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Blood, Vol. 110, Issue 2, 640-650, July 15, 2007
RNAi screen identifies UBE2D3 as a mediator of all-trans retinoic acid-induced cell growth arrest in human acute promyelocytic NB4 cells
Blood Hattori et al.
110: 640
Supplemental materials for Hattori et al, Vol. 110, Issue 2, 640-650
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 73.1 KB)
- Table S1. The complete list of positive colonies from the primary shRNA screen (PDF, 273 KB) -
After the initial screening, 122 authentic ATRA-resistant colonies were obtained. Seventy-four of them were GFP negative and no viral fragment could be recovered from them. Their ATRA resistance is probably caused by somatic mutations occurring on genes involved in ATRA signaling and thus has nothing to do with the viral infection. Fifteen of the positive colonies contained viral sequences (PCR positive); however, the inserted viral DNA could not be sequenced, which might be due to the mixed colonies. The rest are those infected by viruses and their inserted shRNA templates were successfully sequenced. All of the sequenced shRNA templates could be matched to a specific gene.
- Table S2. The list of genes identified from the primary shRNA screen (PDF, 28.7 KB) -
Shown are the full name, standard abbreviation, gene ID, and the corresponding shRNA sequence of each gene. The results of the “liquid culturing confirmation” (“Confirmation of the identified ATRA-resistant colonies by a liquid culturing assay, Materials and methods”) are also indicated. N/A, not available (has not been tested).
- Figure S1. ATRA-induced CD11b expression in NB4 cells (PDF, 359 KB) -
NB4 cells were infected with indicated siRNA viruses and cultured in the absence or presence of 0.5 µM ATRA for indicated time. Cells were preblocked with purified antimouse CD16/CD32 antibody (0.5 µg/0.5 million cells, BD PharMingen, Santa Cruz, CA) for 5 min, and stained with PE anti-mouse CD11b antibody (400:1, 0.2mg/mL, BD PharMingen) for 30 minutes on ice. Fluorescent-assisted cell sorting (FACS) was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 488 nm argon laser. Ten thousand cells were collected and analyzed using the CellQuest software (Becton Dickinson). NCK1 shRNA and UBE2D3 shRNA are two representative positive shRNAs identified from our screening. Essentially the same results were obtained when other positive shRNAs were utilized.
- Figure S2. p53 protein regulation and physical association (PDF, 292 KB) -
(A) The p53 protein level is not regulated by ATRA treatment. NB4 or NB4R2 cells were cultured in the presence of 0.5 µM ATRA for the indicated time. p53 protein were detected by western blot analysis. The relative amounts of p53 protein were quantified using NIH ImageJ software. The p53 signals were normalized to the amount of actin in each sample. All samples were compared to the signal detected in untreated (time point 0) NB4 cells. Data presented are the means (± SD) of three independent experiments. (B) p53 did not physically associate with UBE2D3 in ATRA-treated (48 hr) or -untreated NB4 cells. Cell lysates were immunoprecipitated with UBE2D3 antiserum (Ubc5s antibody from Chemicon, Temecula, CA) or IgG. The precipitates were blotted with monoclonal anti-UBE2D3 as well as anti-p53 antibodies. Lane 1 and 2 contained small aliquots of the whole cell lysates used for co-immunoprecipitation. The experiments were repeated multiple times. The figure shows the results of a representative experiment.
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