation (ThermoFisher, C10418). Cells were analyzed on a BD-FACSymphony Flow Cytometer. For iH2B-FT cell flow analysis, only live cells were processed for cell cycle speed determination to avoid photoconversion ^{45 (link)}. For imaging flow cytometry (IFC), cells were labeled with CD31-FITC and fixed, as described above. Cells were then permeabilized with saponin buffer (0.1% saponin, 0.5% BSA in PBS) and stained for intracellular p-RB1 [Cell Signaling Technology, # 8156; 1:500 in flow buffer (0.5% BSA in PBS)] for 1h. Cells were centrifuged at 1200×g at 4°C and washed twice in ice-cold PBS. Cells were stained with Alexa Fluor 532 secondary antibody (ThermoFisher, A20182; 1:1000 in flow buffer) and centrifuged at 1200×g at 4°C and washed three times in ice-cold PBS before a final resuspension of approximately 100,000 cells/tube in DAPI-supplemented PBS for final analysis. Cells were analyzed on an Amnis ImageStream cytometer. Analyses were performed using IDEAS software and the bright detail intensity feature was used.

Briefly, A375 and SK-MEL-28 cells were treated with 0.625 μM or/and 1.25 μM ABZ for 48 h respectively. Then the total proteins were collected and subjected to 10% SDS–PAGE gel for western blot analysis. For western blot analysis, the following antibody dilutions were used: CDK1 (Santa Cruz, sc-54), Cyclin B1 (Santa Cruz, sc-245), p53 (Santa Cruz, sc-126), p-RB1 (Cell Signaling Technology, 8516), RB1 (Cell Signaling Technology, 9313), Cleaved Caspase-3 (Cell Signaling Technology, 9661), CDK4 (Santa Cruz, sc-238969), Cyclin D (Santa Cruz, sc-8396), phospho Histone H3 (Hunan AiFang biological, Co., Ltd, AF00582) and GAPDH (Santa Cruz, sc-47724).