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Results: 8

1.
Figure 6

Figure 6. KIAA0415 interactors are required for efficient HR-DSBR.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

The relative percentage of GFP positive HeLa cells, normalized to the Rluc transfected cells, is shown for indicated esiRNAs. Error bars indicate standard deviation, *p<0.05, **p<0.01.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
2.
Figure 2

Figure 2. Secondary assays for genes that decrease the frequency of HR-DSBR.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

(A) Effect on the cell viability after transfection of indicated esiRNAs (black) in combination with cisplatin (dark grey), MMC (light grey), and IR (white) treatment are shown. Error bars indicate standard deviation. All results were normalized to esiRNA against Rluc transfections. (B) Effect on percent of gammaH2AX positive cells after transfection of indicated esiRNAs without irradiation (black), 1 h post-irradiation (dark grey), and 6 h post-irradiation (light grey). Error bars indicate standard deviation.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
3.
Figure 5

Figure 5. KIAA0415 interacts with SPG11, SPG15, DKFZp761E198 and C20orf29.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

(A) SDS-PAGE gels obtained from the immunoprecipitation of KIAA0415-LAP and SPG11-LAP. Baits (marked in green) and prey (marked in black) were identified by in-gel digestion and nanoLC-MS/MS analysis (see Figure S3). Bands that are not marked represent unspecific background proteins or bait specific proteins (see Table S3, and Online Methods). (B) The composition of KIAA0415 protein complex analyzed as established by shotgun-LC-MS/MS (see Table S3). The number of matched detected peptides and protein sequence coverage are shown. Results for bait proteins are marked in bold.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
4.
Figure 7

Figure 7. KIAA0415 mutation in HSP patients.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

(A) Schematic representation of the KIAA0415 exon structure. The location of the homozygous mutation and the predicted putative helicase fold (in green) are indicated. (B) Pedigree of KIAA0415 mutation in the FSP-083 family. Square symbols represent men; the circles represent women. Symbols of dead subjects are crossed. The filled symbols indicate affected individuals. The numbers below individuals are an internal reference for each sampled individual. Stars indicate sampled subjects. Segregation of the mutation and of 3 microsatellites markers on chromosome 7p are shown next to the pedigree, relative to their position in Mbases. The alleles of the microsatellites are given in base pairs; M, mutation. (C) Electropherograms of KIAA0415 mutations. Deviating sequences from the wt sequence are underlined.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
5.
Figure 8

Figure 8. Sensitivity of lymphoblast cell lines to DNA damaging agents.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

(A) Growth curves of a control lymphoblast cell line—MHU-2619 (solid line), a cell line carrying the KIAA0415 mutation—FSP-083-4 (dotted line) without drug treatment (black line) or MMC treatment (light grey), are presented. (B and C) Cell lines derived from patients with a mutation in KIAA0415 and SPG15 are more sensitive to DNA damaging drugs. The decrease of viable cells (propidium iodide and Annexin V negative) is shown in percent (grey bar) for the indicated cell lines after MMC treatment (B) and bleomycin treatment (C). *p<0.05.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
6.
Figure 3

Figure 3. Structure-based sequence alignment of the helicase C domains of the SF2 helicases UvrB (2D7D), Hel308 (2P6R), RecG (1GM5), and TRCF (2EYQ).. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

Their consensus secondary structure elements are shown bellow as red spirals (α-helices) and blue arrows (β-strands). The sequence alignment of KIAA0415 obtained from threading and used to build a 3D model of its putative helicase C-like domain based on these structural templates is shown at the top. Sequence conservation of KIAA0415 with respect to the template structures is highlighted in grey (conservative) and yellow (semi-conservative). Gap deletions and insertions are represented by dashed lines and inverted U symbols, respectively. Insertions are labelled with the corresponding N- and C-ending residue numbering (black for KIAA0415, green for UvrB, and blue for Hel208). Regions I, Ia, II, III, IV, and V of consensus SF2 helicase motifs are underlined. Residues involved in ADP- and Mg2+ binding are coloured in blue and red, respectively.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
7.
Figure 4

Figure 4. Functional analysis of KIAA0415.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

(A) Efficiency of KIAA0415 mRNA knockdown with two independent esiRNAs in HeLa cells. Relative levels of mRNA 24 h post-transfection of indicated esiRNAs are shown. Error bars indicate standard deviation, **p<0.01. (B) Efficiency of KIAA0415 protein knockdown with two independent esiRNAs in HeLa cells. KIAA0415-LAP Western blot analysis of HeLa cell extracts 48 h post-transfection of indicated esiRNAs is shown. A blot against tubulin served as the loading control. (C) Two independent KIAA0415 esiRNAs influence the frequency of homologous recombination repair measured utilizing the DR-GFP assay. The relative percentage of GFP positive HeLa cells, normalized to the Rluc transfected cells, is shown for indicated esiRNAs. Error bars indicate standard deviation. **p<0.01. (D) The KIAA0415 knockdown phenotype is not cell line dependent. The relative percentage of GFP positive U2OS cells, normalized to the Rluc transfected cells, is shown for indicated esiRNAs. Error bars indicate standard deviation, **p<0.01. (E) Expression of the mouse KIAA0415 orthologue rescues the KIAA0415 RNAi phenotype. The relative percentage of GFP positive in DR-GFP HeLa cells stably expressing the mouse KIAA0415 from a BAC transfected with indicated esiRNAs are shown. Error bars indicate standard deviation, **p<0.01. (F) Different KIAA0415 isoforms are found in the nucleus and in the cytoplasm. A KIAA0415 Western blot of HeLa cell extracts after cell fractionation is shown. Blots against tubulin and histone H3 served as controls.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.
8.
Figure 1

Figure 1. Genome-scale HR-DSBR esiRNA screen.. From: A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia.

(A) Schematic representation of the DR-GFP assay. The two non-functional GFP alleles and the I-SceI cutting site are shown. The transfected plasmid encoding the I-SceI endonuclease is presented as a red circle. The functional GFP gene that is generated after successful HR-DSBR is shown in green. (B) Immunofluorescence analysis of the DR-GFP HeLa cell line after transfection with or without the I-SceI endonuclease plasmid and indicated esiRNAs. Scale bars represent 10 µm. (C) Analysis of an example plate from the screen. Grey wells indicate knockdowns that did not significantly change the percentage of GFP positive cells observed, and red and green wells denote knockdowns that decreased or increased the percentages of GFP positive cells observed, respectively. Control wells are marked with black frames. On each plate there were four positive controls (esiRNA targeting Rad51) and eight negative controls (esiRNA against Rluc - renilla luciferase). Example FACS histograms for the control transfections are presented. (D) Dot plot of the primary screen. Results are presented as average z-scores derived from two independent replicates. Knockdowns with z-scores below −2 or above 2 are shown in red or green, respectively. (E) Results of the gene ontology enrichment analysis for the primary (black) and validated (grey) hits.

Mikołaj Słabicki, et al. PLoS Biol. 2010 June;8(6):e1000408.

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