Figure 4Model predictions of IFNα - induced dynamics of occupied GAS bindings sites ( OccGASbs ) ( left panel ) , of the sum of the pSTAT1 : pSTAT2 heterodimers and the pSTAT1 : pSTAT2 : IRF9 trimers ( middle panel ) and of the pSTAT1 : pSTAT2 : IRF9 trimers ( right panel ) in untreated Huh7 . 5 cells and in Huh7 . 5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα . Lines with shading represent model predictions with 68 % confidence intervals using the prediction profile likelihood method . For experimental validation of the dynamics of OccGASbs , electrophoretic mobility shift assays ( EMSA ) were performed using nuclear protein lysates obtained from untreated Huh7 . 5 cells or Huh7 . 5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα . Lysates were incubated with radioactively - labeled oligonucleotides probes harboring the GAS - binding region of the human IRF1 promoter . Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments ( left panel ) . For experimental validation of the dynamics of the sum of the pSTAT1 : pSTAT2 heterodimers and the pSTAT1 : pSTAT2 : IRF9 trimers , immunoprecipitations ( IP ) were performed using total cell lysates obtained from untreated Huh7 . 5 cells or Huh7 . 5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα . Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting ( IB ) ( middle panel ) . For experimental validation of the dynamics of the pSTAT1 : pSTAT2 : IRF9 trimers , immunoprecipitations were performed using total cell lysates obtained from untreated Huh7 . 5 cells or Huh7 . 5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα . Lysates were subjected to immunoprecipitation ( IP ) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting ( IB ) ( right panel ) . Antibodies and the corresponding proteins in the complexes are underlined . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 ) using a combined scaling and error model . Model predictions of IFNα - induced dynamics of occupied GAS bindings sites ( OccGASbs ) and of occupied ISRE binding sites ( OccISREbs ) in Huh7 . 5 cells without prestimulation and in cells prestimulated for 24 hours with 28 pM and 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα . Model predictions were performed using the prediction profile likelihood method . Lines with shading represent model predictions with 68 % confidence intervals . For experimental validation , growth factor - depleted Huh7 . 5 cells were prestimulated with 0 pM , 28 pM and 280 pM IFNα . After 24 hours cells were stimulated with 1400 pM IFNα and IFNα - induced expression of target genes was measured by qRT - PCR . RNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP , averaged and displayed as fold change , represented by filled circles with errors representing standard error of the mean calculated from biological replicates ( N = 3 ) . Except for gene - specific parameters ( mRNA synthesis and degradation rates , time - delay parameter and Hill coefficient ) , qRT - PCR data were used for model validation but not for model calibration . Dose - dependent sensitization of signal transduction induced by the therapeutic interferon Roferon . Growth factor - depleted Huh7 . 5 were prestimulated with 0 , with 1 . 2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later . Concentrations of Roferon correspond to equipotent concentrations of IFNα . Nuclear lysates were subjected to quantitative immunoblotting and Roferon - induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD - based camera device ( ImageQuant ) . Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 8 ) using a combined scaling and error model . Model trajectories are represented by lines . Dose - dependent sensitization of signal transduction induced by Roferon in HepG2 - hNTCP . Growth factor - depleted HepG2 - hNTCP were prestimulated with 0 , 1 . 8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later . Nuclear lysates were subjected to quantitative immunoblotting and Roferon - induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD - based camera device ( ImageQuant ) . Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 ) using a combined scaling and error model . Model trajectories are represented by lines .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) (left panel), of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers (middle panel) and of the pSTAT1:pSTAT2:IRF9 trimers (right panel) in untreated Huh7.5 cells and in Huh7.5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Lines with shading represent model predictions with 68% confidence intervals using the prediction profile likelihood method. For experimental validation of the dynamics of OccGASbs, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were incubated with radioactively-labeled oligonucleotides probes harboring the GAS-binding region of the human IRF1 promoter. Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments (left panel). For experimental validation of the dynamics of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations (IP) were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (middle panel). For experimental validation of the dynamics of the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation (IP) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (right panel). Antibodies and the corresponding proteins in the complexes are underlined. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3) using a combined scaling and error model.Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) and of occupied ISRE binding sites (OccISREbs) in Huh7.5 cells without prestimulation and in cells prestimulated for 24 hours with 28 pM and 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Model predictions were performed using the prediction profile likelihood method. Lines with shading represent model predictions with 68% confidence intervals. For experimental validation, growth factor-depleted Huh7.5 cells were prestimulated with 0 pM, 28 pM and 280 pM IFNα. After 24 hours cells were stimulated with 1400 pM IFNα and IFNα-induced expression of target genes was measured by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP, averaged and displayed as fold change, represented by filled circles with errors representing standard error of the mean calculated from biological replicates (N=3). Except for gene-specific parameters (mRNA synthesis and degradation rates, time-delay parameter and Hill coefficient), qRT-PCR data were used for model validation but not for model calibration.Dose-dependent sensitization of signal transduction induced by the therapeutic interferon Roferon. Growth factor-depleted Huh7.5 were prestimulated with 0, with 1.2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later. Concentrations of Roferon correspond to equipotent concentrations of IFNα. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=8) using a combined scaling and error model. Model trajectories are represented by lines.   Dose-dependent sensitization of signal transduction induced by Roferon in HepG2-hNTCP. Growth factor-depleted HepG2-hNTCP were prestimulated with 0, 1.8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3) using a combined scaling and error model. Model trajectories are represented by lines.


Figure 5Knockdown of USP18 results in sustained signaling . USP18 knockdown efficiency determined by quantitative immunoblotting of cytoplasmic lysates of Huh7 . 5 transfected with USP18 siRNA relative to parental Huh7 . 5 cells and Huh7 . 5 cells transfected with non - targeting siRNA . Cells were stimulated with 1400 pM IFNα for 24 hours . Error bars represent 1σ confidence intervals estimated from biological replicates ( N = 6 to N = 10 ) ( left ) . Model fit and experimental data of Huh7 . 5 cells transfected with control siRNA or USP18 siRNA are shown . Cells were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor - depletion . Next , cells were prestimulated with 1400 pM IFNα ( yellow background ) and stimulated with 1400 pM IFNα at 24 hours or untreated ( white background ) . IFNα - induced phosphorylation of STAT1 and STAT2 and induction of USP18 and tSTAT1 , comprising both phosphorylated and unphosphorylated STAT1 protein , are displayed . Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera device ( ImageQuant ) . For model purposes data in control siRNA and untransfected Huh7 . 5 are combined to one condition . Data from multiple time courses scaled together is displayed as filled circles with errors representing 1σ confidence interval estimated from biological replicates ( N = 2 to N = 10 ) using a combined scaling and error model . Lines represent model trajectories . Overexpression of Huh7 . 5 is not sufficient to explain negative memory . Induced expression of USP18 after treatment of Huh7 . 5 - TetON - USP18 and Huh7 . 5 - TetON - control cells with doxycycline for 24 hours in comparison with parental Huh7 . 5 cells treated with 1400 pM IFNα for 24 hours . Analysis was performed by quantitative immunoblotting . Error bars represent 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 5 ) ( left ) . Model fits and experimental data of Huh7 . 5 - TetON - USP18 treated with doxycycline for 24 hours and stimulated with 1400 pM IFNα or parental Huh7 . 5 cells prestimulated with 0 or 1400 pM IFNα and stimulated with 1400 pM IFNα after 24 hours are shown . The dynamics of IFNα - induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted . Experimental data was obtained by quantitative immunoblotting using chemiluminescence and CCD camera device ( ImageQuant ) . For modeling purposes data from Huh7 . 5 - TetON empty vector control and untransduced Huh7 . 5 are combined to one condition . Data is displayed as filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 to N = 4 ) using a combined scaling and error model . Line represents model trajectories .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Knockdown of USP18 results in sustained signaling. USP18 knockdown efficiency determined by quantitative immunoblotting of cytoplasmic lysates of Huh7.5 transfected with USP18 siRNA relative to parental Huh7.5 cells and Huh7.5 cells transfected with non-targeting siRNA. Cells were stimulated with 1400 pM IFNα for 24 hours. Error bars represent 1σ confidence intervals estimated from biological replicates (N=6 to N=10) (left). Model fit and experimental data of Huh7.5 cells transfected with control siRNA or USP18 siRNA are shown. Cells were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor-depletion. Next, cells were prestimulated with 1400 pM IFNα (yellow background) and stimulated with 1400 pM IFNα at 24 hours or untreated (white background). IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 and tSTAT1, comprising both phosphorylated and unphosphorylated STAT1 protein, are displayed. Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For model purposes data in control siRNA and untransfected Huh7.5 are combined to one condition. Data from multiple time courses scaled together is displayed as filled circles with errors representing 1σ confidence interval estimated from biological replicates (N=2 to N=10) using a combined scaling and error model. Lines represent model trajectories.Overexpression of Huh7.5 is not sufficient to explain negative memory. Induced expression of USP18 after treatment of Huh7.5-TetON-USP18 and Huh7.5-TetON-control cells with doxycycline for 24 hours in comparison with parental Huh7.5 cells treated with 1400 pM IFNα for 24 hours. Analysis was performed by quantitative immunoblotting. Error bars represent 1σ confidence intervals estimated from biological replicates (N=3 to N=5) (left). Model fits and experimental data of Huh7.5-TetON-USP18 treated with doxycycline for 24 hours and stimulated with 1400 pM IFNα or parental Huh7.5 cells prestimulated with 0 or 1400 pM IFNα and stimulated with 1400 pM IFNα after 24 hours are shown. The dynamics of IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted. Experimental data was obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For modeling purposes data from Huh7.5-TetON empty vector control and untransduced Huh7.5 are combined to one condition. Data is displayed as filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=4) using a combined scaling and error model. Line represents model trajectories.


Figure 6The number of molecules per cell for STAT1 , STAT2 , IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients . Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting . Detection was performed with antibodies specific to STAT1 , STAT2 , IRF9 or USP18 using chemiluminescense on a CCD camera - based device ( ImageQuant ) . Average of at least N = 3 are displayed with standard deviations . Data was used for calibration of a primary human hepatocyte - specific mathematical model and estimated values are shown with green squares . Experimental data and model fits of IFNα - and Roferon - induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor - depleted primary human hepatocytes prestimulated with 0 , 2 . 8 or 1400 pM IFNα ( patients 1 - 3 ) or 0 , 1 . 2 or 608 pM Roferon ( patients 4 - 6 ) . Primary human hepatocytes from the same patients 1 - 6 as in ( B ) were used . Experimental data is represented by filled circles ( N = 1 per patient ) . Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα . Lines indicate model fits .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients. Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting. Detection was performed with antibodies specific to STAT1, STAT2, IRF9 or USP18 using chemiluminescense on a CCD camera-based device (ImageQuant). Average of at least N=3 are displayed with standard deviations. Data was used for calibration of a primary human hepatocyte-specific mathematical model and estimated values are shown with green squares.Experimental data and model fits of IFNα- and Roferon-induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor-depleted primary human hepatocytes prestimulated with 0, 2.8 or 1400 pM IFNα (patients 1-3) or 0, 1.2 or 608 pM Roferon (patients 4-6). Primary human hepatocytes from the same patients 1-6 as in (B) were used. Experimental data is represented by filled circles (N=1 per patient). Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα. Lines indicate model fits.


Figure 7Plasma levels of IFNα were measured in a cohort of 36 patients with chronic HBV .

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Figure 7Plasma levels of IFNα were measured in a cohort of 36 patients with chronic HBV.


Figure 5Western blot for the protein indicated below the blot , comparing input MCF - 7 cells , and IP with IgG or the indicated antibody . The arrows indicate the expected size of the protein of interest . RIP - qPCR for the indicated RNA ( AsGFP or NORAD ) in cells transfected with the AcGFP bearing NucLibB in the 3 ' UTR , with RIP performed with the indicated antibody and RNA levels determined by qRT - PCR . N = 5 . Error bars - s . e . m . Nuc / Cyto ratios for NucLibB tiles , bound ( + , log2 ( IP / Input ) ≥ 0 . 5 ) or not bound ( - , log2 ( IP / Input ) < 0 . 5 ) by the indicated factors . Boxplots are as in Figure 1C . P values computed using two - sized Wilcoxon rank - sum test .

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Figure 5Western blot for the protein indicated below the blot, comparing input MCF-7 cells, and IP with IgG or the indicated antibody. The arrows indicate the expected size of the protein of interest.RIP-qPCR for the indicated RNA (AsGFP or NORAD) in cells transfected with the AcGFP bearing NucLibB in the 3' UTR, with RIP performed with the indicated antibody and RNA levels determined by qRT-PCR. N=5. Error bars - s.e.m.Nuc/Cyto ratios for NucLibB tiles, bound (+, log2(IP/Input)≥0.5) or not bound (-, log2(IP/Input)<0.5) by the indicated factors. Boxplots are as in Figure 1C. P values computed using two-sized Wilcoxon rank-sum test.


Figure 6mRNA ( top ) and protein ( bottom ) levels of the indicated genes in cells transfected with the indicated siRNAs . n = 8 . Error bars are s . e . m . P - values computed two - sided t - test . * * P ≤ 0 . 01 * * * * P ≤ 0 . 0001 . Differences in the Nuc / Cyto ( log2 ) ratios for tiles containing the SIRLOIN element ( up to 6 mismatches ) and other NucLibB tiles ( only WT sequence tiles were considered ) . Boxplots are as in Figure 1C . P - values computed using two - sided Wilcoxon rank - sum test . IP / Input ( top ) and changes in Nuc / Cyto ( log2 ) ratios upon siRNA targeting of the indicated factor ( top ) for WT NucLibB tiles with the indicated number of the indicated motifs . Number of tiles in each group is indicated in parentheses . * - P < 0 . 05 * * P < 0 . 005 * * * P < 0 . 0005 for comparing the group with the indicated number of matches of the motif with all tiles containing a lower motif count . Boxplots are as in Figure 1C . P - values computed using two - sided Wilcoxon rank - sum test . * - P < 0 . 05 , * * - P < 0 . 005 , * * * - P < 0 . 0005 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6mRNA (top) and protein (bottom) levels of the indicated genes in cells transfected with the indicated siRNAs. n=8. Error bars are s.e.m. P-values computed two-sided t-test. ** P≤0.01 **** P≤0.0001.Differences in the Nuc/Cyto (log2) ratios for tiles containing the SIRLOIN element (up to 6 mismatches) and other NucLibB tiles (only WT sequence tiles were considered). Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test.IP/Input (top) and changes in Nuc/Cyto (log2) ratios upon siRNA targeting of the indicated factor (top) for WT NucLibB tiles with the indicated number of the indicated motifs. Number of tiles in each group is indicated in parentheses. *- P<0.05 ** P<0.005 *** P<0.0005 for comparing the group with the indicated number of matches of the motif with all tiles containing a lower motif count. Boxplots are as in Figure 1C. P-values computed using two-sided Wilcoxon rank-sum test. * - P<0.05, ** - P<0.005, *** - P<0.0005.


Figure 1B ) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells . D ) Agarose gel electrophoresis of RNA extracted from control , 4hr and 24hr ActD treated K562 cells . E ) Barplots showing qRT - PCR gene expression levels ( mean + / - S . D . ) of PTEN , FNDC3B , and STAM genes in control , 4hr and 24hr actinomycin D treated K562 cells . p - value : two - tailed t - test , ( * * * p < 0 . 05 ) . The results represent data from 3 technical replicates from 3 independent biological preparations .

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Figure 1B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells.D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells.E) Barplots showing qRT-PCR gene expression levels (mean +/- S.D.) of PTEN, FNDC3B, and STAM genes in control, 4hr and 24hr actinomycin D treated K562 cells. p-value: two-tailed t-test, (*** p < 0.05). The results represent data from 3 technical replicates from 3 independent biological preparations.


Figure 2Hi - C interaction heatmaps at 500kb ( left ) resolution showing all of chromosome 11 , and at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for aXL control and RNase A treated cells . The arrows represent similar patterning of long - range interactions in samples treated with RNase after crosslinking . Hi - C interaction heatmaps at 500kb ( left ) resolution showing all of chromosome 11 , and at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for bXL control and RNase A treated cells . Black arrow indicates the reduction in long - range interactions in the bXL RNase A dataset . The arrows represent perturbed patterning of long - range interactions in samples treated with RNase before crosslinking . C - D ) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4 , with the 1st principal components ( 1st PC ) below for aXL ( C ) and bXL ( D ) samples . The bXL RNase A sample shows reduced Pearson correlations across the genome . E ) Violin plot showing the first eigenvalues ( calculated by the trans - chromosomal data ) in control and RNase treated cells before and after crosslinking . The bXL RNase A sample show reduction of the negative eigenvalues , indicative of perturbation of B - type compartments . p - value : Wilcoxon rank - sum test . In the violin plots , the horizontal bands represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates . F ) Saddle plots showing the compartmental interactions for both cis - and trans - contacts across the conditions . The bXL RNase A samples display reduced B - B type interactions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for aXL control and RNase A treated cells. The arrows represent similar patterning of long-range interactions in samples treated with RNase after crosslinking.   Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for bXL control and RNase A treated cells. Black arrow indicates the reduction in long-range interactions in the bXL RNase A dataset. The arrows represent perturbed patterning of long-range interactions in samples treated with RNase before crosslinking.  C-D) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal components (1st PC) below for aXL (C) and bXL (D) samples. The bXL RNase A sample shows reduced Pearson correlations across the genome.E) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and RNase treated cells before and after crosslinking. The bXL RNase A sample show reduction of the negative eigenvalues, indicative of perturbation of B-type compartments. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.F) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions. The bXL RNase A samples display reduced B-B type interactions.


Figure 3Interaction heatmaps at 40kb resolution zooming in chr21 : 19 . 5 - 33Mb and showing the TAD structures in aXL and bXL datasets . The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps . Boxplot showing the TAD boundary scores for the aXL and bXL datasets . p - value : Wilcoxon rank - sum test . ( * * * p < 0 . 05 ) . The horizontal bands in the boxplots represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates . C - D ) Mean interactions frequencies + / - 1Mb centered on all TAD boundaries for each dataset at 40kb resolution , as well as the log2 ratios of aXL ( left panel ) or bXL ( right panel ) samples . The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Interaction heatmaps at 40kb resolution zooming in chr21:19.5-33Mb and showing the TAD structures in aXL and bXL datasets. The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps.Boxplot showing the TAD boundary scores for the aXL and bXL datasets. p-value: Wilcoxon rank-sum test. (*** p < 0.05). The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.C-D) Mean interactions frequencies +/- 1Mb centered on all TAD boundaries for each dataset at 40kb resolution, as well as the log2 ratios of aXL (left panel) or bXL (right panel) samples. The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking.


Figure 4A ) Hi - C interaction heatmaps at 500kb ( left ) resolution showing all of chromosome 11 , and at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for control and 24hr ActD treated cells . The arrows point out to perturbed long - range interactions at shorter scales . B ) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4 , with the 1st principal component ( 1st PC ) below for control and 24hr ActD treated cells . C ) Violin plot showing the first eigenvalues ( calculated by the trans - chromosomal data ) in control and ActD treated cells . p - value : Wilcoxon rank - sum test . In the violin plots , the horizontal bands represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates . D ) Saddle plots showing the compartmental interactions for both cis - and trans - contacts across the conditions . E ) Scaling plot generated by 500kb resolution Hi - C data showing the interaction frequency as a function of genomic distance .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for control and 24hr ActD treated cells. The arrows point out to perturbed long-range interactions at shorter scales.B) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal component (1st PC) below for control and 24hr ActD treated cells.C) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and ActD treated cells. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.D) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions.E) Scaling plot generated by 500kb resolution Hi-C data showing the interaction frequency as a function of genomic distance.


Figure 5A ) Interaction heatmaps at 40kb resolution zooming in chr9 : 104 - 114Mb and showing the TAD structures , as well as the log2 ratios , in control and transcriptionally inhibited datasets . Black arrow indicates the increase in inter - TAD interactions . B ) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells . p - value : Wilcoxon rank - sum test . The horizontal bands in the boxplots represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates . C ) Mean interaction frequencies centered on all TAD boundaries + / - 1Mb for each dataset at 40kb resolution , as well as the log2 ratios . The inter - TAD interactions are increased upon transcriptional inhibition . D ) Aggregate Peak Analysis based on the K562 loops coordinates The heatmaps are centered on the loop positions + / - 250kb using 25kb resolution Hi - C data . There is a ~ 50 % reduction of loop intensity as assessed by z - scores and log2 fold change . E ) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells for boundaries that are bound with differing numbers of CTCF binding sites . The reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding . p - value : Wilcoxon rank - sum test . The horizontal bands in the boxplots represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates . F ) Scaling plot generated by 40kb resolution Hi - C data showing the interaction frequency as a function of genomic distance , with upper distance limit of 3Mb . The 24hr ActD treated samples shows a slower rate of decay at shorter distances , correlating with the reduction of insulation scores .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Interaction heatmaps at 40kb resolution zooming in chr9:104-114Mb and showing the TAD structures, as well as the log2 ratios, in control and transcriptionally inhibited datasets. Black arrow indicates the increase in inter-TAD interactions.B) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.C) Mean interaction frequencies centered on all TAD boundaries +/- 1Mb for each dataset at 40kb resolution, as well as the log2 ratios. The inter-TAD interactions are increased upon transcriptional inhibition.D) Aggregate Peak Analysis based on the K562 loops coordinates The heatmaps are centered on the loop positions +/- 250kb using 25kb resolution Hi-C data. There is a ~50% reduction of loop intensity as assessed by z-scores and log2 fold change.E) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells for boundaries that are bound with differing numbers of CTCF binding sites. The reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.F) Scaling plot generated by 40kb resolution Hi-C data showing the interaction frequency as a function of genomic distance, with upper distance limit of 3Mb. The 24hr ActD treated samples shows a slower rate of decay at shorter distances, correlating with the reduction of insulation scores.


Figure 1C . SDS - PAGE showing a representative fraction of the purified spMis18 N - terminal Yippee - like globular domain ( amino acid residues 1 - 120 ; spMis181 - 120 ) . D . Cartoon representation of the crystal structure of spMis181 - 120 in two different orientations , highlighting key residues within the putative substrate - binding pocket . E . Substrate - binding pocket mutations Y90A and Y74A / Y90A / T105A / S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18 - 262 cells to varying degrees . Five - fold serial dilutions of mis18 - 262 cells transformed with plasmids harbouring the indicated spMis18fl constructs , spotted on PMG - uracil + Phloxine B media supplemented with ( repressed ) or without ( expressed ) thiamine , and incubated at the indicated temperatures ; dead cells stain dark pink .

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Figure 1C. SDS-PAGE showing a representative fraction of the purified spMis18 N-terminal Yippee-like globular domain (amino acid residues 1-120; spMis181-120).D. Cartoon representation of the crystal structure of spMis181-120 in two different orientations, highlighting key residues within the putative substrate-binding pocket.E. Substrate-binding pocket mutations Y90A and Y74A/Y90A/T105A/S107K affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-262 cells to varying degrees. Five-fold serial dilutions of mis18-262 cells transformed with plasmids harbouring the indicated spMis18fl constructs, spotted on PMG - uracil + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.


Figure 2A . Cartoon representation of the assembly of spMis18 Yippee - like domains ( spMis181 - 120 ) as observed in the crystal asymmetric unit . Close up views highlight the amino acid composition and interactions stabilizing the dimeric arrangements . Interface - II is composed of conserved residues and an extensive binding surface as compared to Interface - I ( see text for details ) , suggesting Dimer - II to be a physiologically relevant dimer . Residues mutated in this study are highlighted with circles . B . Size Exclusion Chromatography ( SEC ) and SEC - MALS ( SEC combined with Multi Angle Light scattering ) analyses of the recombinant wild - type ( wt ) and dimer disrupting mutant ( I31A ) of spMis181 - 120 . Measured molecular weights from SEC - MALS confirm that spMis181 - 120 wt is a dimer in solution and spMis181 - 120 I31A is a monomer . C . Evaluation of the ability of Yippee - like domains of human Mis18 proteins ( hsMis18α77 - 187 and hsMis18β56 - 183 ) to form a heterodimer . SEC and SEC - MALS analyses demonstrate that while hsMis18α77 - 187 elutes at 15 . 5 ml as a dimer ( 31 . 3 kDa ) , hsMis18β56 - 183 elutes at 17 . 3 ml as a monomer ( 15 kDa ) . Purified hsMis18α77 - 187 and hsMis18β56 - 183 when mixed together elute at 15 . 4 ml as a heterodimer ( 27 . 5 kDa ) . D . Cartoon representation of the structure of the homology - modelled human Mis18α77 - 187 - Mis18β56 - 183 heterodimer using the crystal structure of spMis181 - 120 reported here as a template . Residues mutated in this study are highlighted with circles . Modelling was carried out using Phyre2 web server ( www . sbg . bio . ic . ac . uk / phyre2 / ) . E . SDS - PAGE analysis of the GST - pull down assay where wt and dimer disrupting mutants of hsMis18α77 - 187 and hsMis18β56 - 183 were co - expressed as His - and GST - tagged proteins , respectively in E . coli . While wt GST - hsMis18β56 - 183 showed interaction with wt His - hsMis18α77 - 187 , hsMis18 proteins harbouring dimer - disrupting mutations , GST - hsMis18β56 - 183 V77E / Y172D and His - hsMis18α77 - 187 V82E / Y176D did not show noticeable interaction . The corresponding Ni - NTA pull downs showing the expression of His - tagged proteins are shown in Fig EV2D .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Cartoon representation of the assembly of spMis18 Yippee-like domains (spMis181-120) as observed in the crystal asymmetric unit. Close up views highlight the amino acid composition and interactions stabilizing the dimeric arrangements. Interface-II is composed of conserved residues and an extensive binding surface as compared to Interface-I (see text for details), suggesting Dimer-II to be a physiologically relevant dimer. Residues mutated in this study are highlighted with circles.B. Size Exclusion Chromatography (SEC) and SEC-MALS (SEC combined with Multi Angle Light scattering) analyses of the recombinant wild-type (wt) and dimer disrupting mutant (I31A) of spMis181-120. Measured molecular weights from SEC-MALS confirm that spMis181-120 wt is a dimer in solution and spMis181-120 I31A is a monomer.C. Evaluation of the ability of Yippee-like domains of human Mis18 proteins (hsMis18α77-187 and hsMis18β56-183) to form a heterodimer. SEC and SEC-MALS analyses demonstrate that while hsMis18α77-187 elutes at 15.5 ml as a dimer (31.3 kDa), hsMis18β56-183 elutes at 17.3 ml as a monomer (15 kDa). Purified hsMis18α77-187 and hsMis18β56-183 when mixed together elute at 15.4 ml as a heterodimer (27.5 kDa).D. Cartoon representation of the structure of the homology-modelled human Mis18α77-187 - Mis18β56-183 heterodimer using the crystal structure of spMis181-120 reported here as a template. Residues mutated in this study are highlighted with circles. Modelling was carried out using Phyre2 web server (www.sbg.bio.ic.ac.uk/phyre2/).E. SDS-PAGE analysis of the GST-pull down assay where wt and dimer disrupting mutants of hsMis18α77-187 and hsMis18β56-183 were co-expressed as His- and GST-tagged proteins, respectively in E. coli. While wt GST-hsMis18β56-183 showed interaction with wt His-hsMis18α77-187, hsMis18 proteins harbouring dimer-disrupting mutations, GST-hsMis18β56-183 V77E/Y172D and His-hsMis18α77-187 V82E/Y176D did not show noticeable interaction. The corresponding Ni-NTA pull downs showing the expression of His-tagged proteins are shown in Fig EV2D.


Figure 3A B . SEC - MALS analysis of His - GFP - spMis18fl and His - spMis18ΔC , respectively . Refractive index ( RI , left Y - axis ) and molecular mass ( kDa , right Y - axis ) profiles show that both His - GFP - spMis18fl ( predicted MW of a monomer is 51 . 97 kDa ) and His - spMis18ΔC ( Predicted MW of a monomer 21 . 8 kDa ) predominantly exist as tetramers ( 187 . 6 kDa and 78 . 6 kDa , respectively ) with a small population of dimer ( 128 . 5 kDa and 45 . 8 kDa , respectively ) . C . SEC profile of a sample containing an equimolar mixture of purified His - GFP - spMis18C - term - α and His - spMis18MeDiY ( upper panel ) and SDS - PAGE analysis of SEC - fractions ( bottom panel ) . His - GFP - spMis18C - term - α and His - spMis18MeDiY eluted separately at 12 . 3 and 15 . 3 ml , respectively , demonstrating the inability of these domains to interact with each other . D . SEC - MALS analysis of His - GFP - spMis18C - term - α shows that it exists as a homotrimer with a measured molecular weight of 119 . 8 kDa . ( Theoretically calculated MW of a monomer is 38 . 3 kDa ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A B. SEC-MALS analysis of His-GFP-spMis18fl and His-spMis18ΔC, respectively. Refractive index (RI, left Y-axis) and molecular mass (kDa, right Y-axis) profiles show that both His-GFP-spMis18fl (predicted MW of a monomer is 51.97 kDa) and His-spMis18ΔC (Predicted MW of a monomer 21.8 kDa) predominantly exist as tetramers (187.6 kDa and 78.6 kDa, respectively) with a small population of dimer (128.5 kDa and 45.8 kDa, respectively).C. SEC profile of a sample containing an equimolar mixture of purified His-GFP-spMis18C-term-α and His-spMis18MeDiY (upper panel) and SDS-PAGE analysis of SEC-fractions (bottom panel). His-GFP-spMis18C-term-α and His-spMis18MeDiY eluted separately at 12.3 and 15.3 ml, respectively, demonstrating the inability of these domains to interact with each other.D. SEC-MALS analysis of His-GFP-spMis18C-term-α shows that it exists as a homotrimer with a measured molecular weight of 119.8 kDa. (Theoretically calculated MW of a monomer is 38.3 kDa).


Figure 4A . spMis18fl - Myc co - immunoprecipitates with both GFP - tagged spMis18fl and spMis18MeDiY , but shows reduced association when MeDiY - mediated dimerization is disrupted in spMis18flI31A . The asterisk ( * ) in the top panel denotes the IgG heavy chain . B C . Dimer - II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18 - 818 and mis18 - 262 cells , while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization - dependent manner . Five - fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome , spotted on complete PMG + Phloxine B media supplemented with ( repressed ) or without ( expressed ) thiamine , and incubated at the indicated temperatures ; dead cells stain dark pink . D . Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres . qChIP analyses of spMis18fl - GFP association with centromere 2 ( cc2 ) in the indicated strains when grown in complete PMG media supplemented with ( repressed ) or without ( expressed ) thiamine . Error bars represent standard deviation between at least three biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. spMis18fl-Myc co-immunoprecipitates with both GFP-tagged spMis18fl and spMis18MeDiY, but shows reduced association when MeDiY-mediated dimerization is disrupted in spMis18flI31A. The asterisk (*) in the top panel denotes the IgG heavy chain.B C. Dimer-II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-818 and mis18-262 cells, while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization-dependent manner. Five-fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.D. Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres. qChIP analyses of spMis18fl-GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.


Figure 1 ( C ) Brucella replication in BMMs expressing either GFP or GFP - BspF and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria , measured as number of bacteria per cell at 24 h pi . Data are means ± SD of n = 3 independent experiments . Grey dots represent values from individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons test ) compared to controls .

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Figure 1(C) Brucella replication in BMMs expressing either GFP or GFP-BspF and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments. Grey dots represent values from individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) compared to controls.


Figure 2 ( A ) Quantification of ss - eGFP - FKBPF36M trafficking in HeLa ( M ) - C1 cells transfected for 24 h with pmCherry ( mCherry ) or pmCherry - BspF ( mCherry - BspF ) . Rapamycin was added to initiate secretory traffic of ss - eGFP - FKBPF36M and its colocalization with calnexin ( ER ) , ERGIC - 53 ( ERGIC ) , GM130 ( Golgi ) , p230 ( TGN ) , or secretory vesicles ( SV ) was scored over a 60 min time course . Data are means ± SD from n = 3 independent experiments . Asterisks indicate statistically significant differences between mCherry - and mCherry - BspF - expressing cells as determined by a two - way ANOVA with Sidak ' s multiple comparisons test ( P < 0 . 05 ) . ( F ) Representative confocal fluorescence micrographs of HeLa cells co - transfected for 24 h to produce mCherry - BspF and either GFP - Rab6a , GFP - Rab8a or Arf6 - GFP and treated with Cytochalasin D ( 200 nM ) for 30 min prior to fixation . Scale bars : 10 and 2 µm ( insets ) . ( G ) Quantification of localization of GFP - Rab6a , GFP - Rab8a and Arf6 - GFP on mCherry - BspF - labelled tubules in transfected HeLa cells . Data are means ± SD from n = 3 independent experiments , in which at least 300 individual cells per experiment were analyzed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Quantification of ss-eGFP-FKBPF36M trafficking in HeLa(M)-C1 cells transfected for 24 h with pmCherry (mCherry) or pmCherry-BspF (mCherry-BspF). Rapamycin was added to initiate secretory traffic of ss-eGFP-FKBPF36M and its colocalization with calnexin (ER), ERGIC-53 (ERGIC), GM130 (Golgi), p230 (TGN), or secretory vesicles (SV) was scored over a 60 min time course. Data are means ± SD from n=3 independent experiments. Asterisks indicate statistically significant differences between mCherry- and mCherry-BspF-expressing cells as determined by a two-way ANOVA with Sidak's multiple comparisons test (P<0.05).(F) Representative confocal fluorescence micrographs of HeLa cells co-transfected for 24 h to produce mCherry-BspF and either GFP-Rab6a, GFP-Rab8a or Arf6-GFP and treated with Cytochalasin D (200 nM) for 30 min prior to fixation. Scale bars: 10 and 2 µm (insets). (G) Quantification of localization of GFP-Rab6a, GFP-Rab8a and Arf6-GFP on mCherry-BspF-labelled tubules in transfected HeLa cells. Data are means ± SD from n=3 independent experiments, in which at least 300 individual cells per experiment were analyzed.


Figure 3 ( A ) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry - BspF ( grayscale panels ) , incubated on ice with AlexaFluor ™ 488 - Cholera Toxin subunit B ( CTxB ; green ) and shifted to 37 ˚ C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus ( stained using an anti - GM130 antibody ; purple ) . CTxB accumulation within Golgi structures appears white in overlays . Scale bars : 10 and 2 µm ( insets ) . ( B ) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry , mCherry - BspF or HA - BspF over a 30 min time course , expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker , as in ( A ) . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate statistically significant differences compared to mCherry - producing cells as determined by a two - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( ( D ) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA - mediated depletion of either Arf6 ( siArf6 ) , Rab8a ( siRab8a ) or Rab6a / a ' ( siRab6a / a ' ) after AlexaFluor ™ 488 - CTxB binding on ice followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( E ) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock - infected or infected with wild type ( 2308 ) , ΔbspF , complemented ∆ bspF ( ΔbspF : : bspF ) , ∆ bspB or complemented ∆ bspB ( ΔbspB : : bspB ) bacteria for 24 h , incubated for 30 min with AlexaFluor ™ 488 - CTxB on ice for binding followed by 30 min incubation at 37 ˚ C . Data are means ± SD from n = 3 independent experiments , in which 100 cells were analyzed per experiment . Asterisks indicate a statistically significant difference compared to mock - infected cells as determined by a one - way ANOVA with Tukey ' s multiple comparisons test ( P < 0 . 05 ) . ( ( G - I ) Brucella replication in BMMs treated with non - targeting siRNAs ( siNT ) , or siRNAs against Rab6a / a ' ( siRab6a / a ' ) ( G ) , Rab8a ( siRab8a ) ( H ) or Arf6 ( siArf6 ) ( I ) and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria , measured as number of bacteria per cell at 24 h pi . Data are means ± SD of n = 3 independent experiments in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative confocal fluorescence micrographs of HeLa cells transfected for 24 h to produce either mCherry or mCherry-BspF (grayscale panels), incubated on ice with AlexaFluor™488-Cholera Toxin subunit B (CTxB; green) and shifted to 37˚C for 20 min to allow for CTxB retrograde transport to the Golgi apparatus (stained using an anti-GM130 antibody; purple). CTxB accumulation within Golgi structures appears white in overlays. Scale bars: 10 and 2 µm (insets). (B) Quantification of CTxB transport to the Golgi apparatus in HeLa cells producing either mCherry, mCherry-BspF or HA-BspF over a 30 min time course, expressed as percentages of cells in which CTxB colocalized with the GM130 Golgi marker, as in (A). Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate statistically significant differences compared to mCherry-producing cells as determined by a two-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( (D) Quantification of CTxB transport to the Golgi apparatus in BMMs following siRNA-mediated depletion of either Arf6 (siArf6), Rab8a (siRab8a) or Rab6a/a' (siRab6a/a') after AlexaFluor™488-CTxB binding on ice followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to siNT control cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). (E) Quantification of CTxB transport to the Golgi apparatus in BMMs that were either mock-infected or infected with wild type (2308), ΔbspF, complemented ∆bspF (ΔbspF::bspF), ∆bspB or complemented ∆bspB (ΔbspB::bspB) bacteria for 24 h, incubated for 30 min with AlexaFluor™488-CTxB on ice for binding followed by 30 min incubation at 37˚C. Data are means ± SD from n=3 independent experiments, in which 100 cells were analyzed per experiment. Asterisks indicate a statistically significant difference compared to mock-infected cells as determined by a one-way ANOVA with Tukey's multiple comparisons test (P<0.05). ( (G-I) Brucella replication in BMMs treated with non-targeting siRNAs (siNT), or siRNAs against Rab6a/a' (siRab6a/a') (G), Rab8a (siRab8a) (H) or Arf6 (siArf6) (I) and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria, measured as number of bacteria per cell at 24 h pi. Data are means ± SD of n=3 independent experiments in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed; black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


Figure 4 ( B ) Representative co - immunoprecipitations of HA - BspF and myc - ACAP1 in HeLa cells . HeLa cells were transfected to either co - produce or individually produce HA - BspF and myc - ACAP1 and either HA - BspF or myc - ACAP1 were immunoprecipitated using either anti - HA - conjugated ( upper panel ) or anti - myc - conjugated ( lower panel ) magnetic beads following cross - linking ( + DSP ) or not ( - DSP ) with dithiobis [ succinimidylpropionate ] . Input lysates ( 10 % of the post - nuclear supernatant ) and co - immunoprecipitates were separated by SDS - PAGE and probed for HA - BspF and myc - ACAP1 by Western blotting . ( C ) Representative confocal micrographs of HeLa cells transfected to produce mCherry - BspF and GFP - ACAP1 and treated with Cytochalasin D ( 200 nM ) for 30 min and quantification of colocalization between mCherry - BspF and GFP - ACAP1 . Arrows indicate areas of BspF and ACAP1 colocalization . Scale bars : 10 µm and 2 µm ( insets ) . Data are means ± SD from n = 3 independent experiments in which 10 cells were analyzed per experiment . Pearson ' s correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc _ 2 plug - in .

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Figure 4(B) Representative co-immunoprecipitations of HA-BspF and myc-ACAP1 in HeLa cells. HeLa cells were transfected to either co-produce or individually produce HA-BspF and myc-ACAP1 and either HA-BspF or myc-ACAP1 were immunoprecipitated using either anti-HA-conjugated (upper panel) or anti-myc-conjugated (lower panel) magnetic beads following cross-linking (+DSP) or not (-DSP) with dithiobis[succinimidylpropionate]. Input lysates (10% of the post-nuclear supernatant) and co-immunoprecipitates were separated by SDS-PAGE and probed for HA-BspF and myc-ACAP1 by Western blotting.(C) Representative confocal micrographs of HeLa cells transfected to produce mCherry-BspF and GFP-ACAP1 and treated with Cytochalasin D (200 nM) for 30 min and quantification of colocalization between mCherry-BspF and GFP-ACAP1. Arrows indicate areas of BspF and ACAP1 colocalization. Scale bars: 10 µm and 2 µm (insets). Data are means ± SD from n=3 independent experiments in which 10 cells were analyzed per experiment. Pearson's correlation coefficients were calculated from whole cells using NIH Fiji image analysis software and Coloc_2 plug-in.


Figure 5 ( B ) Representative Western blot analysis of co - immunoprecipitations of myc - ACAP1 and Arf6 - HA in the presence or absence of HA - BspF . HeLa cells were transfected to produce Arf6 - HA and combinations of myc - ACAP1 and HA - BspF , or not , and myc - ACAP1 was immunoprecipitated using anti - myc - conjugated magnetic beads . Input lysates ( 6 % of post - nuclear supernatants ) and co - immunoprecipitates were separated by SDS - PAGE and probed for Arf6 - HA , HA - BspF and myc - ACAP1 by Western blotting . Quantification of the Arf6 / ACAP1 ratio was performed by densitometric analysis . Data are means ± SD of 3 independent experiments . The asterisk indicates a statistically significant difference ( P = 0 . 0017 , unpaired Student ' s t test ) between BspF - producing and control conditions . ( E ) Bacterial replication in BMMs transduced to either produce GFP , GFP - ACAP1 or GFP - ACAP1R448Q and infected with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria for 24 h . Data are means ± SD of n = 3 independent experiments , in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , two - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

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Figure 5(B) Representative Western blot analysis of co-immunoprecipitations of myc-ACAP1 and Arf6-HA in the presence or absence of HA-BspF. HeLa cells were transfected to produce Arf6-HA and combinations of myc-ACAP1 and HA-BspF, or not, and myc-ACAP1 was immunoprecipitated using anti-myc-conjugated magnetic beads. Input lysates (6% of post-nuclear supernatants) and co-immunoprecipitates were separated by SDS-PAGE and probed for Arf6-HA, HA-BspF and myc-ACAP1 by Western blotting. Quantification of the Arf6/ACAP1 ratio was performed by densitometric analysis. Data are means ± SD of 3 independent experiments. The asterisk indicates a statistically significant difference (P = 0.0017, unpaired Student's t test) between BspF-producing and control conditions.(E) Bacterial replication in BMMs transduced to either produce GFP, GFP-ACAP1 or GFP-ACAP1R448Q and infected with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria for 24 h. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, two-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


Figure 6 ( D - F ) Recruitment of Stx6 - positive vesicles to rBCVs in BMMs treated with non - targeting siRNAs ( siNT ) , siRNAs against Arf6 ( siArf6 ) ( D ) , Rab8a ( siRab8a ) ( E ) or Rab6a / a ' ( siRab6a / a ' ) ( F ) and infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria . Data are means ± SD of n = 3 independent experiments , in which 200 BCVs were analyzed per experiment . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions ; ns , not significant . ( H ) Brucella replication in BMMs treated with either non - targeting siRNAs ( siNT ) , or siRNAs against Stx6 ( siStx6 ) and infected for 24 h with either wild type ( 2308 ) , ΔbspF , or complemented ∆ bspF ( ΔbspF : : bspF ) bacteria . Data are means ± SD of n = 3 independent experiments , in which at least 100 cells were analyzed per experiment . Grey dots represent individual cells analyzed ( n > 300 ) ; black dots indicate means of individual experiments . Asterisks indicate statistically significant differences ( P < 0 . 05 , one - way ANOVA followed by Dunnett ' s multiple comparisons test ) between test and control conditions .

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Figure 6(D-F) Recruitment of Stx6-positive vesicles to rBCVs in BMMs treated with non-targeting siRNAs (siNT), siRNAs against Arf6 (siArf6) (D), Rab8a (siRab8a) (E) or Rab6a/a' (siRab6a/a') (F) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which 200 BCVs were analyzed per experiment. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions; ns, not significant.(H) Brucella replication in BMMs treated with either non-targeting siRNAs (siNT), or siRNAs against Stx6 (siStx6) and infected for 24 h with either wild type (2308), ΔbspF, or complemented ∆bspF (ΔbspF::bspF) bacteria. Data are means ± SD of n=3 independent experiments, in which at least 100 cells were analyzed per experiment. Grey dots represent individual cells analyzed (n>300); black dots indicate means of individual experiments. Asterisks indicate statistically significant differences (P<0.05, one-way ANOVA followed by Dunnett's multiple comparisons test) between test and control conditions.


Figure 1 . ( A - D ) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9 - mo - old NTg mouse brains ( A , arrowheads outline normal neurites ) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1 / APP mice ( B , arrowheads outline dystrophic neurite profiles in C ) and biopsied brain material from an AD patient ( B , inset ) . At higher magnification ( C ) , AVs include autophagosomes ( arrows ) and multilamellar bodies ( arrowhead ) . In normal dendrites of PS1 / APP mice , multiple AVs are frequently seen ( D , arrows ) . ( E and F ) LC3 quantification analyzed from immunoblots of LC3 - I and LC3 - II ( top ) in prefrontal cortical homogenates from cases of nonaffected ( Cont ) , early stage ( preclinical ) AD ( AD - ES ) , and moderate AD ( AD - MS ; E ) , and from brains of 18 - 22 - mo - old PS1 / APP ( PA ) mice ( n = 3 ; F ) compared with nontransgenic ( NTg ) controls ( n = 3 ; * , P < 0 . 01 ) . Error bars represent SEM . ( G - L ) LC3 immunofluorescence in 9 - mo - old PS1 / APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex ( G , arrows ) and along adjacent dendrites . LC3 ( H , arrows ) is strong in dystrophic neurites in the periphery ( asterisks ) of a thioflavin S - labeled plaque core ( H , inset ) but is less so in neurites closest ( H , arrowheads ) to the Aβ deposit . LC3 is diffuse and uniform in neurons of NTg mice ( I and J ) but is predominantly vesicular and distributed more to the dendrites ( arrows ) than the cell soma ( arrowheads ) in 9 - mo - old PS1 / APP cortex ( K and L ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(A-D) EM images of cortical neuropil show an absence of AVs and normal neurite profile in 9-mo-old NTg mouse brains (A, arrowheads outline normal neurites) and a marked accumulation of AVs within enlarged or dystrophic neurites in PS1/APP mice (B, arrowheads outline dystrophic neurite profiles in C) and biopsied brain material from an AD patient (B, inset). At higher magnification (C), AVs include autophagosomes (arrows) and multilamellar bodies (arrowhead). In normal dendrites of PS1/APP mice, multiple AVs are frequently seen (D, arrows).(E and F) LC3 quantification analyzed from immunoblots of LC3-I and LC3-II (top) in prefrontal cortical homogenates from cases of nonaffected (Cont), early stage (preclinical) AD (AD-ES), and moderate AD (AD-MS; E), and from brains of 18-22-mo-old PS1/APP (PA) mice (n = 3; F) compared with nontransgenic (NTg) controls (n = 3; *, P < 0.01). Error bars represent SEM.(G-L) LC3 immunofluorescence in 9-mo-old PS1/APP mice can be seen mainly as puncta in dystrophic dendrites of the cortex (G, arrows) and along adjacent dendrites. LC3 (H, arrows) is strong in dystrophic neurites in the periphery (asterisks) of a thioflavin S-labeled plaque core (H, inset) but is less so in neurites closest (H, arrowheads) to the Aβ deposit. LC3 is diffuse and uniform in neurons of NTg mice (I and J) but is predominantly vesicular and distributed more to the dendrites (arrows) than the cell soma (arrowheads) in 9-mo-old PS1/APP cortex (K and L).


Figure 2 . Ultrastructural inspection of brain tissue from PS1 / APP mice ( A - D ) shows that AVs ( A and B , arrows ) are five times more frequent in the dendrites of 8 - wk - old PS1 / APP than in those of age - matched NTg mice . The frequency of AVs per EM field ( C ) and mean number of AVs per EM field ( D ) within the hippocampal molecular layer ( n = 3 ) are shown . LC3immunoblot and analysis ( E ) and immunofluorescent labeling ( F - K ) of the hippocampaldendrites ( brackets ) in 8 - 9 - wk - old PS1 / APP and NTg mice show LC3 - II elevation ( P < 0 . 05 ) in 8 - wk - old PS1 / APP compared with NTg mice ( E ) . ( D ) * , P < 0 . 001 . ( E ) * , P < 0 . 05 . Error bars represent SEM . LC3 immunoreactivity in pyramidal celldendrites is increased in 9 - mo - old ( F - H ) and 9 - wk - old ( I - K ) PS1 / APPmice and frequently exhibits a punctate labeling pattern , which is more evident at 9 mo than at 9 wk ( H and K , arrows ) and is uncommon in NTg mice ( F and I ) . Bars ( F , G , I , and J ) , 20 μm ; ( H and K ) , 10 μm .

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Figure 2.Ultrastructural inspection of brain tissue from PS1/APP mice (A-D) shows that AVs (A and B, arrows) are five times more frequent in the dendrites of 8-wk-old PS1/APP than in those of age-matched NTg mice. The frequency of AVs per EM field (C) and mean number of AVs per EM field (D) within the hippocampal molecular layer (n = 3) are shown.LC3immunoblot and analysis (E) and immunofluorescent labeling (F-K) of the hippocampaldendrites (brackets) in 8-9-wk-old PS1/APP and NTg mice show LC3-II elevation (P < 0.05) in 8-wk-old PS1/APP compared with NTg mice (E). (D) *, P < 0.001. (E) *, P < 0.05. Error bars represent SEM. LC3 immunoreactivity in pyramidal celldendrites is increased in 9-mo-old (F-H) and 9-wk-old (I-K) PS1/APPmice and frequently exhibits a punctate labeling pattern, which is more evident at 9 mo than at 9 wk (H and K, arrows) and is uncommon in NTg mice (F and I). Bars (F, G, I, and J), 20 μm; (H and K), 10 μm.


Figure 3 . Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1 / APP mice . Cingulate cortex from 9 - mo - old PS1 / APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques ( A ) . At higher magnification , anti - PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona ( B ) . PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1 / APP animals ( C and D ) Arrowheads identify tubulovesicular membrane labeling . PS1 ( C - F , arrows ) was localized on the outer limiting membrane of the AV but not in mitochondria ( Mito ) or on plasma membranes ( PM ) . PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain ( E and F ) by IEM . Arrowheads identify tubulovesicular membrane labeling . PS1 ( C - F , arrows ) was localized on the outer limiting membrane of the AV but not in mitochondria ( Mito ) or on plasma membranes ( PM ) . IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD ( F ) .

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Figure 3.Immunolocalization of PS1 in plaques and AVs within dystrophic neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 localized to plaques (A). At higher magnification, anti-PS1 antibodies strongly labeled neuritic profiles that were distributed within the plaque corona (B).PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of PS1/APP animals (C and D)Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM).PS1 immunoreactivity is identified by IEM in AVs within dystrophic neurites of human brain (E and F) by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C-F, arrows) was localized on the outer limiting membrane of the AV but not in mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver stain enhancement for PS1 was performed on a humanbrain that was diagnosed for AD (F).


Figure 4 . Induction of macroautophagy in L / APP , SH - SY5Y , and N2a cells . ( A and B ) EM images showing changes in the number of AVs ( arrows ) in L / APP - overexpressing APP695 ( L / APP cells ) grown in complete medium ( A , top left ) or in medium lacking Leu and His ( A , top right ) for 6 h and in SH - SY5Y cells grown in the presence ( A , bottom left ) or absence ( A , bottom right ) of serum . At higher magnification , early and late AVs with typical morphologies are seen in a Leu / His - deprived L / APP cell ( B ) . ( C ) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine ( 0 . 5 μg / ml for 30 min ; left ) and LC3 antibody ( middle , L / APP ; right , SH - SY5Y ) in macroautophagy - induced cells ( bottom ) , which are much less abundant in cells grown in complete medium ( top ) . ( D ) Western blots confirm the cytochemical evidence for increased LC3 - II levels as well as phospho - mTOR ( P - 2481 ) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin ( Rap ) and macroautophagy inhibition by 5 mM 3MA in L / APP , N2a , and SH - SY5Y cells . Immunoblots for LC3 in SH - SY5Y cells and P - 2481 mTOR in L / APP cells have been spliced but are derived from the same blot .

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Figure 4.Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B).(C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top).(D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.


Figure 5 . Aβ generation in cells after autophagic modulation . Levels of Aβ40 ( A ) , Aβ42 ( B ) , βCTF ( C ) , and APP ( D ) measured by sandwich ELISA after the incubation of L / APP cells ( 6 h ) in conditions that block autophagy ( + Leu , + His , + Leu / + His , and 5 mM 3MA ) , activate macroautophagy ( − Leu , − His , − Leu / − His , and rapamycin ) , or do not affect autophagy ( complete media and enrichment of the deprivation of Gly or Val ) . Values reported as percent difference of control ± SEM ; * , P < 0 . 05 ( at least ) . In similar experiments , Aβ40 and Aβ42 levels from SH - SY5Y ( E and F ) and N2a cells ( G and H ) after various treatments . Error bars represent SEM .

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Figure 5.Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least).In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.


Figure 6 . Immunolocalization of Aβ in AVs from L / APP cells and PS1 / APPbrains and γ - secretase components ( PS1 and nicastrin ) in L / APP cells . Immunogold localization of Aβ40 ( A ) , Aβ42 ( B ) , PS1 ( C ) , nicastrin ( D ) , and in the absence of primary antibody ( E ) in L / APP cells grown for 6 h in the absence of Leu and His . ( F ) Quantification of gold particle frequency in AV or tubulovesicular compartments ( TBV ) , which comprise 27 . 0 ± 11 . 0 and 19 . 2 ± 7 . 2 % , respectively , of the total cell area . Error bars represent SEM . ( G and H ) IEM followed by silver stain enhancement for Aβ40 was performed in 9 - mo - old PS1 / APP mice .

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Figure 6.Immunolocalization of Aβ in AVs from L/APP cells and PS1/APPbrains and γ-secretase components (PS1 and nicastrin) in L/APP cells. Immunogold localization of Aβ40 (A), Aβ42 (B), PS1 (C), nicastrin (D), and in the absence of primary antibody (E) in L/APP cells grown for 6 h in the absence of Leu and His. (F) Quantification of gold particle frequency in AV or tubulovesicular compartments (TBV), which comprise 27.0 ± 11.0 and 19.2 ± 7.2%, respectively, of the total cell area. Error bars represent SEM.(G and H) IEM followed by silver stain enhancement for Aβ40 was performed in 9-mo-old PS1/APP mice.


Figure 7 . ( A and B ) Ultrastructure of AVs ( A1 and A2 ) and lysosomes in subcellular fractions from serum - deprived L / APP cells ( A ) and Western blot analysis ( B ) for LC3 - II , rab24 , APP , βCTF , PS1 ( PS1 amino - terminal fragment ) , and nicastrin ( NCT ) in L / APP subcellular fractions . AVs , A1 and A2 ; L , lysosomes ; E , tubulovesicular compartments ( Golgi / ER / endosomes ) ; P , postnuclear pellet ; C , cytosol ; M , mitochondria . Empty lanes in the original blot have been removed from the figure and noted with a white line . ( C ) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L / APP cells grown in − serum media ( left ) or − Leu / − His media ( right ) . PNP , postnuclear pellet . ( D and E ) Proportions of the total recovered cell γ - secretase activity in different subcellular fractions after serum deprivation ( D ) or in uninduced ( + serum ) or induced ( − Leu / − His ) cells ( E ) . Comparison of + serum versus − Leu and − His conditions shows the redistribution of γ - secretase activity from the tubulovesicular to AV fractions after macroautophagic induction . G , Golgi ; A , AV . ( F ) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs . Selected ion chromatogram corresponding to different cleavage products is displayed . Numbers in graph refer to the cleavage / amino acid site from the Aβ peptide . ( G ) γ - Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted ( PS KO ; BD8 ) or in which human PS1 was stably transfected into the PS KO blastocysts ( hPS1 ; BD8 / hPS1 ) . Numbers on x axis are in minutes . ( H ) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA . Values are given as means ± SEM ( error bars ) .

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Figure 7.(A and B) Ultrastructure of AVs (A1 and A2) and lysosomes in subcellular fractions from serum-deprived L/APP cells (A) and Western blot analysis (B) for LC3-II, rab24, APP, βCTF, PS1 (PS1 amino-terminal fragment), and nicastrin (NCT) in L/APP subcellular fractions. AVs, A1 and A2; L, lysosomes; E, tubulovesicular compartments (Golgi/ER/endosomes); P, postnuclear pellet; C, cytosol; M, mitochondria. Empty lanes in the original blot have been removed from the figure and noted with a white line.(C) Rates of cleavage of the fluorogenic substrate in subcellular fractions from L/APP cells grown in −serum media (left) or −Leu/−His media (right). PNP, postnuclear pellet.(D and E) Proportions of the total recovered cell γ-secretase activity in different subcellular fractions after serum deprivation (D) or in uninduced (+serum) or induced (−Leu/−His) cells (E). Comparison of +serum versus −Leu and −His conditions shows the redistribution of γ-secretase activity from the tubulovesicular to AV fractions after macroautophagic induction. G, Golgi; A, AV.(F) Liquid chromatography mass spectrometry analysis of the fluorogenic substrate after incubation with purified AVs. Selected ion chromatogram corresponding to different cleavage products is displayed. Numbers in graph refer to the cleavage/amino acid site from the Aβ peptide.(G) γ-Secretase activity in AV and lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted (PS KO; BD8) or in which human PS1 was stably transfected into the PS KO blastocysts (hPS1; BD8/hPS1). Numbers on x axis are in minutes.(H) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA. Values are given as means ± SEM (error bars).


Figure 1 ( E ) Quantitative RT - PCR ( qRT - PCR ) of μGLT , αGLT , and AID from siControl - and siPhf5a - treated samples . The values are presented as mean ± sd ( n = 3 ) .

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Figure 1(E) Quantitative RT-PCR (qRT-PCR) of μGLT, αGLT, and AID from siControl- and siPhf5a-treated samples. The values are presented as mean ± sd (n=3).


Figure 2 ( B ) Southern blots of the IgH / c - Myc translocation assay using genomic DNA extracted from CIT - stimulated CH12F3 - 2A cells treated with the siRNAs shown at the bottom of each panel . Numbers on the top indicate gel lanes . ( C ) Frequency of IgH / c - Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample ( Boboila et al . , 2012b ) . The values are presented as mean ± sd ( n = 3 ) . Statistical significance was assessed by two tailed unpaired Student ' s t - test ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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Figure 2(B) Southern blots of the IgH/c-Myc translocation assay using genomic DNA extracted from CIT-stimulated CH12F3-2A cells treated with the siRNAs shown at the bottom of each panel. Numbers on the top indicate gel lanes. (C) Frequency of IgH/c-Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample (Boboila et al., 2012b). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (** p ≤ 0.01, *** p ≤ 0.001).


Figure 3 ( D - E ) Detection of Sμ DNA double - strand breaks by LM - PCR in CH12F3 - 2A cells transfected with the indicated siRNA combinations . The DNA samples were either left untreated ( none ) or treated with indicated end processing enzymes prior to linker ligation .

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Figure 3(D-E) Detection of Sμ DNA double-strand breaks by LM-PCR in CH12F3-2A cells transfected with the indicated siRNA combinations. The DNA samples were either left untreated (none) or treated with indicated end processing enzymes prior to linker ligation.


Figure 4 ( B ) Effect of Phf5a knockdown on I - SceI - induced NHEJ . Plot represents the GFP - positive cells obtained by FACS analysis after co - transfection of the I - SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct . The values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated against corresponding siControl by two - tailed unpaired Student ' s t - test ( * * * p ≤ 0 . 001 ) .

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Figure 4(B) Effect of Phf5a knockdown on I-SceI-induced NHEJ. Plot represents the GFP-positive cells obtained by FACS analysis after co-transfection of the I-SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001).


Figure 5 ( C ) Top : schematic diagram of the position of the ChIP assay PCR products specific to the S region . Bottom : ChIP assay of the indicated DNA repair proteins using CIT - stimulated CH12F3 - 2A cells transfected with siControl or siPhf5a . The values are presented as mean ± sd ( n = 3 ) . Statistical significance was evaluated in reference to siControl by two - tailed unpaired Student ' s t - test ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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Figure 5(C) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).


Figure 6 ( C ) Western blot analysis of the whole cell extract from CIT - stimulated CH12F3 - 2A cells transfected with the indicated siRNAs . Expression of various proteins including AID , variant H2As and Ku80 were not decresed by Phf5a KD . ( ns , non - specific )

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Figure 6(C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)