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 .

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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.


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

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


Western 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 .

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Western 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 .

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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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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.


mRNA ( 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 .

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mRNA (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 .

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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 .

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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.


B ) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells .

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B) 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 .

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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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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.


Hi - 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 .

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Hi-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 .

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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 .

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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 .

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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.


Interaction 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 .

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Interaction 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 .

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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 .

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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.


A ) 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 .

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A) 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 .

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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 .

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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 .

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 .

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E) Scaling plot generated by 500kb resolution Hi-C data showing the interaction frequency as a function of genomic distance.


A ) 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 .

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A) 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 .

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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 .

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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 .

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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 .

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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 .

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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.


C . SDS - PAGE showing a representative fraction of the purified spMis18 N - terminal Yippee - like globular domain ( amino acid residues 1 - 120 ; spMis181 - 120 ) .

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C. 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 .

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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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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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 .

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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 ) .

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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 / ) .

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 .

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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.


A 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 ) .

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A 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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


A . 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 .

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A. 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 .

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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 .

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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.


( 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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(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.


( 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 ) .

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(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 .

(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.


( 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 ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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.


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O 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) 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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(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.


( 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 .

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(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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(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.


( 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 .

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(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 ) .

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(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 .

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(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 ) .

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(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).


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 .

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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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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.


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 ) .

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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 ) .

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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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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).


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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

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 ) .

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(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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(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.


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 ) .

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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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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.


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 .

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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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(G and H) IEM followed by silver stain enhancement for Aβ40 was performed in 9-mo-old PS1/APP mice.


( 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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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(H) Aβ40 and Aβ42 levels in medium from these cells as detected by sandwich ELISA. Values are given as means ± SEM (error bars).


( 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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(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).


( 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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(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).


( 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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(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.


( 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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(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).


( 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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(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).


( 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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(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)


( C ) CH12F3 - 2A cells were transfected with WT Phf5a or its mutants ( Phf5aR - MF ) along with siRNA against Phf5a . An anti - Flag antibody was used to immunoprecipitate and immunodetect Phf5aR - MF . An anti - Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a . The antibodies used to detect the co - associated proteins are indicated next to the respective blot .

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(C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot.


( B ) Western blot showing expression of endogenous and exogenously introduced Phf5a .

(B) Western blot showing expression of endogenous and exogenously introduced Phf5a.


( C ) H2A . Z ChIP assay was performed using CH12F3 - 2A cells transfected with indicated siRNA and the Phf5a expression constructs . The data are compiled from 2 experiments and presented relative to siControl treated sample , which was set as 100 . The error bars represent the mean ± sd .

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(C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.


C . Western blot ( WB ) analysis of splicing factors for Vector and 2FLAG - eIF4E U2OS cell lines . Myc , Mcl1 , and cyclin D1 ( CCND1 ) served as positive controls , and β - actin as a loading control . Both 2FLAG - eIF4E ( 2Flag - eIF4E ) and endogenous eIF4E ( endog - eIF4E ) are shown . Each β - actin blot corresponds to the above western blots . Experiments were carried out at least three independent times , and one representative experiment is shown .

C. Western blot (WB) analysis of splicing factors for Vector and 2FLAG-eIF4E U2OS cell lines. Myc, Mcl1, and cyclin D1 (CCND1) served as positive controls, and β-actin as a loading control. Both 2FLAG-eIF4E (2Flag-eIF4E) and endogenous eIF4E (endog-eIF4E) are shown. Each β-actin blot corresponds to the above western blots. Experiments were carried out at least three independent times, and one representative experiment is shown.


D . WB analysis of splicing factors as a function of eIF4E reduction using CRISPR - eIF4E and CRISPR - Ctrl U2OS cell lines . Myc , Mcl1 and CCND1 served as positive controls , and β - actin a loading control . Each β - actin blot corresponds to the western blots immediately above . Experiments were carried out at least three independent times , and one representative experiment is shown .

D. WB analysis of splicing factors as a function of eIF4E reduction using CRISPR-eIF4E and CRISPR-Ctrl U2OS cell lines. Myc, Mcl1 and CCND1 served as positive controls, and β-actin a loading control. Each β-actin blot corresponds to the western blots immediately above. Experiments were carried out at least three independent times, and one representative experiment is shown.


H . Total RNA levels monitored by RT - qPCR corresponding to mRNA export assays shown in G . Data were normalized to Vector control to calculate fold change . The mean and standard deviation , , as well as p - values ( using Student t - test ) , were derived from three biological experiments , each carried out in technical triplicate . Data information : Student t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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H. Total RNA levels monitored by RT-qPCR corresponding to mRNA export assays shown in G. Data were normalized to Vector control to calculate fold change. The mean and standard deviation, , as well as p-values (using Student t-test), were derived from three biological experiments, each carried out in technical triplicate. Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001


A . WB analysis of the impacts of S53A 2FLAG - eIF4E ( 2Flag - S53A ) relative to WT 2FLAG - eIF4E ( 2Flag - eIF4E ) on SF production . CCND1 served as positive controls , and β - actin or HSP90 as loading controls . Both 2FLAG - eIF4E ( 2Flag - eIF4E ) and endogenous eIF4E ( endog - eIF4E ) are shown . Each β - actin or HSP90 blot corresponds to the above western blots . S53A eIF4E protein levels are indistinguishable from eIF4E . Experiments were carried out using three separate clones for eIF4E , S53A and Vector controls , and one representative experiment is shown . ( Right ) Western blots were quantified by FIJI , intensities normalized to Vector control and plotted using PRISM . The mean , standard deviation , and p - values are shown . Student t - test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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A. WB analysis of the impacts of S53A 2FLAG-eIF4E (2Flag-S53A) relative to WT 2FLAG-eIF4E (2Flag-eIF4E) on SF production. CCND1 served as positive controls, and β-actin or HSP90 as loading controls. Both 2FLAG-eIF4E (2Flag-eIF4E) and endogenous eIF4E (endog-eIF4E) are shown. Each β-actin or HSP90 blot corresponds to the above western blots. S53A eIF4E protein levels are indistinguishable from eIF4E. Experiments were carried out using three separate clones for eIF4E, S53A and Vector controls, and one representative experiment is shown. (Right) Western blots were quantified by FIJI, intensities normalized to Vector control and plotted using PRISM. The mean, standard deviation, and p-values are shown. Student t-test: * p<0.05, **p<0.01, ***p<0.001.


B . Polysomal loading analysis was measured to assess the relevance of eIF4E ' s capacity to increase translation efficiency on SF protein production . Top panel shows the polysomal profile ( at 254 nm ) demonstrating that 2FLAG - eIF4E overexpression did not alter the profile relative to Vector controls , consistent with previous studies Polysomes were isolated by size exclusion chromatography ( SEC ) and thus the heaviest polysomes elute first and monosomes last . ( Middle and Bottom panels ) RNAs were monitored on heavy , light or monosome polysomes using RT - qPCR and presented as a fraction for the given RNA . Known translation targets of eIF4E , MYC and VEGF was shifted to higher polysomes in eIF4E overexpressing cells as well as SF - encoding RNAs with altered polysomes ( middle ) or those that were unchanged by eIF4E including the negative control ACTB are in the bottom panel Experiments were carried out in biological duplicates using different clones . Means are shown and p - values calculated using ANOVA ( PRISM ) . Data information : ANOVA test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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B. Polysomal loading analysis was measured to assess the relevance of eIF4E's capacity to increase translation efficiency on SF protein production. Top panel shows the polysomal profile (at 254 nm) demonstrating that 2FLAG-eIF4E overexpression did not alter the profile relative to Vector controls, consistent with previous studies Polysomes were isolated by size exclusion chromatography (SEC) and thus the heaviest polysomes elute first and monosomes last. (Middle and Bottom panels) RNAs were monitored on heavy, light or monosome polysomes using RT-qPCR and presented as a fraction for the given RNA. Known translation targets of eIF4E, MYC and VEGF was shifted to higher polysomes in eIF4E overexpressing cells as well as SF-encoding RNAs with altered polysomes (middle) or those that were unchanged by eIF4E including the negative control ACTB are in the bottom panel Experiments were carried out in biological duplicates using different clones. Means are shown and p-values calculated using ANOVA (PRISM) . Data information: ANOVA test, * p<0.05, **p<0.01, ***p<0.001


B . Western blot analysis of untreated ( Ctrl ) and Ribavirin treated ( Rib ) Vector and eIF4E NOMO - 1 cell lines . Myc , and Mcl1 served as positive controls , and β - actin as a loading control . Each β - actin blot corresponds to the above WBs . Experiments were carried out at least three independent times , and one representative experiment is shown . Ribavirin dose was 10uM which is clinically

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B. Western blot analysis of untreated (Ctrl) and Ribavirin treated (Rib) Vector and eIF4E NOMO-1 cell lines. Myc, and Mcl1 served as positive controls, and β-actin as a loading control. Each β-actin blot corresponds to the above WBs. Experiments were carried out at least three independent times, and one representative experiment is shown. Ribavirin dose was 10uM which is clinically achievable


C . WB analysis of Splicing Factors levels in primary AML samples with high ( AML - H ) or normal ( AML - N ) eIF4E levels , as well as bone marrow mononuclear cells from healthy volunteers ( Norm for normal ) . Numbers refer to different individuals . β - actin was used as a loading control . ( * = degraded sample ) .

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C. WB analysis of Splicing Factors levels in primary AML samples with high (AML-H) or normal (AML-N) eIF4E levels, as well as bone marrow mononuclear cells from healthy volunteers (Norm for normal). Numbers refer to different individuals. β-actin was used as a loading control. (* = degraded sample).


E . Total levels of UsnRNA in NOMO - 1 Vector and eIF4E cells monitored by RT - qPCR . Data were normalized to Vector control and shown as a fold change . The mean and standard deviation , as well as p - values ( using Student t - test ) were derived from three biological replicates each carried out in triplicate for all panels .

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E. Total levels of UsnRNA in NOMO-1 Vector and eIF4E cells monitored by RT-qPCR. Data were normalized to Vector control and shown as a fold change. The mean and standard deviation, as well as p-values (using Student t-test) were derived from three biological replicates each carried out in triplicate for all panels.


A . Endogenous eIF4E co - immunoprecipitated with SFs in the nuclear fractions of U2OS cells . Immunoprecipitations ( IP ) were carried out using U2OS nuclear lysates and anti - eIF4E antibody ( eIF4E - IP ) or appropriate IgG control ( IgG - IP ) . IP samples along with Input ( 2 % ) and supernatants ( Sn ) after IPs were analyzed by WB using antibodies as indicated . B . eIF4E co - immunoprecipitated with SFs in the nuclear fractions of NOMO - 1 cells . IPs were carried out using eIF4E overexpressing NOMO - 1 nuclear lysates and anti - eIF4E antibody ( eIF4E - IP ) or appropriate IgG control ( IgG - IP ) . IP samples were analyzed by WB as above and carried out in three biological replicates .

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A. Endogenous eIF4E co-immunoprecipitated with SFs in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates and anti-eIF4E antibody (eIF4E-IP) or appropriate IgG control (IgG-IP). IP samples along with Input (2%) and supernatants (Sn) after IPs were analyzed by WB using antibodies as indicated. B. eIF4E co-immunoprecipitated with SFs in the nuclear fractions of NOMO-1 cells. IPs were carried out using eIF4E overexpressing NOMO-1 nuclear lysates and anti-eIF4E antibody (eIF4E-IP) or appropriate IgG control (IgG-IP). IP samples were analyzed by WB as above and carried out in three biological replicates.


C . eIF4E interaction with splicing factors depends on the presence of RNAs . The eIF4E IP was divided into two and treated with buffer or RNase . After elution , 2 % of the nuclear extract and the IPs were resolved on SDS - PAGE and analyzed by western blotting with indicated antibodies . Representative gels of three biological replicates are shown ( middle panel ) and intensities measured using FIJI are shown in scatter plot ( bottom panel ) generated in PRISM with means depicted as horizontal lines and standard deviation as vertical lines . Each point represents an independent experiment . Fraction purity was assessed with the indicated antibodies ( C top panel ) . Data information : ( C , The number of independent experiments for each target is shown which varies from n = 3 - 4 replicates . Error bars representative standard deviations ( vertical line ) .

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C. eIF4E interaction with splicing factors depends on the presence of RNAs. The eIF4E IP was divided into two and treated with buffer or RNase. After elution, 2% of the nuclear extract and the IPs were resolved on SDS-PAGE and analyzed by western blotting with indicated antibodies. Representative gels of three biological replicates are shown (middle panel) and intensities measured using FIJI are shown in scatter plot (bottom panel) generated in PRISM with means depicted as horizontal lines and standard deviation as vertical lines. Each point represents an independent experiment. Fraction purity was assessed with the indicated antibodies (C top panel). Data information: (C, The number of independent experiments for each target is shown which varies from n=3-4 replicates. Error bars representative standard deviations (vertical line).


C . Survival analyses for of High - eIF4E patients and Normal - eIF4E patients for which specimens were studied The rest of the cohort is shown for comparison . Log Rank test was performed and shows a significant difference in survival between the High - eIF4E group ( 10 specimens ) and the rest of the cohort ( 432 specimens ) , as well as between High - eIF4E and Normal - eIF4E groups ( 11 specimens ) ( p - value < 0 . 05 )

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C. Survival analyses for of High-eIF4E patients and Normal-eIF4E patients for which specimens were studied The rest of the cohort is shown for comparison. Log Rank test was performed and shows a significant difference in survival between the High-eIF4E group (10 specimens) and the rest of the cohort (432 specimens), as well as between High-eIF4E and Normal-eIF4E groups (11 specimens) (p-value < 0.05)


C . Validation of splicing targets identified by rMATs in U2OS ( upper panel ) and NOMO - 1 ( lower panel ) cell lines . RT - qPCR analysis using specific primers for each splicing event normalized to the corresponding total levels of that given transcript Data were normalized to Vector control to calculate fold change . The mean and standard deviation , as well as p - values were derived from three biological replicates . P - values were calculated between eIF4E overexpressing and Vector cells ( when the asterisk is over eIF4E ) , and eIF4E cells treated with Ribavirin vs eIF4E overexpressing untreated cells ( for the asterisk over Ribavirin ) . Data information : Student t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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C. Validation of splicing targets identified by rMATs in U2OS (upper panel) and NOMO-1 (lower panel) cell lines. RT-qPCR analysis using specific primers for each splicing event normalized to the corresponding total levels of that given transcript Data were normalized to Vector control to calculate fold change. The mean and standard deviation, as well as p-values were derived from three biological replicates. P-values were calculated between eIF4E overexpressing and Vector cells (when the asterisk is over eIF4E), and eIF4E cells treated with Ribavirin vs eIF4E overexpressing untreated cells (for the asterisk over Ribavirin). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001


D . eIF4E associates with both pre - mRNA splicing substrates and their spliced products . The enrichment of mRNAs for splicing targets in eIF4E - RIPs versus input mRNAs from the nuclear fractions of 2FLAG - eIF4E U2OS cells monitored by RT - qPCR using primers for the specific splice event ( S for " spliced " ) , pre - mRNA specific primers to the relevant intron or intron - exon boundary ( NS for " unspliced " ) , and primers specific to common regions present in all spliced and not - spliced mRNAs ( All ) . Data were normalized to input samples and presented as a fold change . The mean , standard deviation and p - values ( Student t - test ) were derived from three independent experiments ( each carried out in triplicate ) . Data information : Student t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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D. eIF4E associates with both pre-mRNA splicing substrates and their spliced products. The enrichment of mRNAs for splicing targets in eIF4E-RIPs versus input mRNAs from the nuclear fractions of 2FLAG-eIF4E U2OS cells monitored by RT-qPCR using primers for the specific splice event (S for "spliced"), pre-mRNA specific primers to the relevant intron or intron-exon boundary (NS for "unspliced"), and primers specific to common regions present in all spliced and not-spliced mRNAs (All). Data were normalized to input samples and presented as a fold change. The mean, standard deviation and p-values (Student t-test) were derived from three independent experiments (each carried out in triplicate). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001