ShRNA CD24 Lentiviral Transduction Particles (CD24-V2LHS_71908)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed cells in 10-cm plates and cultured with puromycin	Human CD24-shRNA S1 TGCCTCGACACACATAAAC Human CD24- shRNA S2 TTGCATTGACCACGACTAA

Upstream tips
Seed cells in 10-cm plates and cultured with puromycin
Protocol tips
Human CD24-shRNA S1 TGCCTCGACACACATAAAC Human CD24- shRNA S2 TTGCATTGACCACGACTAA

Publication protocol

Prostate cancer cell lines DU145, PC3 and LNCaP were obtained from the American Type Culture Collection. Antibodies specific for the following targets were used for this study: human CD24 (BD Pharmingen, ML5, Cat #:555426), mouse Cd24 (BD Pharmingen, M1/69, Cat #: 562360), CD24 (Santa Cruz Biotechnology, Cat #: sc-11406 and Cat #: sc-53660, and SN3b), NPM1 (Abcam, Cat #: ab10530), p14 (Santa Cruz Biotechnology, Cat #: sc-8340; Cell Signaling, Cat #:2407), p53 (Santa Cruz Biotechnology, DO-1, Cat #: sc-126; FL-393, Cat #sc-6243), MDM2 (Santa Cruz Biotechnology, Cat #: sc-7918), p21 (Cell Signaling, Cat #: 2946), p27 (Cell Signaling, Cat #: 2946), Cyclin B1 (Santa Cruz Biotechnology, Cat #: H-433), GFP (Cell Signaling, Cat #: 2946), Lamin B1 (Santa Cruz Biotechnology, sc-56145), H1.5 (Abcam ab18208), β-actin (Sigma-Aldrich, Cat #: A5441) and anti-IgG (Santa Cruz Biotechnology), phospho-ATM (Ser1981) (Rabbit mAb D6H9, Cat #5883, Cell Signaling) and ATM (Rabbit mAb D2E2, Cell Signaling, Cat #2873).

The two CD24 shRNA constructs were CD24-V2LHS_71908 (Open Biosystems, Cat #: RHS4430-99291677, GenBank accession #: NM_013230) and CD24-V2LHS_71909 (Open Biosystems, Cat #: RHS4430-98854299, GenBank accession #: L33930). DU145 and PC3 cells were infected with lentivirus expressing either control shRNA or CD24 shRNA vectors. The sequence of shRNA and primers used for PCR are listed in Supplementary Table 3.

Gene expression profiling and pathway analysis
DU145 cells stably transduced with lentivirus expressing either control shRNA or CD24 shRNA were seeded onto 10-cm plates and cultured with puromycin. Individual clones were isolated and tested for CD24 expression. Clones with stable CD24 silencing were used for gene expression analysis, usually 2 months after the initial transduction. The stable clones were seeded again in 10-cm plates and at 3–5 days after culture, when the cells reached 50% confluency, the cells were washed with ice-cold PBS twice and RNA extraction was performed with the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Contaminated genomic DNA was eliminated with DNase I (Invitrogen) according to the manufacturer’s protocol. We conducted mRNA microarray analysis using HG-U133 Plus 2.0 (Affymeatrix) was performed according to the manufacturer’s protocol. The microarray data are submitted to NCBI GEO (accession No. GSE46708). dChip software (University of California at Los Angeles Clinical Microarray Core) was used to generate a heat map of the microarray profile. Gene expression profiles of cells were compared with and without CD24. Differences in mRNA expression levels between CD24+ and CD24− cells were calculated by Student’s t-test. Ingenuity pathways analysis (IPA) for CD24-related genes was performed using IPA software (version 7.1, Ingenuity Systems, Inc.).

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