Supplementary Protocol. sirna transfection methodology and performance

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1 Supplementary Protocol sirna transfection methodology and performance sirna oligonucleotides, DNA construct and cell line. Chemically synthesized 21 nt RNA duplexes were obtained from Ambion Europe, Ltd. (Supplementary Table 1) designed with a proprietary algorithm from Cenix Bioscience GmbH. Lyophilized oligonucleotide duplexes were resuspended in water at 30µM. H2B-GFP was as described 20. A strongly adherent HeLa isolate Kyoto was a kind gift from Prof. Shuh Narumiya (Kyoto University). Production of transfected cell microarrays for live cell imaging. The sirna-gelatin transfection solution was prepared in 384 well plates (NalgeNunc) as described 8 using a Microlab STAR (Hamilton) liquid handling robot with the following modifications. 5µl of sirna solution (30µM), 3µl Opti-MEM (Invitrogen) containing 0.4M sucrose and 3.5µl Lipofectamine 2000 (Invitrogen) were mixed and incubated for 20 minutes at room temperature. After incubation 7.25µl of 0.2% gelatin and 3.5x10-4 % fibronectin (both Sigma-Aldrich) were added. In some experiments 0.5µl of a 40µM marker solution of Cy3 labeled DNA oligonucleotide was added together with the sirna to judge transfection efficiency. These final sirna transfection cocktails were then arrayed onto single-well chambered LabTek coverglass live cell imaging dishes (NalgeNunc) using a ChipWriter Compact Robot (Bio-Rad) with solid pins (Point Technologies) resulting in a spot volume of ~4nl (containing ~5ng RNA) and a spot diameter of ~400µm and a spot to spot distance of 900 µm mirroring one 384 well plate per chamber. sirna microarrays were printed in 48 replicates then dried and stored in plastic boxes containing silica gel (Merck) at least over night. After drying, 1.5x10 5 HeLa-H2B-GFP cells were plated on the microarrays in a total volume of 1.5 ml culture medium (DMEM containing 10% heat-inactivated fetal calf serum, 2mM glutamine, 100U/ml penicillin and 100µg/ml streptomycin), and incubated for 20 hours at 37 o C and 5% CO 2. Cells were transfected simply by growing on sirna spots without further manipulation. After 20h incubation the 1.5ml culture medium was removed and replaced by 5ml preheated CO 2 -independent imaging medium (Invitrogen) and live cell microarrays were transferred to the environmental microscope incubators.

2 Uptake of labeled sirna. HeLa-Kyoto (human cervix carcinoma cells), A549 (epithelial human lung carcinoma cells), RPE (retinal pigment epithelia cells), PHSF (primary human skin fibroblasts) or U2OS (osteosarcoma cells) cells were seeded onto RNAi microarrays printed with a rhodamine labeled SCRAMBLED sirna. 24h after seeding, cells were fixed with PFA and images were taken with a 10x objective (Plan10x, NA 0.4; Olympus-Europe). The experimental set up for all cell lines including HeLa- Kyoto cells were the same. Source of cell lines: HeLa-Kyoto (Prof. Shuh Narumiya, Kyoto University and Toru Hirota, IMP Vienna), A549 (Cenix Bioscience GmbH, Dresden), RPE (Jan-Michael Peters, IMP Vienna), U2OS cells (Ingrid Hoffmann, DKFZ Heidelberg) PHSF (Heiko Runz, University of Heidelberg). Protein knock down of COPB and TPX2. 48h or 24h hours after seeding on sirna microarrays containing sirnas directed against COPB and TPX2, HeLa Kyoto cells were fixed in -20C 0 C methanol for 10 min, followed by 2 washes of PBS. After blocking with 2% BSA for 30 min the monoclonal against COPB (7a) was applied for 4 h followed by washing the cells and incubation with anti-mouse Alexa488 (Molecular Probes, Eugene, OR,USA). (7b) HeLa Kyoto cells were incubated with a polyclonal Antibody against TPX2 (kind gift from Oliver Gruss) for 4 h followed by a incubation with a anti-rabbit Alexa650 (Molecular Probes, Eugene, OR,USA). For both stainings Hoechst (1µg/ml final concentration, Sigma-Aldrich) was added for 15 min to visualize the cell nuclei. COPB and KIF11 RNAi. U2OS, A549 or RPE cells were seeded onto RNAi microarrays which were spotted with sirna directed against KIF11 or COPB. 18 h after seeding Hoechst (1µg/ml final concentration) was added. After 1h incubation the 1.5ml culture medium was removed and replaced by 5ml preheated CO 2 -independent imaging medium (Invitrogen) without Hoechst and live cell microarrays were transferred to the environmental microscope incubators. For the imaging procedure see methods in the main article (High-throughput time-lapse imaging).

3 sirna uptake by different human cell lines Uptake of rhodamine labeled SCRAMBLED sirna by different human cell lines. HeLa-Kyoto, A549, RPE, U2OS and primary human skin fibroblasts show efficient uptake of rhodamine labeled SCRAMBLED sirna within spot area on sirna microarrays. Spot areas were imaged by phase contrast and fluorescence 24 hours after seeding the different cell lines onto microarrays. The Cy3 channel also illustrates the spot location on the sirna microarray and the merged images show the uptake of the labeled sirna into characteristic punctate cytoplasmic structures by the cells.

4 Quantification of sirna uptake in different human cell lines Quantitation of sirna uptake. A high percentage (>90%) of HeLa-Kyoto, A549, RPE, U2OS cells and primary human skin fibroblasts (PHSF) take up the rhodamine labeled SCRAMBLED sirna. Cells were seeded on microarrays spotted with rhodamine labeled SCRAMBLED sirna. 24h after seeding cells were fixed. The mean percentage and standard deviation of cells containing fluorescent sirna from 6 different spot areas per cell line are shown.

5 Specific knock down of COPB and TPX2 proteins in HeLa cells within the spot area Specific down regulation of two endogenous proteins. HeLa-Kyoto cells growing on sirna microarrays containing six spots targeting the Golgi protein COPB, the mitotic spindle protein TPX2, the mitotic spindle protein KIF11 or SCRAMBLED sirna were fixed and processed for immunofluorescence with the DNA dye Hoechst and antibodies against COPB or Tpx2. (a) Down regulation of COPB. Loss or strong reduction of specific anti COPB staining in the vast majority cells on sicopb versus scrambled sirna as a control 48 hours after seeding. Remaining but reduced COPB levels in some cells is likely due to the known high stability of COPB in the coatamer complex. (b) Loss of specific anti TPX2 staining in cells on sitpx2 versus sikif11 as a control (that also arrests cells in prometaphase of mitosis allowing us to compare cells in exactly the same mitotic phase) 24 hours after seeding. Cells growing on si KIF11 show clear TPX2 staining at the monopolar spindle whereas none of the prometaphase arrested cells caused by a sirna against TPX2 show detectable spindle staining.

6 Phenotypic efficiency of sirna microarrays after long term storage Performance of sirna microarrays after storage. Percentage of prometaphase arrested cells 24h after a siplk1 knock-down is shown after one day, two or seven months of dry sirna microarray storage. Shown are averages and standard deviations of three transfections. Proliferation plot of HeLa-Kyoto cells Proliferation of cells on SCRAMBLED sirna spots. Cells transfected by SCRAMBLED sirna without a gene target in the human genome were imaged under high-throughput conditions for 44 hours and cells counted automatically in each frame after segmentation. Plot shows average nuclear counts normalized to 1 at the beginning of the experiment 20 hours after cell plating. Error bars are standard deviations of three experiments on one microarray.

7 COPB phenotype in U2OS, A549 and RPE cells. U2OS, A549 and RPE cells show a cell death phenotype 66h after seeding the cells. The three different cell lines were seeded on sirna microarrays, containing sirna directed against COPB, stained with the vital DNA dye Hoechst and automatically time-lapse imaged in both phase contrast and fluorescence every 30 min for 48 h starting 20 h after seeding. Cells of all three cell types growing on a spot area of sicopb show an apoptotic phenotype 66h after seeding the cells. Comparable results were obtained on replicate spots (not shown).

8 KIF11 phenotype in U2OS, A549 and RPE cells. Images analogous to the previous figure. Times lapse images of the three different cell lines growing on spots of sirna directed against KIF11. U2OS and A549 show a prometaphase arrest phenotype while RPE cells exhibit only a prometaphase delay. Comparable results were obtained on replicate spots (not shown).

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