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Non-apoptotic role for Apaf-1 in the DNA damage checkpoint

By Pedro Williams,2014-02-10 03:06
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Non-apoptotic role for Apaf-1 in the DNA damage checkpoint

LEGENDS TO SUPPLEMENTARY FIGURES

    Supplementary Fig. 1: Dissociation of autophagy and NF-B activation in HeLa cells. Cells were

    transfected with a GFP-LC3-encoding construct for 18 h, followed by the addition of the indicated autophagy inducers for 2 h (A,C) or the indicated time (B). TNFα was used as a positive control for

    NF-κB activation. Thereafter, cells were fixed, permeabilized and stained for the

    ? immunofluorescence detection of p65/RelA (revealed with an AlexaFluor 568-coupledsecondary

    antibody, which emits in red). Hoechst 33342 (emitting in blue) was employed for nuclear counterstaining. Representative images are shown in A. In B and C, columns illustrate the percentage of cells (mean ? SEM, n = 3,* = p < 0.05) manifesting GFP-LC3 aggregation in

    vacnuccytoplasmic dots (% GFP-LC3 cells) or p65/RelA nuclear translocation (% p65 cells),

    respectively.

    Supplementary Fig. 2: Failure of autophagy induction to activate NF-B-dependent genes.

    Hierarchical clustering of 70 human NF-kB signaling pathway-specific genes expressed in HeLa

    -;;CT cells is shown. The log fold changes relative to untreated control (2 method(Livak and

    Schmittgen, 2001), with ß2-microglubulin as endogenous control) of transcripts detected by real-

    time polymerase chain reaction (RT2 Profiler PCR Array PAHS-025 SABiosciences performed

    on a StepOnePlus system Applied Biosystems) were clustered using the Cluster and TreeView programs (Eisen et al, 1998) (average linkage clustering using Pearson’s uncentered correlation as similarity metric). Each row represents a gene and each column represents a sample. Red and green indicate expression levels above and below the untreated control value, respectively. The dendrogram of genes (at the left of the matrix) represents overall similarities in gene expression profiles. Arrows indicate target genes of NF-kB based on the Rel/NF-kB transcription factors website of TD Gilmore (http://people.bu.edu/gilmore/nf-kb/target/index.html), the survey paper by

    Pahl (Pahl, 1999), and additional search on PubMed.

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    Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci USA 95: 14863-14868

    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408

    Pahl HL (1999) Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18: 6853-6866

    Supplementary Fig. 3: Kinetics of autophagy induction and nuclear GFP-p65 translocation. Human non-small cell lung cancer (NSCLC) A549 cells that stably express a GFP-p65/RelA fusion protein were exposed to the indicated autophagy inducers or to TNFα (positive control for NF-κB

    activation) for the indicated time, followed by monitoring of the nuclear translocation of GFP-p65/RelA (A). Representative images of GFP-p65/RelA fluorescence are shown before (left) and after addition of TNFα (60 min) in B. The autophagy-associated lipidation of LC3 (generating the LC3II isoform) was monitored by immunoblot at 4 hours after addition of the indicated stimuli (C). Supplementary Fig. 4: Impact of autophagy inducers on the NF-B activation pathway in mouse

    embryonic fibroblasts (MEFs). MEFs isolate from C57Bl/6 mice were exposed to nutrient starvation, rapamycin or pifithrin-α for the indicated time, followed by immunoblotting for the

    assessment of the maturation of LC3 and the degradation of IBα (A). In parallel, IKK activation

    was measured in vitro, by assessing the phosphorylation status of the semi-synthetic IB-derived

    substrate GST-IB (1-54) (B); NF-B DNA binding activity was monitored by EMSA (C); and

    gel supershift assays were performed with the indicated antibodies (D), confirming that NF-B

    activation was accounted for by p65/RelA and p50. Results are representative of three independent experiments.

    Supplementary Fig. 5: CA-IKK and MN-NEMO enhance autophagic vacuolization but have no

    effect on vacuole-lysosome fusion. The pcDNA3.1 vector or wild type (WT) IKK-, CA-IKK-, WT

    NEMO- or MN-NEMO-encoding constructs were co-transfected with a plasmid for the expression GFP-LC3 into HeLa cells that were cultured in the absence or presence of 10 nM bafilomycin A1

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    (BafA1) for 24 h. Thereafter, cells were fixed, permeabilized and stained with an antibody that

    ? 568-conjugated secondary antibody), specifically recognizes Lamp2 (revealed with an AlexaFluor

    in order to visualize the cytoplasmic colocalization between GFP-LC3 (green fluorescence) and Lamp2 (red fluorescence). Nuclei were counterstained with Hoechst 33342 (blue fluorescence). Representative confocal microphotographs are shown together with the colocalization profiles (A) within the area of interest, indicated by the orientation of the arrow. In B, columns report the percentage of colocalization between the GFP-LC3 and Lamp2 signals (mean ? SEM; n = > 50

     cells per condition; * p < 0.05). The kinetics of GFP-LC3aggregation was monitored at the

    indicated time points by conventional fluorescence microscopy (mean ? SEM; n = 3; * p < 0.05)

    (C).

    +Supplementary Fig. 6: CA-IKK and MN-NEMO do not colocalize with GFP-LC3

    SQSTM1compartments nor with p62. HeLa cells were transfected for 12 h with CA-IKK- or MN-

    NEMO-encoding constructs, alone or in combination with a plasmid for the expression of GFP-LC3, and then processed to assess the colocalization between CA-IKK or MN-NEMO and GFP-LC3 or

     SQSTM1p62. The colocalization profiles within the area of interest (defined by the α-ω arrow) are

    reported.

    Supplementary Fig. 7: p65 is not required for autophagy induction by physiological and

    -/-pharmacological triggers. Wild type (WT) or p65 murine NIH-3T3 cells were transfected with the

    construct encoding GFP-LC3 for 12 h and treated with the indicated autophagic stimuli during the last hours of the experiment (starvation and pifithrin- = 2 h, rapamycin = 4 h, lithium and

    tunicamycin = 6 h). Thereafter, cells were subjected to conventional fluorescence microscopy for

     the assessment of GFP-LC3aggregation. Columns report the percentage of cells (mean ? SEM, n =

    vac3, * = p < 0.05) exhibiting GFP-LC3 in cytoplasmic dots (% GFP-LC3 cells).

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Supplementary Fig. 8: siRNA-mediated protein knockdown efficiency. HeLa cells were

    transfected with a control siRNA (Co.) or with siRNAs targeting JNK-1 (A), MKK4 or MKK7 (B), or c-Jun (C) for 48 h, followed by immunoblotting for the detection of protein levels. The abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was monitored to ensure equal loading of lanes.

    Supplementary Fig. 9: Impact of IKK constituents and autophagy-relevant proteins on cell viability in conditions of autophagy induction. A. Mouse embryonic fibroblasts (MEFs) of the indicated genotype were cultured in the presence of the indicated autophagy inducers for 24 h,

    )-sensitive dye followed by co-staining with the mitochondrial transmembrane potential (;;m

    DiOC(3) and the vital dye propidium iodide (PI) for the cytofluorometric determination of 6

    apoptosis-related parameters. Black and white columns illustrate the percentage of cells (mean ?

    +low-SEM, n = 3) characterized by plasma membrane breakdown (PI) or loss of ;; (DiOC(3)/PI), m6

    respectively.

    Supplementary Fig. 10: Representative pictures of LC3-specific staining patterns (as assessed by

    ;hepmunohistochemistry) in different organs from control and IKK mice, in normal feeding im

    conditions or after 24 h of starvation.

    Supplementary Fig. 11: Representative transmission electron microscopy pictures of different

    ;heporgans from control and IKK mice, in normal feeding conditions or after 24 h of food

    deprivation.

    Supplementary Fig.12: Differential activation of NF-B by starvation and rapamycin in different

    ;heporgans. Control and IKK mice were maintained in normal feeding conditions or subjected to food deprivation for 24 h, followed by sacrifice and processing of livers and spleens for immunofluorescence microscopy to determine the subcellular distribution of p65/RelA. Nuclei were counterstained with Hoechst 33342 (blue fluorescence). Representative immunofluorescence

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microscopy pictures are shown (A). For all conditions, columns illustrate the percentage of cells

nuc cells) (B). (mean ? SEM, n = 3,* = p < 0.05) exhibiting nuclear p65/RelA (% p65

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