Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-content imaging and RNAi screens for investigating kinase network plasticity

doi: 10.1007/978-1-4939-7154-1_10

Figure Lengend Snippet: Pipetting scheme for reverse transfection of adherent tissue culture cells with arrayed siRNA oligonucleotides. Steps either side of the dashed line represent preparation of parallel copies of the library siRNA transfections combined with either non-targeting (NT) siRNA or a specific additional modifier siRNA (siMOD). This siRNA screen can either look for epistatic modifiers of the siMOD target gene or, vice versa, to test a candidate siMOD target for epistatic impact on the various targets in the library. Values in bold and in parentheses indicate the current volume per well of liquid in the plates upon completion of the indicated steps. (A) A plate of an siRNA library arrayed in a 96 well format is diluted in siRNA buffer to yield a 200 nM copy on a new plate containing 70 ul final volume. The siRNA dilution buffer is supplemented with either 55.5 nM NT or siMOD siRNA, or non-supplemented if the screen does not involve a modifier (see Note 1). (B) Lipid-siRNA complexes are generated by adding diluted lipid mix to the plate(s). (C) The resulting complexes are then sub-divided into three copies on new plates, each with opaque walls and a transparent base, to which the cells are subsequently added. Final oligonucleotide concentrations once the cells have been added are 20 nM for the library siRNA and 5 nM for the modifier siRNA.

Article Snippet: HiPerfect siRNA transfection lipid (Qiagen, catalogue number 301704).

Techniques: Transfection, Generated

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-content imaging and RNAi screens for investigating kinase network plasticity

doi: 10.1007/978-1-4939-7154-1_10

Figure Lengend Snippet: Example scatter plots of data produced using this analysis strategy. The R-script in Fig. 15 has been used in conjunction with an output Nuclei.csv file from a siRNA screen. The x-axes display GFP-ratio values calculated from the GFP-CDK2 reporter and the y-axes the mean nuclear fluorescence intensity of CyclinA antibody staining in arbitrary units. Positions of the assay thresholds (bold white lines) are set based on observations of a relevant test set of wells present on each plate of the screen and numbers in each of the quadrants represent the percent cells of the plotted population contained therein. (A) Individual cell assay values from a test set of siRNA representing NT oligonucleotide, siRNA targeting Cyclin A and a siRNA mixture targeting the G1 phase cyclin-dependent kinases CDK6 and CDK4. The Cyclin A knockdown data was used to set an arbitrary threshold for Cyclin A expression positivity/negativity (horizontal white line) whereas the rightwards trend of the double CDK4 and CDK6 knockdown data, resulting in enrichment of CDK2 negative G1 cells, was used to estimate a suitable value for the vertical threshold of the GFP-CDK2 reporter. (B) Example screen data where a candidate epistatic modifier (siMOD) is shown cancelling loss of CDK2 activity and Cyclin A in cells, otherwise driven by loss of CDK6. Non-targeting siRNA control and CDK6 siRNA data values in the absence (upper panels) and presence (lower panels) of a candidate epistatic siMOD siRNA are shown, respectively. NT siRNA substitutes for the absent siMOD siRNA in the upper panel data such that the total siRNA concentration is constant across all transfection conditions in the screen.

Article Snippet: HiPerfect siRNA transfection lipid (Qiagen, catalogue number 301704).

Techniques: Produced, Fluorescence, Staining, Expressing, Activity Assay, Concentration Assay, Transfection

Journal: Cell & Bioscience

Article Title: Impaired mitophagy induces antimicrobial responses in macrophages infected with Mycobacterium tuberculosis

doi: 10.1186/s13578-023-01107-2

Figure Lengend Snippet: Regulation of BNIP3 controls the intracellular survival of Mtb in vivo . A C57BL/6 WT mice were intraperitoneally injected with siControl or siBINP3 (1.8 mg/kg) using the LIPID-based in vivo transfection reagent (6 mice per group). After 2 days, mice were intratracheally infected with Mtb (1 × 10 6 CFU), and then injected with siRNA (0.6 mg/kg) for 15 consecutive days, once every 3 days. B The levels of BNIP3, LC3, HIF1α, and β-actin were evaluated using western blotting. C Bacterial burden was measured in the lungs 15 days after infection ( n = 6 mice per group). D, E Levels of MCP-1 and TNF-α in the sera of Mtb-infected mice were analyzed by ELISA ( n = 6 mice per group). The data are presented as means ± SD of at least three independent experiments; * p < 0.05, *** p < 0.001

Article Snippet: Delivery of siRNA in vivo was intraperitoneally injected with negative control siRNA or mouse BNIP3 siRNA (Bioneer) following the instructions of the LIPID-based in vivo transfection reagent kit (Altogen Biosystems).

Techniques: In Vivo, Injection, Transfection, Infection, Western Blot, Enzyme-linked Immunosorbent Assay