A - C Representative shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) α - Sma ( red ) collagen deposition by PSR ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete within scar is shown for CCl4 BDL in Sox9fl / fl ; AlbCre + mice ( A ) .

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A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).


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D , E . of surface covered by PSR in ( C ) .

D, E. Quantification of surface area covered by the PSR staining in (C).


F Expression of SOX9 by immunohistochemistry in quiescent ( Q ) activated ( A ) HSCs extracted from Sox9fl / fl ; AlbCre + mice .

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F Expression of SOX9 protein by immunohistochemistry in quiescent (Q) and activated (A) HSCs extracted from Sox9fl/fl;AlbCre+ mice.


G channels showing localization expression of α - SMA ( left panel ) , SOX9 ( middle panel ) composite image for α - SMA ( green ) SOX9 ( red ) in right panel in activated Sox9fl / fl ; AlbCre + HSCs ( genotyping shown in Appendix Figure S9 ) . Two - unpaired t - was used for statistical . Data in bar charts show means ± s . e . m . bars = 100 μm ( A ) , 10 μm ( B ) 200 μm ( C ) , 25 μm ( G ) .

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G Individual fluorescent channels showing localization and expression of α-SMA (left panel), SOX9 (middle panel) and composite image for α-SMA (green) and SOX9 (red) in right panel in activated Sox9fl/fl;AlbCre+ HSCs (genotyping shown in Appendix Figure S9). Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. Size bars = 100 μm (A), 10 μm (B) 200 μm (C), 25 μm (G).


A , B Flow cytometry of CD45 + CD11B + in control ( Sox9fl / fl ; RosaCre - ; A ) Sox9 - null ( Sox9fl / fl ; RosaCre + ; B ) livers stained for Ly6C MHCII ( far left ) to identify four populations of with differences in their expression of MerTK , CD64 , F4 / 80 MHCII .

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A, B Flow cytometry of CD45+CD11B+ cells in control (Sox9fl/fl;RosaCre-; A) and Sox9-null (Sox9fl/fl;RosaCre+; B) livers stained for Ly6C and MHCII (far left) to identify four populations of cells with differences in their expression of MerTK, CD64, F4/80 and MHCII.


C Graphical representation of Ly6C F4 / 80 in macrophages from population 3 4 .

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C Graphical representation of Ly6C and F4/80 staining in macrophages from population 3 and 4.


D , E Graphical representation of numbers in four Ly6C MHCII populations in control Sox9 - null livers . Data are representative of four independent experiments .

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D, E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9-null livers. Data are representative of four independent experiments.


F F4 / 80 immunohistochemistry ( brown ) in control Sox9 - null mice livers following 4 - CCl4 induced fibrosis . bar = 100μm . Two - unpaired t - was used for statistical . Data in bar charts show means ± s . e . m . P values indicated . experiments are n = 4 .

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F F4/80 immunohistochemistry (brown staining) in control and Sox9-null mice livers following 4-weeks CCl4 induced fibrosis. Size bar = 100μm. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated. All experiments are n=4.


A , B Immunofluorescence of activated mouse HSCs cultured on plastic ( A ) or hydrogels ( A , B ) . channels showing localization expression of SOX9 ( left ) , YAP ( middle ) composite image for SOX9 ( red ) YAP ( green ) in right panel . ( B ) composite for αSMA ( green ) TAZ ( red ) or TEAD ( red ) . bar , 25μm .

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A, B Immunofluorescence of activated mouse HSCs cultured on plastic (A) or hydrogels (A, B). Individual fluorescent channels showing localization and expression of SOX9 (left), YAP (middle) and composite image for SOX9 (red) and YAP (green) in right panel. (B) Individual and composite images for αSMA (green) and TAZ (red) or TEAD (red). Size bar, 25μm.


C Quantified decrease in SOX9 following inhibition of YAP using verteporfin ( VP ) in activated mouse HSCs expressed relative to DMSO control ( n = 3 ) .

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C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3).


D Immunohistochemistry for SOX9 ( brown ) following CCl4 or BDL induced fibrosis in mice treated with DMSO ( CCl4 n = 4 , BDL n = 7 ) or verteporfin ( CCl4 n = 3 , BDL n = 5 ) .

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D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5).


E , F of SOX9 numbers in HSC / Hep population . bar , 50 μm .

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E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.


C E139R , E143R , combination ER / ER mutant substitutions in UBE2S increase amount of automodification , monitored by presence of * Ub ( * denotes fluorescently labeled ) using SDS - PAGE scanning .

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C E139R, E143R, and the combination ER/ER mutant substitutions in UBE2S increase the amount of automodification, monitored by the presence of *Ub (* denotes fluorescently labeled protein) and using SDS-PAGE and fluorescent scanning.


D ER / ER substitutions increase Ub chain formation in multiple UBE2S constructs ( WT , UBC only , UBC - UBD fusion ) , monitored by Coomassie - of non - reduced SDS - PAGE gels .

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D ER/ER substitutions increase Ub chain formation in multiple UBE2S constructs (WT, UBC only, and UBC-UBD fusion), monitored by Coomassie-staining of non-reduced SDS-PAGE gels.


E , F , G , H - chase diUb synthesis assays using different E2s , UBE2S ( E ) , UBE2C ( F ) , UBE2D2 ( G ) , or UBE2R2 ( H ) . DiUb formation was monitored by scanning ( top ) , quantitated ( bottom ) , assessed for statistical significance by either an unpaired Welch ' s t - ( UBE2C UBE2R2 ) or one - way ANOVA ( UBE2S UBE2D2 ) ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates for E2s ) . Error bars : . Data normalized to maximal WT diUb formation . # denotes a contaminant band .

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E, F, G, H Pulse-chase diUb synthesis assays using different E2s, UBE2S (E), UBE2C (F), UBE2D2 (G), or UBE2R2 (H). DiUb formation was monitored by fluorescent scanning (top), quantitated (bottom), and assessed for statistical significance by either an unpaired Welch's t-test (UBE2C and UBE2R2) or one-way ANOVA (UBE2S and UBE2D2) (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates for all E2s). Error bars: SEM. Data normalized to the maximal WT diUb formation. # denotes a contaminant band.


C E139R substitution decreases stability of UBE2S ~ Ub compared to WT , revealed by scanning of a lysine discharge ( top ) its ( bottom ) . Statistical comparison was performed using one - way ANOVA ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , n = 4 independent , technical replicates ) . Error bars : . Data normalized to initial timepoint of each respective E2 ~ Ub band .

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C The E139R substitution decreases the stability of UBE2S~Ub compared to WT, revealed by fluorescent scanning of a lysine discharge assay (top) and its quantification (bottom). Statistical comparison was performed using one-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, n=4 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.


D UBE2R2 R149E has a similar E2 ~ Ub stability as WT , monitored by scanning ( top ) of lysine discharge its through an unpaired Welch ' s t - ( bottom ) ( n = 3 independent , technical replicates ) . Error bars : . Data normalized to initial timepoint of each respective E2 ~ Ub band .

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D UBE2R2 R149E has a similar E2~Ub stability as WT, monitored by fluorescent scanning (top) of the lysine discharge assay and its quantification through an unpaired Welch's t-test (bottom) (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the initial timepoint of each respective E2~Ub band.


E scans ( top ) ( bottom ) of a * LRLRGG peptide transfer comparing UBE2S WT ER / ER reveals no significant difference through an unpaired Welch ' s t - ( n = 3 independent , technical replicates ) . Error bars : . Data normalized to maximal * LRLRGG - Ub formation by WT UBE2S .

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E Fluorescent scans (top) and quantification (bottom) of a *LRLRGG peptide transfer assay comparing UBE2S WT and ER/ER reveals no significant difference through an unpaired Welch's t-test (n=3 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2S.


F UBE2R2 R149E is still defective in transfer of * LRLRGG peptide to an acceptor Ub compared to WT , probed by scanning ( top ) its quantitation ( bottom ) . Statistical comparison was performed using an unpaired Welch ' s t - ( * * p ≤ 0 . 01 , n = 5 independent , technical replicates ) . Error bars : . Data normalized to maximal * LRLRGG - Ub formation by WT UBE2R2 .

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F UBE2R2 R149E is still defective in the transfer of the *LRLRGG peptide to an acceptor Ub compared to WT, probed by fluorescent scanning (top) and its quantitation (bottom). Statistical comparison was performed using an unpaired Welch's t-test (** p ≤ 0.01, n=5 independent, technical replicates). Error bars: SEM. Data normalized to the maximal *LRLRGG-Ub formation by WT UBE2R2.


Role of UBE2RHTH in diUb formation is determined from kinetic parameters ( G - H ) for mutants upon titrating acceptor Ub in - chase reactions following UBE2R2 ~ * Ub Apparent Km Vmax were determined using Michaelis - Menten equation ( n ≥ 3 independent , technical replicates ) . Error bars : . Data normalized to WT apparent Vmax .

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Role of the UBE2RHTH in diUb formation is determined from kinetic parameters (G-H) for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub Apparent Km and Vmax were determined using the Michaelis-Menten equation (n≥3 independent, technical replicates). Error bars: SEM. Data normalized to the WT apparent Vmax.


Role of UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating acceptor Ub in - chase reactions following UBE2R2 ~ * Ub monitored by scanning of nonreducing SDS - PAGE

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Role of the UBE2RHTH in diUb formation is determined from kinetic parameters for mutants upon titrating the acceptor Ub in pulse-chase reactions following UBE2R2~*Ub monitored by fluorescent scanning of nonreducing SDS-PAGE gels


B Reducing positive charge of * Ub reduces autoubiquitination of E139R - substituted UBE2S variant , monitored by scanning of an SDS - PAGE gel .

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B Reducing the positive charge of *Ub reduces the autoubiquitination of the E139R-substituted UBE2S variant, monitored by fluorescent scanning of an SDS-PAGE gel.


D Plot of 15N - labeled UBE2SUBC WT ER / ER 1H linewidth changes in presence of Ub revealing ER / ER variant has a more dynamic active - site cysteine HTH , but a more stabilized crossover helix , indicative of " closed " state . Shown are best - fit values standard deviations ( SD ) estimated from covariance matrix of NMR spectra with n = 2048 measurement points for 16 ppm in 1H dimension .

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D Plot of 15N-labeled UBE2SUBC WT and ER/ER 1H linewidth changes in the presence of Ub revealing the ER/ER variant has a more dynamic active-site cysteine and HTH, but a more stabilized crossover helix, indicative of the "closed" state. Shown are the best-fit values and standard deviations (SD) estimated from the covariance matrix analysis of NMR spectra with n = 2048 measurement points for 16 ppm in the 1H dimension.


F C118A substitutions decrease UBE2S autoubiquitination , measured by scanning of SDS - PAGE gels , indicating significance of " closed " formation of UBE2S ~ Ub for activity .

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F C118A substitutions decrease UBE2S autoubiquitination, measured by fluorescent scanning of SDS-PAGE gels, indicating the significance of the "closed" formation of the UBE2S~Ub for activity.


C Securin ubiquitination by APC / C either UBE2C WT or K157E , resolved by SDS - PAGE scanning . of 4 or more ubiquitins added on substrate ( right ) are calculated as a fraction of total ubiquitination analyzed by an unpaired Welch ' s t - ( * * * * p ≤ 0 . 0001 , n = 5 independent , technical replicates ) . Error bars : . D Similar to C , Ub - securin ubiquitination by APC / C either UBE2D2 WT or charge - swap variants . of 4 or more ubiquitins ( right ) added on substrate are calculated as a fraction of total ubiquitination analyzed by one - way ANOVA ( * * * * p ≤ 0 . 0001 , n = 4 independent , technical replicates ) . Error bars : .

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C Securin ubiquitination by APC/C and either UBE2C WT or K157E, resolved by SDS-PAGE and fluorescent scanning. Quantification of 4 or more ubiquitins added on the substrate (right) are calculated as a fraction of total ubiquitination and analyzed by an unpaired Welch's t-test (**** p ≤ 0.0001, n=5 independent, technical replicates). Error bars: SEM. D Similar to C, Ub-securin ubiquitination by APC/C and either UBE2D2 WT or charge-swap variants. Quantification of 4 or more ubiquitins (right) added on the substrate are calculated as a fraction of total ubiquitination and analyzed by one-way ANOVA (**** p ≤ 0.0001, n=4 independent, technical replicates). Error bars: SEM.


F recombinant UBE2S ER / ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild - type ( WT ) , monitored by immunoblot . G1 extracts were prepared from HeLa S3 treated with either control firefly luciferase siRNA ( siFF ) or two UBE2S siRNAs ( siUBE2S ) .

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F The recombinant UBE2S ER/ER variant increases Cyclin B degradation in G1 extracts compared to UBE2S wild-type (WT), monitored by immunoblot. G1 extracts were prepared from HeLa S3 cells treated with either control firefly luciferase siRNA (siFF) or two UBE2S siRNAs (siUBE2S).


B Representative scan of an SDS - PAGE gel ( left ) its ( right ) where charged - swapped variants of UBE2SHTH enhance substrate ubiquitination of APC / Cs harboring APC2 substitutions . Statistical significance was assessed by two - way ANOVA ( * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , n = 3 independent , technical replicates ) . Error bars : . Data normalized to negative control substrate band from reactions lacking an E2 .

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B Representative fluorescent scan of an SDS-PAGE gel (left) and its quantification (right) where the charged-swapped variants of the UBE2SHTH enhance substrate ubiquitination of APC/Cs harboring APC2 substitutions. Statistical significance was assessed by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to the negative control substrate band from reactions lacking an E2.


C APC / C - UBE2S - dependent diUb synthesis was monitored by scanning ( left ) , quantitated ( right ) , assessed for statistical significance by two - way ANOVA ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) , demonstrating that UBE2SHTH substitutions rescue defects in diUb synthesis created by mutating APC2 . Error bars : . Data normalized to WT APC / C - UBE2S diUb reactions . D APC / C Platform - dependent activation of UBE2S - mediated diUb synthesis was monitored by scanning ( left ) , quantitated ( right ) , assessed for statistical significance by two - way ANOVA ( * p ≤ 0 . 05 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) . Error bars : . Data normalized to WT APC / C Platform - UBE2S diUb reactions .

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C APC/C-UBE2S-dependent diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates), demonstrating that the UBE2SHTH substitutions rescue the defects in diUb synthesis created by mutating APC2. Error bars: SEM. Data normalized to WT APC/C-UBE2S diUb reactions. D APC/C Platform-dependent activation of UBE2S-mediated diUb synthesis was monitored by fluorescent scanning (left), quantitated (right), and assessed for statistical significance by two-way ANOVA (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT APC/C Platform-UBE2S diUb reactions.


E Similar to B , role of " closed " conformation in UBE2S - dependent Ub chain elongation of APC / C substrate Ub - CycB * , monitored by incorporation of C118A - substituted UBE2S variants ( top ) . of ubiquitinated Ub - CycB * was assessed ( bottom ) statistical significance by one - way ANOVA between C118A WT , C118A / E139R , C118A / E143R ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 independent , technical replicates ) . Error bars : . Data normalized to WT UBE2S 4 + Ub additions .

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E Similar to B, the role of the "closed" conformation in UBE2S-dependent Ub chain elongation of the APC/C substrate Ub-CycB*, monitored by the incorporation of C118A-substituted UBE2S variants (top). Quantification of ubiquitinated Ub-CycB* was assessed (bottom) and statistical significance by one-way ANOVA between C118A and WT, C118A/E139R, and C118A/E143R (** p ≤ 0.01, **** p ≤ 0.0001, n=3 independent, technical replicates). Error bars: SEM. Data normalized to WT UBE2S 4+ Ub additions.


A Quantitative RT - PCR ( RT - qPCR ) results showing relative expression of mouse Pabpn1l in somatic tissues , oocytes , preimplantation . n = 3 biological replicates . Error bars indicate S . E . M .

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A Quantitative RT-PCR (RT-qPCR) results showing relative expression levels of mouse Pabpn1l in somatic tissues, oocytes, and preimplantation embryos. n = 3 biological replicates. Error bars indicate S.E.M.


B Western blot results of PABPN1L , BTG4 , phosphorylated ERK1 / 2 ( pERK1 / 2 ) in oocytes . Total from 400 oocytes or is loaded in each lane . damage - binding 1 ( DDB1 ) is blotted as a loading control .

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B Western blot results of PABPN1L, BTG4, and phosphorylated ERK1/2 (pERK1/2) in oocytes and embryos. Total protein from 400 oocytes or embryos is loaded in each lane. DNA damage-binding protein 1 (DDB1) is blotted as a loading control.


C Western blot results of PABPN1L in MII oocytes of wild type ( WT ) Pabpn1l − / − . DDB1 is blotted as a loading control .

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C Western blot results of PABPN1L in MII oocytes of wild type (WT) and Pabpn1l−/− females. DDB1 is blotted as a loading control.


D Cumulative pup numbers of WT Pabpn1l - / - mice . n = 5 for each genotype . Error bars , S . E . M . * * * P < 0 . 001 by two - student ' s t - .

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D Cumulative pup numbers of WT and Pabpn1l-/- female mice. n = 5 for each genotype. Error bars, S.E.M. ***P < 0.001 by two-tailed student's t-test.


E of preimplantation derived from WT Pabpn1l - / - when WT reached corresponding stages . number of analyzed is indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - Student ' s t - .

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E Quantification of preimplantation embryos derived from WT and Pabpn1l-/- females when WT embryos reached the corresponding stages. The number of analyzed embryos is indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.


F Representative of collected from oviducts of mated WT Pabpn1l - / - at indicated time points after hCG injection . Scale bar = 100 μm .

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F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l-/- females at the indicated time points after hCG injection. Scale bar = 100 μm.


A Scatter plot comparing transcripts of GV oocytes zygotes from WT Pabpn1l - / - . Transcripts that increased or decreased by more than 3 - fold in Pabpn1l - deleted GV oocytes or zygotes are highlighted in red or blue , respectively .

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A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted GV oocytes or zygotes are highlighted in red or blue, respectively.


D RT - qPCR results for relative expression of indicated maternal transcripts in oocytes zygotes from WT Pabpn1l − / − . n = 3 biological replicates . Error bars , . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - Student ' s t - .

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D RT-qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l −/− females. n = 3 biological replicates. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test.


E Poly ( A ) tail results showing poly ( A ) - tail of indicated transcripts in WT Pabpn1l - deleted oocytes . poly ( A ) tails of Actin are used as an internal control . Each sample was prepared from 100 oocytes or . Plots show averaged relative signal ( y - axis ) of PCR products on mobility ( x - axis ) .

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E Poly(A) tail assay results showing poly(A)-tail length of indicated transcripts in WT and Pabpn1l-deleted oocytes and embryos. The poly(A) tails of Actin are used as an internal control. Each sample was prepared from 100 oocytes or embryos. Plots show the averaged relative signal intensity (y-axis) and length of the PCR products based on mobility (x-axis).


A Scatter plot comparing transcripts of 2 - from WT Pabpn1l - / - . Transcripts that increased or decreased by more than 3 - fold in Pabpn1l - deleted oocytes or are highlighted in red or blue , respectively .

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A Scatter plot comparing the transcripts of 2-cell embryos from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted oocytes or embryos are highlighted in red or blue, respectively.


C 5 - Ethynyl uridine ( EU ) ( green ) showing transcription in WT Pabpn1l ♀ − / ♂ + 2 - . Some WT were treated with α - amanitin as early as zygote stage cultured to 2 - stage . phosphorylated polymerase II CTD repeat YSPTSPS ( pS2 ) ( red ) is co - stained to label polymerase II activity . Nuclei are labeled by DAPI ( blue ) . Scale bar = 20 μm .

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C 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm.


D of EU pS2 signals in ( A ) . numbers of analyzed are indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - Student ' s t - .

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D Quantification of EU and pS2 signals in (A). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.


E Relative of tdTomato signals in ( F ) . numbers of analyzed are indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - Student ' s t - .

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E Relative intensity of tdTomato signals in (F). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.


F Representative of MuERV - L : : tdTomato relative to GFP signal in same when WT reached corresponding stages . Zygotes were injected with MuERV - L : : tdTomato reporter plasmid polyadenylated Gfp mRNA ( as a positive control of microinjection ) , then allowed to develop in vitro . Cultured were imaged at 24 h 40 h after hCG injection . DIC , differential interference contrast . Scale bar = 100 μm .

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F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.


A immunoprecipitation ( RIP ) results showing interaction of BTG4 with indicated transcripts , in presence or absence of PABPN1L ( full , RRM - deleted , or R171A mutant ) . HeLa were co - transfected with plasmids expressing FLAG - tagged PABPN1L HA - tagged BTG4 for 48 h before immunoprecipitating with an anti - HA antibody . RNAs recovered from immunoprecipitants were subjected to RT - qPCR of indicated transcripts . Fold change values of both input IP samples were normalized by RIP results of HA - BTG4 FLAG - PABPN1L co - expression groups . n = 3 biological replicates . Error bars , S . E . M . P - value represents two - Student ' s t - comparing RIP results of BTG4 with indicated transcripts in presence of PABPN1L , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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A RNA immunoprecipitation (RIP) results showing the interaction of BTG4 with indicated transcripts, in the presence or absence of PABPN1L (full length, RRM-deleted, or R171A mutant). HeLa cells were co-transfected with plasmids expressing FLAG-tagged PABPN1L and HA-tagged BTG4 for 48 h before immunoprecipitating with an anti-HA antibody. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both input and IP samples were normalized by RIP results of HA-BTG4 and FLAG-PABPN1L co-expression groups. n = 3 biological replicates. Error bars, S.E.M. The P-value represents the two-tailed Student's t-test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L, *P < 0.05, **P < 0.01, and ***P < 0.001.


Co - IP western blot results showing interactions between PABPN1L BTG4 . Lysates from HeLa expressing HA - BTG4 ( WT or mutants shown in ( B ) ) FLAG - PABPN1L were immunoprecipitated with an anti - HA antibody . immunoprecipitated are detected by western blot with indicated antibodies .

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Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.


D Co - IP western blot results showing interactions between PABPN1L BTG4 . Lysates from HeLa expressing HA - BTG4 ( WT or mutants shown in ( B ) ) FLAG - PABPN1L were immunoprecipitated with an anti - HA antibody . immunoprecipitated are detected by western blot with indicated antibodies .

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D Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.


E Diagrams of mouse BTG2 BTG4 constructs , co - IP results showing that PABPN1L binds to BTG4 but not BTG2 .

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E Diagrams of mouse BTG2 and BTG4 constructs, and co-IP results showing that PABPN1L binds to BTG4 but not BTG2.


F Diagrams co - IP results showing BTG4 binding to PABPN1L . Lysates from HeLa expressing HA - BTG4 FLAG - PABPN1L ( full C - terminal deleted ) were immunoprecipitated with an anti - FLAG antibody . immunoprecipitated are detected by western blot with indicated antibodies .

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F Diagrams and co-IP results showing BTG4 binding to PABPN1L. Lysates from HeLa cells expressing HA-BTG4 and FLAG-PABPN1L (full length and C-terminal deleted) were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.


G Diagrams of PABPN1 PABPN1L constructs , co - IP results showing that PABPN1L , but not PABPN1 , binds to BTG4 .

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G Diagrams of PABPN1 and PABPN1L constructs, and co-IP results showing that PABPN1L, but not PABPN1, binds to BTG4.


B RT - qPCR results showing relative expression level changes ( MII / GV ) of indicated transcripts . Fully grown GV oocytes of Pabpn1l - / - mice were microinjected with mRNAs encoding PABPN1L or BTG4 were released from meiotic arrest at 12 h after microinjection . Total was extracted from 10 oocytes in each sample . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - Student ' s t - compared to first column . ns : non - significant .

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B RT-qPCR results showing the relative expression level changes (MII/GV) of indicated transcripts. Fully grown GV oocytes of Pabpn1l-/- mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection. Total RNA was extracted from 10 oocytes in each sample. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test compared to the first column. ns: non-significant.


C Western blot results showing expression of exogenous PABPN1L ( full , RRM - deleted , or R171A mutants ) BTG4 in Pabpn1l - / - oocytes at MII stage . total from 100 oocytes is loaded in each lane . pERK1 / 2 is blotted to indicate developmental stages . DDB1 is blotted as a loading control . At least three independent experiments were done with consistent results .

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C Western blot results showing the expression of exogenous PABPN1L (full length, RRM-deleted, or R171A mutants) and BTG4 in Pabpn1l-/- oocytes at the MII stage. The total protein from 100 oocytes is loaded in each lane. pERK1/2 is blotted to indicate the developmental stages. DDB1 is blotted as a loading control. At least three independent experiments were done with consistent results.


Diagrams averaged traces of thermal stability assays of PABPN1L in presence absence of poly ( A ) variants ( n = 6 ) . substrate comprised 20 non - poly ( A ) nucleotides followed by a 10 / 20 / 30 / 60 - adenosine poly ( A ) tail . PABPN1L - bound substrates were prepared with 2 / 1 / 0 . 5 / 0 . 2 / 0 . 1 molecules per PABPN1L . midpoint of unfolding curve was determined as melting ( Tm ) .

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Diagrams and averaged fluorescence traces of the thermal stability assays of PABPN1L in the presence and absence of poly(A) variants (n = 6). The RNA substrate comprised 20 non-poly(A) nucleotides followed by a 10/20/30/60-adenosine poly(A) tail. PABPN1L-bound substrates were prepared with 2/1/0.5/0.2/0.1 RNA molecules per PABPN1L. The midpoint of the unfolding curve was determined as the melting temperature (Tm).


D immunoprecipitation results showing interaction of PABPN1L with indicated transcripts . Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L ( WT , RRM - deleted , or R171A mutant ) , were released from meiotic arrest at 12 h after microinjection . Total RNAs were extracted from 350 MI stage oocytes in each sample . RNAs recovered from immunoprecipitants were subjected to RT - qPCR of indicated transcripts . Fold change values of both Input IP samples were normalized by RIP results of FLAG - PABPN1L microinjection group . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by two - Student ' s t - comparing with pulled - down by PABPN1L WT in second column .

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D RNA immunoprecipitation results showing the interaction of PABPN1L with indicated transcripts. Fully grown GV oocytes of WT mice were microinjected with mRNAs encoding PABPN1L (WT, RRM-deleted, or R171A mutant), and were released from meiotic arrest at 12 h after microinjection. Total RNAs were extracted from 350 MI stage oocytes in each sample. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both Input and IP samples were normalized by RIP results of the FLAG-PABPN1L microinjection group. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test comparing with RNA levels pulled-down by PABPN1L WT in the second column.


A Western blot results showing of indicated in WT Pabpn1l - / - oocytes . Total from 100 oocytes is loaded in each lane . DDB1 is blotted as a loading control .

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A Western blot results showing the levels of the indicated proteins in WT and Pabpn1l-/- oocytes. Total protein from 100 oocytes is loaded in each lane. DDB1 is blotted as a loading control.


B HeLa were transfected with plasmids expressing HA - BTG4 FLAG - PABPN1L for 12 h then treated by cycloheximide ( CHX , 10 μM ) . were harvested at indicated time points for western blots . DDB1 is a loading control .

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B HeLa cells were transfected with plasmids expressing HA-BTG4 and FLAG-PABPN1L for 12 h and then treated by cycloheximide (CHX, 10 μM). Cells were harvested at the indicated time points for western blots. DDB1 is a loading control.


C Co - IP western blot results showing interaction of βTrCP1 with BTG4 in presence or absence of PABPN1L .

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C Co-IP and western blot results showing the interaction of βTrCP1 with BTG4 in the presence or absence of PABPN1L.


D Co - IP western blot results showing BTG4 polyubiquitination . HeLa transfected with plasmids encoding indicated were lysed subjected to IP with an anti - HA affinity gel . Input lysates precipitates are immunoblotted with indicated antibodies .

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D Co-IP and western blot results showing BTG4 polyubiquitination. HeLa cells transfected with plasmids encoding the indicated proteins were lysed and subjected to IP with an anti-HA affinity gel. Input cell lysates and precipitates are immunoblotted with the indicated antibodies.


E Western blot results showing endogenous BTG4 in WT Pabpn1l - / - oocytes at MII stage . FLAG - PABPN1L was expressed in Pabpn1l - / - oocytes by mRNA microinjected at GV stage . Total from 100 oocytes are loaded in each lane . ERK1 / 2 is blotted as a loading control .

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E Western blot results showing endogenous BTG4 levels in WT and Pabpn1l-/- oocytes at the MII stage. FLAG-PABPN1L was expressed in Pabpn1l-/- oocytes by mRNA microinjected at the GV stage. Total proteins from 100 oocytes are loaded in each lane. ERK1/2 is blotted as a loading control.


( B ) Growth inhibition upon overexpression of HOS3 - NLS driven by GAL1 promoter is suppressed by overexpression of KAT Esa1 . 10 - fold serial dilutions of indicated strains transformed with an empty vector or with indicated plasmids , were spotted onto SC - Glu SC - Gal medium ( to activate GAL1 promoter ) incubated at 25 ° C for 3 days . Note that HOS3 - NLS ( under control of native HOS3 promoter ) does not affect growth .

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(B) Growth inhibition upon overexpression of HOS3-NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1. 10-fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids, were spotted onto SC-Glu and SC-Gal medium (to activate the GAL1 promoter) and incubated at 25 °C for 3 days. Note that HOS3-NLS (under the control of the native HOS3 promoter) does not affect cell growth.


( C ) esa1 - ts gcn5Δ esa1 - ts mutants have bud emergence defects . ( Top ) of indicated strains were arrested in G1 by treatment with ɑ - factor for 2 . 5 h at 25 ºC , shifted to 37 ºC for 1 h released from G1 arrest at 37 ºC . were fixed at indicated times presence of buds was assessed by microscopy . Mean are derived from n = number of independent experiments . At least 200 were scored for each time point . ( Bottom ) Bright field of indicated strains 60 min after ɑ - factor washout . Arrowheads point to buds . Scale bar , 4 µm .

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(C) esa1-ts and gcn5Δ esa1-ts mutants have bud emergence defects. (Top) Cells of the indicated strains were arrested in G1 by treatment with ɑ-factor for 2.5 h at 25 ºC, shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC. Cells were fixed at the indicated times and the presence of buds was assessed by microscopy. Mean and SEM are derived from n = number of independent experiments. At least 200 cells were scored for each strain and time point. (Bottom) Bright field images of the indicated strains 60 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.


( D ) Inactivation of ESA1 GCN5 delays replication . of indicated genotypes were synchronised as in panel C , content was evaluated by flow cytometry . Numbers indicate time in minutes after release . This experiment was repeated three times with similar results ; one experiment is shown .

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(D) Inactivation of ESA1 and GCN5 delays DNA replication. Cells of the indicated genotypes were synchronised as in panel C, and DNA content was evaluated by flow cytometry. Numbers indicate time in minutes after the release. This experiment was repeated three times with similar results; one experiment is shown.


( A ) Overexpression of Esa1 Gcn5 KATs leads to increased acetylation of basket nucleoporin Nup60 . ( Top ) Nup60 - GFP was immunoprecipitated from extracts of indicated strains , its acetylation state probed with anti - AcLys antibodies . ( Bottom ) Total extracts probed with anti - HA antibodies to verify KAT overexpression .

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(A) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60. (Top) Nup60-GFP was immunoprecipitated from extracts of the indicated strains, and its acetylation state probed with anti-AcLys antibodies. (Bottom) Total extracts probed with anti-HA antibodies to verify KAT overexpression.


( B ) nup60 - KN partially rescues budding defect of esa1 - ts . ( Left ) Budding of of indicated strains were determined as in Figure 1C . At least 200 were scored for each time point . Data from 3 independent experiments is represented as mean ( esa1 - ts , esa1 - ts nup60 - KN ) . ( Right ) Bright field of indicated strains 40 min after ɑ - factor washout . Arrowheads point to buds . Scale bar , 4 µm .

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(B) nup60-KN partially rescues the budding defect of esa1-ts cells. (Left) Budding of cells of the indicated strains were determined as in Figure 1C. At least 200 cells were scored for each strain and time point. Data from 3 independent experiments is represented as mean and SEM (esa1-ts, esa1-ts nup60-KN). (Right) Bright field images of the indicated strains 40 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.


( C ) mRNA of CLN2 , CDC21 , SVS1 , RNR1 were determined for of indicated strains after G1 arrest release at restrictive , with samples collected at indicated times . Data from 3 independent experiments is represented as mean .

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(C) mRNA levels of CLN2, CDC21, SVS1, and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature, with samples collected at indicated times. Data from 3 independent experiments is represented as mean and SEM.


( D ) nup60 - KN mutation partially rescues delay in synthesis of G1 / S cyclin Cln2 in esa1 - ts . of indicated strains were processed as in ( B ) amount of Cln2 - HA at indicated times was assessed by western blot . % of budded is indicated below for each corresponding time point . Note slow , inefficient budding in esa1 - ts mutants . In WT , reduction of Cln2 at 60 min its increase at 90 min reflects start of a second cycle , which is absent in esa1 - ts .

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(D) nup60-KN mutation partially rescues the delay in synthesis of the G1/S cyclin Cln2 in esa1-ts cells. Cells of the indicated strains were processed as in (B) and the amount of Cln2-HA protein at the indicated times was assessed by western blot. % of budded cells is indicated below for each corresponding strain and time point. Note the slow, inefficient budding in esa1-ts mutants. In WT, the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle, which is absent in esa1-ts cells.


( E ) nup60 - KN partially rescues Whi5 export defect of esa1 - ts daughter . Composite of bright field Whi5 - mCherry ( left ) of Whi5 export ( right ) in mother ( M ) daughter ( D ) of indicated strains . Whi5 export ( arrows ) is delayed in esa1 - ts mothers daughters compared to WT ( p < 000 . 1 , Log - rank Mantel - Cox ) ; esa1 - ts nup60 - KN advances Whi5 export relative to esa1 - ts in daughters ( p = 0 . 0105 ) , but not in mothers ( p > 0 . 05 ) . 3 z - confocal slices spaced 0 . 5 µm were acquired every 3 min ; maximum projections of selected timepoints are shown . Time is indicated in minutes ; t = 0 marks Whi5 import . Scale bar , 5 µm . n = number of , pooled from two independent experiments with similar results .

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(E) nup60-KN partially rescues the Whi5 export defect of esa1-ts daughter cells. Composite of bright field and Whi5-mCherry (left) and quantification of Whi5 nuclear export (right) in mother (M) and daughter (D) cells of the indicated strains. Whi5 export (arrows) is delayed in esa1-ts mothers and daughters compared to WT (p < 000.1, Log-rank Mantel-Cox test); esa1-ts nup60-KN advances Whi5 export relative to esa1-ts in daughters (p=0.0105), but not in mothers (p > 0.05). 3 z-confocal slices spaced 0.5 µm were acquired every 3 min; maximum projections of selected timepoints are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. n = number of cells, pooled from two independent experiments with similar results.


( A ) Composite of bright field Whi5 - tdTomato ( BF / Whi5 ) CLN2 - PP7 mRNA labelled with PCP - NLS - GFP ( CLN2 mRNA ) in mother ( M ) daughter ( D ) of indicated strains . Arrows indicate foci . Brighter GFP nucleoplasmic areas may correspond to nucleolus . Insets show enhanced - contrast to visualise mRNA particles ( arrowheads ) . Numbers indicate minutes relative to Whi5 import . Maximum projections of whole - Z - stacks are shown for Whi5 CLN2 mRNA except in inset in selected esa1 - ts , where single Z - slices are shown for clarity . Scale bar : 4 µm ( inset : 1 µm ) .

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(A) Composite of bright field and Whi5-tdTomato (BF/Whi5) and CLN2-PP7 mRNA labelled with PCP-NLS-GFP (CLN2 mRNA) in mother (M) and daughter (D) cells of the indicated strains. Arrows indicate nuclear foci. Brighter GFP nucleoplasmic areas may correspond to the nucleolus. Insets show enhanced-contrast images to visualise cytoplasmic mRNA particles (arrowheads). Numbers indicate minutes relative to Whi5 nuclear import. Maximum projections of whole-cell Z-stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1-ts images, where single Z-slices are shown for clarity. Scale bar: 4 µm (inset: 1 µm).


( B ) Fraction of of indicated strains with CLN2 - PP7 mRNA foci , aligned relative to Whi5 import . Symbols represent values ; lines were generated by smoothing of nearest 3 neighbouring values ( 0th order polynomial ) . ( C ) Fraction of of indicated strains with CLN2 - PP7 mRNA foci , aligned relative to Whi5 export .

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(B) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 import. Symbols represent individual values; lines were generated by smoothing of the nearest 3 neighbouring values (0th order polynomial). (C) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 export.


( D ) Mean of CLN2 mRNA foci at time of Whi5 export , normalised relative to background . Data information boxes include 50 % of data points , lines represent median whiskers extend to maximum minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons . Adjusted p - values in ( D ) : WT vs . esa1 - ts p < 0 . 0001 , WT vs . esa1 - ts nup60 - KN p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 9845 Foci were scored in Z - slices spanning entire volume . n = number of , pooled from two independent experiments with similar results .

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(D) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export, normalised relative to the nuclear background. Data information boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (D): WT vs. esa1-ts p < 0.0001, WT vs. esa1-ts nup60-KN p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.9845 Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.


( E ) Number of CLN2 - PP7 mRNA foci in early G1 ( 5 minutes after Whi5 import ) at G1 / S transition ( 5 min after Whi5 export ) . Data information : boxes include 50 % of data points , lines represent median whiskers extend to maximum minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons . Adjusted p - values in ( E ) : WT vs . esa1 - ts p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 002 . Foci were scored in Z - slices spanning entire volume . n = number of , pooled from two independent experiments with similar results .

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(E) Number of cytoplasmic CLN2-PP7 mRNA foci in early G1 (5 minutes after Whi5 import) and at the G1/S transition (5 min after Whi5 export). Data information: boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (E): WT vs. esa1-ts p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.002. Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.


( F ) position of CLN2 - PP7 foci ( marked with arrowheads in examples ) was scored relatively to periphery ( visualised with PCP - NLS - GFP ) in indicated strains . fraction of non - perinuclear CLN2 - PP7 foci is significantly increased in esa1 - ts compared to WT rescued by nup60 - KN mutation ( two - sided Fisher ' s exact p = 0 . 0209 p = 0 . 0227 ) . Scale bar , 1 µm .

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(F) The position of nuclear CLN2-PP7 foci (marked with arrowheads in the examples) was scored relatively to the nuclear periphery (visualised with PCP-NLS-GFP) in the indicated strains. The fraction of non-perinuclear CLN2-PP7 foci is significantly increased in esa1-ts cells compared to the WT and rescued by nup60-KN mutation (two-sided Fisher's exact test p = 0.0209 and p = 0.0227). Scale bar, 1 µm.


( B ) Overexpression of MEX67 MTR2 rescues lethality of Hos3 - NLS overexpression . 10 - fold serial dilutions of indicated strains transformed with indicated plasmids spotted onto SC - Glu SC - Gal medium incubated at 25 ° C for 3 days .

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(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.


( C ) A high - copy plasmid containing SAC3 rescues lethality of Hos3 - NLS overexpression . Strains carrying a high copy plasmid containing SAC3 ORF ( nucleotides 467 - 3905 ) , or empty vector , were

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(C) A high-copy plasmid containing SAC3 rescues the lethality of Hos3-NLS overexpression. Strains carrying a high copy plasmid containing the SAC3 ORF (nucleotides 467-3905), or the empty vector, were grown


( D ) Overexpression of Hos3 - NLS promotes accumulation of mRNA . ( Left ) Cultures of indicated strains were treated with galactose overnight to induce HOS3 - NLS expression , were fixed , FISH was performed using Cy3 - Oligo ( dT ) . ( Right ) fraction of with mRNA accumulation was determined for indicated strains conditions . Data information : , arrows point to polyadenylated in nucleus , which was visualised by DAPI . Data from 3 independent biological replicates ( 7h30 ) is represented as mean s . d . , data from 2 independent biological replicates ( 0 3h30 ) as mean range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - unpaired t - . At least 200 were scored for each time - point condition . Scale bar , 4 µm .

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(D) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions. Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


( E ) Inactivation of Esa1 impairs export of poly ( A ) . ( Left ) Cultures of indicated strains were incubated at 37 ºC . ( Right ) fraction of with mRNA accumulation was determined for indicated strains conditions Data information : , arrows point to polyadenylated in nucleus , which was visualised by DAPI . Data from 3 independent biological replicates ( 7h30 ) is represented as mean s . d . , data from 2 independent biological replicates ( 0 3h30 ) as mean range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - unpaired t - . At least 200 were scored for each time - point condition . Scale bar , 4 µm .

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(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


( F ) nup60 - KN mutation partially rescues mRNA export defects of esa1 - ts . of indicated strains were processed Data information : arrows point to polyadenylated in nucleus , which was visualised by DAPI . Data from 3 independent biological replicates ( 7h30 ) is represented as mean s . d . , data from 2 independent biological replicates ( 0 3h30 ) as mean range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - unpaired t - . At least 200 were scored for each time - point condition . Scale bar , 4 µm .

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(F) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed Data information: arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


( A ) Depletion of Hos3 or expression of acetyl - mimic Nup60 ( nup60 - KN ) increases localisation of Sac3 in G1 . of indicated strains were imaged by time - lapse microscopy of indicated were determined in G1 ( after cytokinesis ) . NPC component Nup49 was used as a control for Pore . was measured in sum projections of whole - Z - stacks , by segmentation of either in mCherry channel , or of whole in brightfield channel . of Sac3 / Nup49 intensities was then normalised relative to mean of wild - type mothers . Data information arrowheads point to daughter boxes include 50 % of data points , represents median whiskers extend to maximum minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - unpaired t - . Scale bar , 2 µm . n = number of , pooled from three independent experiments with similar results .

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(A) Depletion of Hos3 or expression of acetyl-mimic Nup60 (nup60-KN) increases the nuclear localisation of Sac3 in G1. Cells of the indicated strains were imaged by time-lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 (after cytokinesis). The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels. Fluorescence intensity was measured in sum projections of whole-cell Z-stacks, by segmentation of either the nuclear area in the mCherry channel, or of the whole cell in the brightfield channel. The ratio of Sac3/Nup49 intensities was then normalised relative to the mean intensity of wild-type mothers. Data information arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.


( B ) Inactivation of Esa1 decreases Sac3 . Wild - type ( WT ) esa1 - ts were arrested in mitosis by treatment with nocodazole at 25 ºC , shifted to 37 ºC , released from mitosis block in fresh medium at 37 ºC , imaged by time - lapse microscopy . were quantified in G1 Data information : arrowheads point to daughter boxes include 50 % of data points , represents median whiskers extend to maximum minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - unpaired t - . Scale bar , 2 µm . n = number of , pooled from three independent experiments with similar results .

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(B) Inactivation of Esa1 decreases Sac3 nuclear levels. Wild-type (WT) and esa1-ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC, shifted to 37 ºC, released from the mitosis block in fresh medium at 37 ºC, and imaged by time-lapse microscopy. Fluorescence levels were quantified in G1 Data information: arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.


( C ) Rapamycin - dependent dimerisation abolishes Sac3 mother / daughter asymmetries . NUP60 - mCherry - FKBP SAC3 - GFP - FRB were incubated with rapamycin ( RAPA ) to trigger FRB - FKBP heterodimerization , or with DMSO as control . were quantified in G1 15 to 30 minutes after addition of drug . Data information arrowheads point to daughter , boxes include 50 % of data points , represents median whiskers extend to maximum minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - unpaired t - . Scale bar , 2 µm . n = number of , pooled from three independent experiments with similar results .

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(C) Rapamycin-dependent dimerisation abolishes Sac3 mother/daughter asymmetries. NUP60-mCherry-FKBP SAC3-GFP-FRB cells were incubated with rapamycin (RAPA) to trigger FRB-FKBP heterodimerization, or with DMSO as control. Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug. Data information arrowheads point to daughter cells, boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.


( D ) Sac3 anchoring to basket advances Start in esa1 - ts daughter . Composite of bright field Whi5 - mGFP ( top ) of Whi5 export timing ( bottom ) in wild - type ( WT ) esa1 - ts mother ( M ) daughter ( D ) treated with either rapamycin ( RAPA ) or DMSO expressing Nup60 - FRB Sac3 - mCherry - FKBP . Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter ( DMSO vs RAPA , p > 0 . 05 , Log - rank Mantel - Cox ) , but it advances Whi5 export in esa1 - ts daughters ( p = 0 . 0001 ) . Whi5 export efficiency was slightly improved also in mother ( p = 0 . 0374 ) . 8 z - confocal slices spaced 0 . 4 µm were acquired every 3 min ; maximum projections are shown . Time is indicated in minutes ; t = 0 marks Whi5 import . Scale bar , 5 µm . Data information : arrowheads point to Whi5 export .

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(D) Sac3 anchoring to the nuclear basket advances Start in esa1-ts daughter cells. Composite of bright field and Whi5-mGFP (top) and quantification of Whi5 nuclear export timing (bottom) in wild-type (WT) and esa1-ts mother (M) and daughter (D) cells treated with either rapamycin (RAPA) or DMSO and expressing Nup60-FRB and Sac3-mCherry-FKBP. Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells (DMSO vs RAPA, p > 0.05, Log-rank Mantel-Cox test), but it advances Whi5 export in esa1-ts daughters (p = 0.0001). Whi5 export efficiency was slightly improved also in mother cells (p = 0.0374). 8 z-confocal slices spaced 0.4 µm were acquired every 3 min; maximum projections are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. Data information: arrowheads point to Whi5 export.


( A ) Time Lapse microscopy of WT nup60 - KN expressing GAL1pr : sfGFP Nup60 - mCherry at indicated times of galactose induction . Scale bar , 4 µm .

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(A) Time Lapse microscopy of WT and nup60-KN cells expressing GAL1pr:sfGFP and Nup60-mCherry at the indicated times of galactose induction. Scale bar, 4 µm.


( B ) Depletion of Hos3 , expression of acetyl - mimic Nup60 ( nup60 - KN ) enhances GAL1 expression . WT , hos3Δ , nup60 - KN nup60 - KN hos3Δ were shifted to galactose imaged by time - lapse microscopy to monitor GAL1pr : sfGFP expression during 7 hours . was scored by segmentation of in mCherry channel mean of GFP Nup60 - mCherry was quantified from sum projections of whole - Z - stacks at indicated times . At least 200 were scored for each time point . Shaded areas indicate .

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(B) Depletion of Hos3, and expression of acetyl-mimic Nup60 (nup60-KN) enhances GAL1 expression. WT, hos3Δ, nup60-KN and nup60-KN hos3Δ cells were shifted to galactose and imaged by time-lapse microscopy to monitor GAL1pr:sfGFP expression during 7 hours. Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60-mCherry was quantified from sum projections of whole-cell Z-stacks at the indicated times. At least 200 cells were scored for each strain and time point. Shaded areas indicate the SEM.


( C ) mRNA of GAL1 were determined for wild type ( WT ) nup60 - KN at indicated times after galactose addition . One of two independent experiments with similar results is shown ( mean from 3 technical replicates ) .

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(C) mRNA levels of GAL1 were determined for wild type (WT) and nup60-KN cells at the indicated times after galactose addition. One of two independent experiments with similar results is shown (mean and SEM from 3 technical replicates).


( D ) GFP of mother / daughter pairs for in B , at 5 - minute intervals after galactose addition ( left ) 425 min after galactose addition ( right ) . Boxes include 50 % of data points , represents median whiskers extend to maximum minimum values . * * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - paired t - . n = number of . One of two independent experiments with similar results is shown .

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(D) The GFP intensity of mother/daughter pairs for cells in B, at 5-minute intervals after galactose addition (left) and 425 min after galactose addition (right). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.001; ns, p > 0.05, two-tailed paired t-test. n = number of cells. One of two independent experiments with similar results is shown.


( E ) Expression of GAL1pr : sfGFP induced with galactose ( left ) or ß - estradiol ( right ) , in presence of ß - estradiol - dependent transactivator Gal4 - ER - VP16 . Smooth lines show GFP ( left x axis ) ; lines with circles show difference in GFP between indicated strains ( right x axis ) . difference between wild type hos3 ∆ , between wild type nup60 - KN , increases continuously over time in response to galactose , but not to ß - estradiol .

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(E) Expression of GAL1pr:sfGFP induced with galactose (left) or ß-estradiol (right), in the presence of the ß-estradiol-dependent transactivator Gal4-ER-VP16. Smooth lines show GFP fluorescence intensity (left x axis); lines with circles show the difference in GFP intensity between the indicated strains (right x axis). The difference between wild type and hos3∆, and between wild type and nup60-KN, increases continuously over time in response to galactose, but not to ß-estradiol.


( A ) of indicated strains were incubated with galactose in presence of either rapamycin ( RAPA ) to induce FRB - FKBP heterodimerization , or DMSO as control . GAL1pr : sfGFP expression was monitored over

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(A) Cells of the indicated strains were incubated with galactose in the presence of either rapamycin (RAPA) to induce FRB-FKBP heterodimerization, or DMSO as control. GAL1pr:sfGFP expression was monitored over time


( B ) ( Top ) Representative of indicated in rapamycin or DMSO , 250 min after galactose addition . Scale bar 4 µm . ( Bottom ) Mother / daughter pairs were quantified as in Figure 6D at 250 min after galactose addition ( pink shaded in A ) . Boxes include 50 % of data points , represents median whiskers extend to maximum minimum values . * * , p ≤ 0 . 01 ; * , p ≤ 0 . 05 ; ns , p > 0 . 05 , two - paired t - for M - D comparisons , unpaired for comparisons between strains . n = number of . One of two independent experiments with similar results is shown .

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(B) (Top) Representative images of the indicated cells in rapamycin or DMSO, 250 min after galactose addition. Scale bar 4 µm. (Bottom) Mother/daughter pairs were quantified as in Figure 6D at 250 min after galactose addition (pink shaded area in A). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. **, p ≤ 0.01; *, p ≤ 0.05; ns, p > 0.05, two-tailed paired t-test for M-D comparisons, unpaired for comparisons between strains. n = number of cells. One of two independent experiments with similar results is shown.


( a ) Induction of GFP + dots in differentiated THP - 1 stably expressing GFP - LC3 , left untransfected or transfected for 6 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , then exposed to control antibody ( Control Ab ) , antibody to neutralize IL - 1β ( Anti - IL - 1β ) , dimethyl sulfoxide ( DMSO ) or 3 - MA ( 5 mM ) ; results are presented as of GFP - LC3 + dots ( top ) of ( middle ) . Scale bars , 10 μm .

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(a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.5 μg/ml), then exposed to control antibody (Control Ab), antibody to neutralize IL-1β (Anti-IL-1β), dimethyl sulfoxide (DMSO) or 3-MA (5 mM); results are presented as quantification of GFP-LC3+ dots (top) and images of individual cells (middle). Scale bars, 10 μm.


( b ) Expression of AIM2 beclin - 1 in transfected with control siRNA or siRNA targeting AIM2 or beclin - 1 ; numbers below lanes indicate band ( of transfected with targeting siRNA to those transfected with control siRNA ) .

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(b) Expression of AIM2 and beclin-1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin-1; numbers below lanes indicate band intensity (ratio of cells transfected with targeting siRNA to those transfected with control siRNA).


( c ) Induction of GFP + dots in differentiated THP - 1 stably expressing GFP - LC3 treated with various siRNAs ( below graph ) , quantified 6 h after transfection with poly ( dA : dT ) ( + ) or no transfection ( - ) .

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(c) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3 and treated with various siRNAs (below graph), quantified 6 h after transfection with poly(dA:dT) (+) or no transfection (-).


( d ) Immunoblot of expression of LC3 - I LC3 - II ( above ) in wild - type ( WT ) Aim2 − / − BMDMs left untransfected or transfected for 6 h with poly ( dA : dT ) ; numbers below lanes indicate division of of induced LC3 - I to induced LC3 - II by of basal LC3 - I to basal LC3 - II ( band intensities ) . Below , immunoblot of IL - 1β in supernatants after overnight culture .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

(d) Immunoblot analysis of the expression of LC3-I and LC3-II (above) in wild-type (WT) and Aim2−/− BMDMs left untransfected or transfected for 6 h with poly(dA:dT); numbers below lanes indicate division of the ratio of induced LC3-I to induced LC3-II by the ratio of basal LC3-I to basal LC3-II (band intensities). Below, immunoblot analysis of IL-1β in supernatants after overnight culture.


( e ) Immunoblot of LC3 - I LC3 - II in or wild - type , ASC - deficient ( Pycard - / - ; called ' Asc - / - ' here ) or caspase - 1 - deficient ( Casp1 - / - ) BMDMs primed with LPS ( 20 ng / ml ) then left untransfected or transfected with poly ( dA : dT ) or left untreated or treated with nigericin for 4 h ; numbers below lanes as in d . Actin serves as a loading control throughout . NS , not significant ; * P 0 . 05 * * P 0 . 01 ( unpaired t - ) . Data are from three

O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O

(e) Immunoblot analysis of LC3-I and LC3-II in or wild-type, ASC-deficient (Pycard-/-; called 'Asc-/-' here) or caspase-1-deficient (Casp1-/-) BMDMs primed with LPS (20 ng/ml) and then left untransfected or transfected with poly(dA:dT) or left untreated or treated with nigericin for 4 h; numbers below lanes as in d. Actin serves as a loading control throughout. NS, not significant; *P 0.05 and **P 0.01 (unpaired t-test). Data are from three individual experiments


( a ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 transfected for 3 h with Cy3 - labeled poly ( dA : dT ) ( 1 . 5 μg / ml ) . Yellow dots ( Merge ) indicate colocalization of green ( LC - 3 ) red ( Cy3 ) signals . Scale bars , 10 μm .

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(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected for 3 h with Cy3-labeled poly(dA:dT) (1.5 μg/ml). Yellow dots (Merge) indicate colocalization of green (LC-3) and red (Cy3) signals. Scale bars, 10 μm.


( b ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 transfected to express Cy3 - poly ( dA : dT ) , imaged every 15 min starting 1 h after transfection ( numbers in bottom left corners indicate time in min ( m ) ) . Arrow indicates coalescence of Cy3 GFP signals ( also in bottom right image ) .

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(b) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected to express Cy3-poly(dA:dT), imaged every 15 min starting 1 h after transfection (numbers in bottom left corners indicate time in min (m)). Arrow indicates coalescence of the Cy3 and GFP signals (also in bottom right image).


( c ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 sham - transfected with no poly ( dA : dT ) ( Control ) or transfected with poly ( dA : dT ) , then fixed immunostained for ASC ( red ) . Insets ( bottom right ) , 5 × enlargement of areas indicated by arrowheads at left . Scale bars , 10 μm ( top ) or 20 μm ( bottom ) .

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(c) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells sham-transfected with no poly(dA:dT) (Control) or transfected with poly(dA:dT), then fixed and immunostained for ASC (red). Insets (bottom right), 5× enlargement of areas indicated by arrowheads at left. Scale bars, 10 μm (top) or 20 μm (bottom).


( d ) Confocal microscopy of differentiated THP - 1 transfected with poly ( dA : dT ) immunostained for LAMP - 1 ASC ; below , z - axis projections . Scale bars , 10 μm .

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(d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA:dT) and immunostained for LAMP-1 and ASC; below, z-axis projections. Scale bars, 10 μm.


( e ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 treated for 2 h with LPS ( 500 ng / ml ) ATP ( 3 mM ) , then immunostained for ASC ( red ) : left image in each pair , three - dimensional volume renderings reconstructed from z - stack ; right image in each pair , surface - segmentation models ( 2 × enlargement ) . Scale bars , 10 μm .

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(e) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for ASC (red): left image in each pair, three-dimensional volume renderings reconstructed from z-stack images; right image in each pair, surface-segmentation models (2× enlargement). Scale bars, 10 μm.


( f ) Electron microscopy of differentiated THP - 1 stimulated with LPS ATP ( left ) or transfected with poly ( dA : dT ) ( right ) for 2 h immunostained for ASC , visualized with 15 - nm gold - conjugated A . Red arrows indicate ASC immunostaining . Right image in each pair , 4 . 5 × enlargement of outlined at left . Scale bars , 500 nm . Data are representative of three experiments ( a , c ) or two experiments ( b , d - f ) .

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(f) Electron microscopy of differentiated THP-1 cells stimulated with LPS and ATP (left) or transfected with poly(dA:dT) (right) for 2 h and immunostained for ASC, visualized with 15-nm gold-conjugated protein A. Red arrows indicate ASC immunostaining. Right image in each pair, 4.5× enlargement of outlined area at left. Scale bars, 500 nm. Data are representative of three experiments (a,c) or two experiments (b,d-f).


( a ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 left untransfected ( Control ) or transfected for 2 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , then immunostained for endogenous

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(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells left untransfected (Control) or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), then immunostained for endogenous AIM2


( b ) Confocal microscopy of differentiated THP - 1 transfected for 3 h with poly ( dA : dT ) , then immunostained for LAMP - 1 AIM2 .

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(b) Confocal microscopy of differentiated THP-1 cells transfected for 3 h with poly(dA:dT), then immunostained for LAMP-1 and AIM2.


( c ) Confocal microscopy of differentiated THP - 1 left untreated or treated for 2 h with LPS ( 500 ng / ml ) ATP ( 3 mM ) , then immunostained for endogenous LC3 NLRP3 . Scale bars , 10 μm . Data are representative of at least three experiments .

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(c) Confocal microscopy of differentiated THP-1 cells left untreated or treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for endogenous LC3 and NLRP3. Scale bars, 10 μm. Data are representative of at least three experiments.


( a ) Immunoblot of AIM2 , ASC LC3 - II in lysates of differentiated THP - 1 left untransfected or transfected with poly ( dA : dT ) ( 1 . 5 μg / ml ) left untreated or treated with 3 - MA , rapamycin or - acid starvation , then separated into an inflammasome - enriched fraction ( top ) or an autophagosome - enriched fraction ( middle ) . Bottom , immunoblot of total lysates .

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(a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.5 μg/ml) and left untreated or treated with 3-MA, rapamycin or amino-acid starvation, then separated into an inflammasome-enriched fraction (top) or an autophagosome-enriched fraction (middle). Bottom, immunoblot analysis of total cell lysates.


( b ) Immunoblot of unprocessed processed caspase - 1 IL - 1β in lysates supernatants of differentiated THP - 1 treated as in a .

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(b) Immunoblot analysis of unprocessed and processed caspase-1 and IL-1β in lysates and supernatants of differentiated THP-1 cells treated as in a.


( c ) Immunoblot of LC3 - I LC3 - II ( top ) in lysates of mouse BMDMs primed with LPS ( 20 ng / ml ) left untreated or treated with alum , then left unexposed or exposed for 5 h to live ( Live TB ) or dead ( Dead TB ) M . tuberculosis ( numbers below lanes as in Fig . 1d ) . Below , immunoblot of IL - 1β in supernatants of LPS - primed mouse BMDMs infected overnight with live M . tuberculosis in presence or absence of 3 - MA ( 2 mM ) ; treatment with LPS ATP serves as a positive control . Data are representative of at least two experiments .

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(c) Immunoblot analysis of LC3-I and LC3-II (top) in lysates of mouse BMDMs primed with LPS (20 ng/ml) and left untreated or treated with alum, then left unexposed or exposed for 5 h to live (Live TB) or dead (Dead TB) M. tuberculosis (numbers below lanes as in Fig. 1d). Below, immunoblot analysis of IL-1β in supernatants of LPS-primed mouse BMDMs infected overnight with live M. tuberculosis in the presence or absence of 3-MA (2 mM); treatment with LPS and ATP serves as a positive control. Data are representative of at least two experiments.


( a ) Immunoblot of ASC immunoprecipitates ( IP ; left ) total lysates ( right ) of differentiated THP - 1 left untransfected or transfected for 2 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for beclin - 1 ASC ( below ) , then stripped reprobed for p62 ( above ) .

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(a) Immunoblot analysis of ASC immunoprecipitates (IP; left) and total lysates (right) of differentiated THP-1 cells left untransfected or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), probed for beclin-1 and ASC (below), then stripped and reprobed for p62 (above).


( b ) Confocal microscopy of differentiated THP - 1 treated as in a , immunostained for p62 ( green ) ASC ( red ) ; arrows indicate colocalized .

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(b) Confocal microscopy of differentiated THP-1 cells treated as in a, immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins.


( c ) Confocal microscopy of differentiated THP - 1 treated for 2 h with LPS ( 500 ng / ml ) ATP ( 3mM ) , immunostained for p62 ( green ) ASC ( red ) : left two , merged channel volume surface rendering of a three - dimensional reconstruction ; right four , enlargement of at left ( yellow arrow ) , with arrows indicating removal of part of signal to reveal red signal ( ASC ) inside green - stained ( p62 ) .

O O O O O 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-MEASURED_VAR O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O

(c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62).


( d ) Immunoblot of lysates of transfected twice with siRNA with scrambled ( − ) or siRNA specific for beclin - 1 or p62 ; numbers below lanes indicate band ( as in Fig . 1b ) .

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

(d) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence (−) or siRNA specific for beclin-1 or p62; numbers below lanes indicate band intensity (as in Fig. 1b).


( e ) Immunoblot of inflammasome autophagosome fractions total lysates of differentiated THP - 1 transfected as in d , with poly ( dA : dT ) included in second transfection ; lysates were prepared 4 h after final transfection .

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

(e) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP-1 cells transfected as in d, with poly(dA:dT) included in the second transfection; lysates were prepared 4 h after the final transfection.