Journal: Scientific Reports

Article Title: Functional kinomics establishes a critical node of volume-sensitive cation-Cl − cotransporter regulation in the mammalian brain

doi: 10.1038/srep35986

Figure Lengend Snippet: ( A ) Characterization of HEK293 cells with doxycycline (dox)-inducible MYC-KCC3 expression used in the RNAi screen. KCC3 wild type (WT) and KCC3 Thr 991 Ala/Thr 1048 Ala protein expression was induced by 0.1 μg/ml doxycycline in the culture medium for 24 hours . Cell lysates were subjected to Western immunoblot (IB) analysis with the indicated antibodies ( B ) Characterization of anti-KCC3 P-Thr 991 and anti-KCC3 P-Thr 1048 phospho-specific antibodies. 36 hours post-transfection with the indicated FLAG-tagged constructs, HEK293 cells were treated for 30 min with either isotonic conditions or hypotonic high K + conditions. Total cell extracts were subjected to IB analysis with the indicated antibodies. Mutation of these residues to alanine (Ala 991 and Ala 1048 ) prevented phosphorylation and eliminated the phospho-specific antibody signal at both sites. ( C ) Scheme of the RNAi screen using the human Dharmacon SMARTpool siRNA kinome library to identify essential kinase regulators of KCC3 Thr 991 phosphorylation. ( D ) Example of results from the primary siRNA screen. Band density of KCC3 P-Thr 991 from Western blots was quantitated by ImageJ software, and these values were used to calculate the magnitude of KCC3 P-Thr 991 increase or decrease by comparing to values derived from Firefly (FF) luciferase negative controls. The heat map depicts the average scores for each kinase siRNA pool in the screen that decreased (green) or increased (red) the signal of KCC3 P-Thr 991 relative to that of the FF siRNA. See Methods for further details. ( E ) Scattered and sorted robust z-scores of kinase hits from the siRNA primary screen. Several siRNA pools led to a significant decrease in the KCC3 P-Thr 991 signal (>50%, p < 0.01 compared to FF siRNA negative control). ( F ) Summary of kinase hits from the secondary siRNA screen. siRNAs targeting primary screen hits were analyzed for their ability to decrease KCC3 P-Thr 991 without affecting total KCC3 level. The (KCC3 P-Thr 991 )/(total MYC-KCC3) ratio was calculated for each target based on the quantification of immuno-reactive signals in triplicate Western blots, with a value of 100% for FF. Ratios were compared by one-way ANOVA (n = 3, mean ± SEM), with p < 0.01 considered statistically significant.

Article Snippet: To identify genes required for KCC3 P-Thr 991 phosphorylation, a high-throughput RNAi screen was performed in 24-well plates with the human Dharmacon SMARTpool siRNA kinome library targeting 541 kinases and kinase-related genes in which each mRNA is targeted by a pool of siRNAs consisting of a combination of four siRNA duplexes directed at different regions of the gene.

Techniques: Expressing, Western Blot, Transfection, Construct, Mutagenesis, Phospho-proteomics, Software, Derivative Assay, Luciferase, Negative Control

Journal: PLoS Genetics

Article Title: Identification of a Tissue-Selective Heat Shock Response Regulatory Network

doi: 10.1371/journal.pgen.1003466

Figure Lengend Snippet: (A–D) Nomarski and fluorescent images corresponding to the phsp70::gfp reporter strain. (A) Control animals show little reporter expression and only faint autofluorescence of the intestine. (B) Reporter induction in animals exposed to heat shock at 33°C for 1 hour. (C) RNAi knockdown of hsf-1 decreases induction of the reporter by heat shock. (D) RNAi knockdown of hsp-1 causes constitutive induction of the reporter in the absence of heat shock. The scale bar corresponds to 100 µm. (E–F) Quantitation of the effects on endogenous hsp70 and hsp-16.2 genes using qRT-PCR. (E) HSR positive regulators normalized to the heat shocked empty vector control. (F) HSR negative regulators normalized to empty vector control. Averages are from at least three biological replicates and error bars represent SEM.

Article Snippet: Genome-wide RNAi screening was performed using a bacterial feeding approach with a library targeting approximately 86% of the C. elegans genome (MRC Geneservice, Cambridge, U.K.).

Techniques: Control, Expressing, Knockdown, Quantitation Assay, Quantitative RT-PCR, Plasmid Preparation

Journal: PLoS Genetics

Article Title: Identification of a Tissue-Selective Heat Shock Response Regulatory Network

doi: 10.1371/journal.pgen.1003466

Figure Lengend Snippet: Nomarski and fluorescent images corresponding to whole animals and fluorescent images of the spermatheca, intestine, and muscle tissue are shown. The boundary of the animals, intestine and spermatheca taken from Nomarski images are added as a visual aide to some images. (A–E) RNAi knockdown of daf-21 leads to induction of the reporter in all three tissues. (F–J) RNAi knockdown of cct-1 causes induction only in muscle. (K–O) knockdown of F38A1.8 causes induction in the intestine and spermatheca. Images are taken at different exposures to maximize fluorescence of each image. Scale bars of whole animal images correspond to 100 µm, while scale bars of the images depicting specific tissues correspond to 50 µm. Asterisks denote only autofluorescence.

Article Snippet: Genome-wide RNAi screening was performed using a bacterial feeding approach with a library targeting approximately 86% of the C. elegans genome (MRC Geneservice, Cambridge, U.K.).

Techniques: Knockdown, Fluorescence

Journal: PLoS Genetics

Article Title: Identification of a Tissue-Selective Heat Shock Response Regulatory Network

doi: 10.1371/journal.pgen.1003466

Figure Lengend Snippet: A) Incubation with 100 µM MG132, a small molecule inhibitor of the proteasome, but not DMSO alone, causes tissue-selective induction of the phsp70::gfp reporter in the intestine and spermatheca (arrows), similar to RNAi knockdown of proteasomal subunits. The scale bar corresponds to 200 µm. Asterisks denote only autofluorescence. B) Mutations in T24H7.2, sgt-1, cyn-11, and unc-45 cause induction of the HSR in whole worms measured using qRT-PCR. C) Mutation of T24H7.2 , but not unc-45 , causes induction in the intestine relative to N2 control animals, measured by qRT-PCR analysis of hsp70 in dissected intestinal tissue. Averages shown are from at least two biological replicates.

Article Snippet: Genome-wide RNAi screening was performed using a bacterial feeding approach with a library targeting approximately 86% of the C. elegans genome (MRC Geneservice, Cambridge, U.K.).

Techniques: Incubation, Knockdown, Quantitative RT-PCR, Mutagenesis, Control

Journal: PLoS Genetics

Article Title: Identification of a Tissue-Selective Heat Shock Response Regulatory Network

doi: 10.1371/journal.pgen.1003466

Figure Lengend Snippet: The effects of HSR positive regulator knockdown on induction of the reporter by negative HSR regulator knockdown were measured using the phsp70::gfp reporter. (A) Images showing the results from double RNAi with each positive regulator and the negative regulator hsp-1 . In each case, knockdown of the positive regulator decreased reporter fluorescence compared to knockdown of hsp-1 alone. (B) Quantitation of the effects of HSR positive regulator knockdown using RNAi on induction of endogenous HSR genes by the HSR negative regulator T24H7.2 mutant reveals that the positive regulators are epistatic to T24H7.2 .

Article Snippet: Genome-wide RNAi screening was performed using a bacterial feeding approach with a library targeting approximately 86% of the C. elegans genome (MRC Geneservice, Cambridge, U.K.).

Techniques: Knockdown, Fluorescence, Quantitation Assay, Mutagenesis