B . Scatterplot of genomic locations of accessibility ( ca ) QTL ( x - axis , LOD > 5 ) versus location of ATAC peak being regulated ( y - axis ) for ESCs . Genetic effects that act locally ( n = 3 , 973 ) , i . e . in cis , fall on diagonal , while distal acting QTL ( n = 13 , 767 ) lie off diagonal . Several prominent distal QTL hotspots appear as vertical lines . C . Similar to B , plotting expression ( e ) QTL position versus gene location ( LOD > 5 , n = 701 local - eQTL , n = 357 distal - eQTL ) .

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B. Scatterplot of genomic locations of individual chromatin accessibility (ca)QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines. C. Similar to B, plotting expression (e)QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL). D


G . Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3 , ATAC , read depth from merged replicates . Locations of Chr 7 trans caQTL targets are indicated with black bars LOD scores listed below .

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G. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below.


F . Left - Coverage profiles ( averaged biological replicates , N = 3 ) for ChIP factor occupancy at Cd59a on Chr 2 under trans - regulation by Chr 13d QTL . Distal ca - QTL target region used for is indicated by a black box with LOD score below . Right - Scatterplots for ChIP factors comparing quantitative level of modification / binding for independent replicates between B6 D2 ESCs .

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F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs.


G . difference in occupancy of P300 in parental strains is correlated to difference in accessibility in 33 BXDs on genotype of BXD at QTL for Chr 13 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 61 ) . H . TRIM28 occupancy at same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal ( Pearson ' s r = - 0 . 64 ) .

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G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61). H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64). I


I . Similar to F , coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932 , targeted by Chr 12 trans caQTL , showed greater occupancy of POU5F1 increased open compared to D2 . Distal ca - QTL target region used for is indicated by a black box with LOD score below .

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I. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below.


J . difference in occupancy of POU5F1 in parental strains is correlated to difference in accessibility in 33 BXDs on genotype of BXD at QTL for Chr 12 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 66 ) . K . Similar to J showing negative correlation between TRIM28 occupancy accessibility ( Pearson ' s r = - 0 . 51 ) .

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J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66). K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51). D


A . Coverage profile for H3K4me3 , ATAC , P300 , TRIM28 ChIP at a target locus for QTL on Chr 4 for both accessibility ( black boxes ) gene expression ( Riok3 Rmc1 ) . LOD scores are listed under each target locus .

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A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus.


E . Box whisker plot indicating change in H3K9me3 between WT Chr 4 KO mESCs grouped by whether accessibility is higher when QTL is B6 ( left ) or D2 ( right ) . Dots represent mean values from biological duplicate experiments lines connect means . Orange indicates loci for which H3K9me3 signal decreases in KO ( p - value indicated on top , two - sided paired Wilcoxon ) .

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E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test).


H . MA - plot similar to D showing change in gene expression between WT Chr 4 KO mESCs ( n = 2 ) . Chr 4 QTL targets are indicated in magenta increase expression in KO .

H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO.


Flag - INVS interacted with HA - Akt ( lanes 1 - 3 ) , but not with HA - PDK1 ( lanes 4 - 6 ) or HA - PrKA ( lanes 7 - 9 ) in HEK293T . Expression of HA - Akt , PDK1 , PrKA , Flag - INVS were similar , as determined by immunoblotting ( HA : anti - HA antibody ; F : anti - Flag antibody ; C : control antibody ) . results presented ( A - E ) are representative of at least two independent experiments .

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Flag-INVS interacted with HA-Akt (lanes 1-3), but not with HA-PDK1 (lanes 4-6) or HA-PrKA (lanes 7-9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A-E) are representative of at least two independent experiments.


B . Flag - tagged INVS interacted with HA - tagged Akt1 ( lane 1 - 3 ) , Akt2 ( lane 4 - 6 ) , Akt3 ( lane 7 - 9 ) in co - immunoprecipitationassays . Expression of each Akt isoform INVS were similar .

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B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar.


C . INVS was co - immunoprecipitated with endogenous Akt from HEK293 , in which INVS is also endogenously expressed ( lane 2 ) .

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C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2).


D . To determine Akt domain responsible for interacting with INVS , Akt subfragments were generated . C - terminus of Akt is domain for INVS interaction ( lanes 7 - 9 ) . Similar of each Akt fragment were immunoprecipitated using anti - HA antibody .

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D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7-9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody.


E . Schematic showing structure functional domains of Flag - tagged full - fragmented INVS in mammalian expression vectors ( N - Term : 1 - 421 , Intermediate domain : 421 - 678 , C - Term : 678 - 1065 ) used in current study . Intermediate ( lanes 6 - 9 ) C - terminal ( lanes 10 - 12 ) fragments of INVS were important for interaction with Akt .

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E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1-421, Intermediate domain: 421-678, and C-Term: 678-1065) used in the current study. Intermediate (lanes 6-9) and C-terminal (lanes 10-12) fragments of INVS were important for interaction with Akt.


A . indicated recombinant INVS ( lanes 1 - 4 ) were used for IVK . Akt phosphorylates WT ( lane5 ) , 1 - 970 ( lane 6 ) , 1 - 898 ( lane 7 ) INVS , but failed to phosphorylate 1 - 670 ( lane 8 ) INVS .

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A. The indicated recombinant INVS proteins (lanes 1-4) were used for IVK. Akt phosphorylates WT (lane5), 1-970 (lane 6), and 1-898 (lane 7) INVS, but failed to phosphorylate 1-670 (lane 8) INVS.


indicated recombinant INVS ( lanes 1 - 10 ) were used for IVK . Akt phosphorylates WT ( lane 6 ) 1 - 898 ( lane7 ) INVS , but failed to phosphorylate other subfragments of INVS ( 1 - 822 , 1 - 746 , 1 - 670 , in lane 8 , 9 , 10 , respectively ) , as detected using a phospho - Akt substrate antibody . .

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The indicated recombinant INVS proteins (lanes 1-10) were used for IVK. Akt phosphorylates WT (lane 6) and 1-898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1-822, 1-746, and 1-670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody..


C . Using indicated subfragments of INVS for IVK , we confirmed that Akt phosphorylates 675 - 1065 ( lane 4 ) , but not 675 - 822 ( lane 5 ) , or 675 - 746 ( lane 6 ) INVS .

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C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675-1065 (lane 4), but not 675-822 (lane 5), or 675-746 (lane 6) INVS.


E . Purified recombinant WT or 3A INVS were generated ( top panel , lanes 1 - 4 ) . In IVK , Akt phosphorylated WT fragment ( 824 - 898 , middle panel , lane 3 ) , but failed to phosphorylate 3A INVS ( middle panel , lane 4 ) , as detected by using a phospho - Akt substrate antibody .

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E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1-4). In IVK, Akt phosphorylated the WT fragment (824-898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody.


F . Akt phosphorylated WT ( lane 10 ) , 1 - 898 ( lane 12 ) 675 - 1065 ( lane 15 ) INVS , but failed to phosphorylate both full - 675 - 1065 , ∆ 727 - 896 , 1 - 857 , or 1 - 822 , 3A INVS ( lanes 16 , 11 , 14 , 15 , respectively ) , of which lacked T864 / S865 / T866 target acid .

F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.


A . Confocal microscopy shows that active Akt ( phospho - S473Akt ) is localized in basal of cilia ( right side panels show higher magnification ) . B . INVS is localized in proximal region of INVS compartment in cilia ( INVS compartment , right side panels show higher magnification ) . C . INVS co - localizes with γ - tubulin , a centrosome marker . D . Anti - phospho - Akt substrates stain positively at basal region of cilia ( arrows ) , as determined by anti - acetylated tubulin ( right side panels show higher magnification ) .

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A. Confocal microscopy shows that active Akt (phospho-S473Akt) is localized in the basal body of primary cilia (right side panels show higher magnification).B. INVS is localized in the proximal region of the INVS compartment in primary cilia (INVS compartment, right side panels show higher magnification).C. INVS co-localizes with γ-tubulin, a centrosome marker.D. Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin (right side panels show higher magnification).


E . Silver - intensified immunogold electron microscopy shows that both phospho - Akt ( left side panels ) INVS ( right side panels ) were localized to ciliary pocket of cilia in presence of PDGF - AA ( arrow ) .

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E. Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left side panels) and INVS (right side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrow).


PDGF - AA stimulation enhances localization of active Akt ( phospho - S473Akt ) at basal of cilia ( right side panels show higher magnification ) .

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PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification).


of co - localization of phosphorylated Akt ( green ) INVS ( red ) at indicated time points after PDGF - AA stimulation revealed a time - dependent increase . Colocalization was measured by counting yellow pixels ( colocalized ) using Imaris software ( Bitplane AG ) . Results presented are means ± SE ( n = 31 at time 0 , n = 35 at 1 min . , n = 33 at 3 min , respectively ) . Three independent experiments were analyzed with similar results . Statistical significance was determined by student ' s t .

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Quantification of co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student's t test.


Co - immunoprecipitation assays using HEK293T show that INVS preferentially binds to WT - Akt ( second panel , lane1 ) or phospho - mimetic Akt ( T308D / S473D , lane2 ) , but fails to interact with phosphorylation - defective forms of Akt ( T308A / S473A , lane 3 ) . PDGF - AA stimulation of HEK293T results in augmented interaction of endogenous INVS with Akt ( top panel , comparison between lane 1 lane 2 , untreated PDGF - AA - stimulated conditions , respectively ) .

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Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively).


Both WT 3A - INVS , which are localized at INVS compartment of cilia under serum - starved condition ( left side panels , lower panels show higher magnification ) , translocate to basal of cilium after PDGF - AA stimulation ( right side panels , lower panels show higher magnification ) . Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP - INVS ( green ) with γ - tubulin ( red ) in hTERT - RPE1 ( n = 28 ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t . intensities of INVS ( green ) , acetylated - α - tubulin ( blue ) , γ - tubulin ( red ) along ( a - b ) are plotted underneath .

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Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student's t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.


A . PDGF - AA stimulation resulted in phosphorylation of WT INVS ( top panel , lane 2 ) , but not 3A INVS ( top panel , lane 6 ) . PDGF - AA - stimulated INVS phosphorylation is inhibited by Akt inhibitors ( top panel , LY294002 , MK2206 , GSK690693 , lanes 3 , 4 , 5 , respectively ) .

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A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively).


B . Myr - Akt , a constitutively active form of Akt , was expressed together with WT or 3A INVS in HEK293 INVS phosphorylation was quantified . WT , but not 3A INVS , can be phosphorylated in presence of Myr - Akt .

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B. Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt.


C . 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS ( top panel , compare lanes 1 2 , WT phospho - defective mutant , respectively ) . Expression of Akt INVS are shown underneath . D . 3A INVS exhibited weaker dimerization as compared to WT INVS ( top panel , compare lanes 1 2 , WT 3A , respectively ) . Expression of Akt INVS are shown below each .

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C. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath.D. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each.


E . of cilia in confluent hTERT - RPE was analyzed after siRNA - mediated knock down of Akt1 / 2 . Ciliary decreased in expressing siRNA . Results presented are means ± SE of longitudinal of acetylated - tubulin ( red ) , a marker of cilia , in siRNA - transfected ( green ) hTERT - RPE1 ( n = 175 for control siRNA Akt siRNA , n = 195 for Akt siRNA plus siRNA resistant Akt , respectively ) . Three independent experiments showed similar results . Statistical significance was analyzed by student ' s t . Note that re - introduction of siRNA - resistant Akt ( Matsuda - Lennikov et al . , 2014 ) in Akt knockdown rescued effect on ciliary .

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E. Length of primary cilia in confluent hTERT-RPE cells was analyzed after siRNA-mediated knock down of Akt1/2. Ciliary length decreased in cells expressing siRNA. Results presented are means ±SE of the longitudinal length of acetylated-tubulin (red), a marker of primary cilia, in siRNA-transfected (green) hTERT-RPE1 cells (n=175 for control siRNA and Akt siRNA, and n=195 for Akt siRNA plus siRNA resistant Akt, respectively). Three independent experiments showed similar results. Statistical significance was analyzed by student's t test. Note that re-introduction of siRNA-resistant Akt (Matsuda-Lennikov et al., 2014) in Akt knockdown cells rescued the effect on ciliary length.


F . Expression of 3A INVS ( bottom panels ) but not WT INVS ( middle panels ) inhibited development of cilia as compared to control ( top panels ) .

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F. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels).


of ciliary . Results are mean ± SE of longitudinal of acetylated - tubulin ( blue ) , a marker of cilia , in EGFP - INVS ( green ) transfected in hTERT - RPE1 ( n = 58 for EGFP vector for EGFP - INVS WT , n = 52 for EGFP - INVS 3A mutant , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t .

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Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


Schematic describing measurement of spindle angle ( θ ) ( Kikuchi et al . , 2010 ) . Z - stack were obtained from 0 . 45 mm - thick sections of metaphase angle between axis of metaphase spindle that of substrate surface , which is termed spindle angle , was calculated . Results presented are scatter plots of angle ( θ ) of mitotic spindle ( n = 28 for samples of vector , HA - WT , - 3D , - 3A , - R899X , - Q671X , - R603X INVS stably transfected MDCK ) . Three independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median .

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Schematic describing the measurement of spindle angle (θ) (Kikuchi et al., 2010). Z-stack images were obtained from 0.45 mm-thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface, which is termed the spindle angle, was calculated. Results presented are scatter plots of the angle (θ) of the mitotic spindle (n=28 for all samples of vector, HA-WT, -3D, -3A, -R899X, -Q671X, and -R603X INVS stably transfected MDCK cells). Three independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


Spindle angles were measured in MDCK transfected with indicated INVS plasmids ( + ) . spindle axis was misaligned in expressing mutant forms of INVS as compared to expressing WT INVS or sham - transduced . Expression of 3A INVS of clinically relevant truncated forms of INVS but not WT INVS , resulted in disorientation of spindle axis during metaphase . Similar expression of INVS constructs was verified by immunoblotting ( Appendix figure S4A ) .

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Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids (+). The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham-transduced cells. Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS, resulted in disorientation of the spindle axis during metaphase. Similar expression of all INVS constructs was verified by immunoblotting (Appendix figure S4A).


WT INVS robustly inhibited transactivation of INVS in a luciferase reporter , which could be reverted by C - terminal truncated INVS ( R899X , Q671X , or Q603X ) , but not by a phospho - mimetic mutant or 3A INVS . Results presented are mean ± SE of of triplicates between sequentially measured firefly TCF / LEF signal Renilla luciferase signal in HA - INVS transfected 293T . Dual - Luciferase reporter ® System ( Promega ) was used experiments were performed in triplicates . Two independent experiments showed similar results . Statistical significance was determined by student ' s t .

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WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay, which could be reverted by C-terminal truncated INVS (R899X, Q671X, or Q603X), but not by a phospho-mimetic mutant or 3A INVS. Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF/LEF signal and Renilla luciferase signal in HA-INVS transfected 293T cells. Dual-Luciferase reporter® Assay System (Promega) was used and experiments were performed in triplicates. Two independent experiments showed similar results. Statistical significance was determined by student's t test.


expression of HA - Dvl ( upper panels ) indicated forms of Flag - INVS ( lower panels ) are shown by immunoblots ; α - tubulin was used as an internal control .

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The expression of HA-Dvl (upper panels) and the indicated forms of Flag-INVS (lower panels) are shown by immunoblots; α-tubulin was used as an internal control.


WT INVS - transduced MDCK in 3D cultures form uniform round tubules . However , expression of 3A or NPHP2 - related truncated INVS mutants , which lack Akt phosphorylation site ( s ) , disrupts this uniformity . Results presented are representative of three independent experiments show mean ± SE of normal acini in vector - or WT INVS - transduced MDCK ( n = 422 for Vector , n = 398 for HA - WT , n = 401 for - 3D , n = 365 for - 3A , n = 344 for - R899X , n = 331 for - Q671X , n = 325 for - R603X INVS stably transfected MDCK , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t .

WT INVS- transduced MDCK cells in 3D cultures form uniform round tubules. However, expression of 3A or NPHP2-related truncated INVS mutants, which lack Akt phosphorylation site (s), disrupts this uniformity. Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=422 for Vector, n=398 for HA-WT, n=401 for -3D, n=365 for -3A, n= 344 for -R899X, n=331 for -Q671X, and n=325 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


WT transduced MDCK form a relatively smaller lumen ; however , expression of 3A INVS or indicated NPHP2 - related truncation mutants result in relatively larger acini . Both truncation mutants ( Q671X or R603X ) lack Akt phosphorylation site exhibit a larger lumen . Three independent experiments showed similar results .

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WT transduced MDCK cells form a relatively smaller lumen; however, expression of 3A INVS or the indicated NPHP2-related truncation mutants result in relatively larger acini. Both truncation mutants (Q671X or R603X) lack the Akt phosphorylation site and exhibit a larger lumen size. Three independent experiments showed similar results.


C . of normal lumen in transduced with WT or mutant INVS . Results presented are mean ± SE of normal acini in vector - or WT INVS - transduced MDCK ( n = 166 for Vector , n = 204 for HA - WT , n = 233 for - 3D , n = 150 for - 3A , n = 164 for - R899X , n = 128 for - Q671X , n = 137 for - R603X INVS stably transfected MDCK , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t .

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C. Quantification of normal lumen in cells transduced with WT or mutant INVS. Results presented are mean ±SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=166 for Vector, n=204 for HA-WT, n=233 for -3D, n=150 for -3A, n=164 for -R899X, n=128 for -Q671X, and n=137 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


D . of lumen in transduced with WT or mutant INVS is shown . Results presented are scatter plots of acini ( n = 205 for Vector , n = 143 for HA - WT , n = 131 for - 3D , n = 167 for - 3A , n = 144 for - R899X , n = 128 for - Q671X , n = 144 for - R603X INVS stably transfected MDCK , respectively ) . Two independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median .

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D. Size of lumen in cells transduced with WT or mutant INVS is shown. Results presented are scatter plots of acini size (n=205 for Vector, n=143 for HA-WT, n=131 for -3D, n=167 for -3A, n=144 for -R899X, n=128 for -Q671X, and n=144 for -R603X INVS stably transfected MDCK cells, respectively). Two independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


B ) Electrophoretic mobility shift ( EMSA ) examination of binding affinity of CENPC - CT CM peptide to a CENP - A nucleosome . Binding to a canonical H3 nucleosome was also examined using EMSA .

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B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA.


C ) EMSA results of binding affinities of CENPC - CT WT , CENPC - CT 3A679 - 681 , CENPC - CT ∆ 678 - 683 to CENP - A nucleosome .

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C) EMSA results of the binding affinities of CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 to the CENP-A nucleosome.


D ) Stable expression of GFP - fused CENPC - CT its mutants ( shown in ( B ) ) in CENP - C knockout chicken DT40 . α - Tubulin ( Tub ) was probed as a loading control . Parental CENP - C knockout ( parental ) were also examined .

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D) Stable expression of GFP-fused CENPC-CT and its mutants (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. Parental CENP-C knockout cells (parental) were also examined.


E ) Localization of GFP - fused CENPC - CT WT , CENPC - CT 3A679 - 681 , CENPC - CT ∆ 678 - 683 ( green ) on mitotic chromosomes in chicken DT40 . CENP - T ( red ) was used as a centromere marker . was stained using 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar indicates 10 μm .

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E) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm.


F ) Localization of CENP - C in CENP - A knockout DT40 stably expressing GFP - fused gCENP - A WT , RG - AA , or ∆ RG . Scale bar indicates 10 μm . CENP - C was stained by an anti - CENP - C antibody ( red ) was stained by DAPI ( blue ) . CENP - C signals on kinetochores in mitotic were quantified in each . Bar graph indicates mean ± SD ( n = 7 ; * * * * , P < 0 . 0001 , unpaired t , two ) .

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F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).


C ) EMSA results of binding affinities of CENP - C fragments indicated in ( B ) to CENP - A nucleosome . fragments were phosphorylated by CDK1 .

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C) EMSA results of the binding affinities of CENP-C fragments indicated in (B) to the CENP-A nucleosome. All fragments were phosphorylated by CDK1.


D ) Stable expression of GFP - fused CENPC - CT CENPC643 - 864 ( shown in ( B ) ) in CENP - C knockout chicken DT40 . α - Tubulin ( Tub ) was probed as a loading control .

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D) Stable expression of GFP-fused CENPC-CT and CENPC643-864 (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control.


E ) Localization of GFP - fused CENPC - CT CENPC643 - 864 lacking CM upstream region ( green ) on mitotic chromosomes in CENP - C knockout DT40 . CENP - T ( red ) was used as a centromere marker . was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

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E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) position of a CDK1 phosphorylation site , T651CENP - C , is indicated in diagram of chicken CENPC - CT . EMSA was performed to examine binding affinities of phosphorylated or non - phosphorylated CENPC - CT ( left panel ) CM peptide ( right panel ) to CENP - A nucleosome .

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A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome.


D ) constructs analyzed in ( E ) were stably expressed in CENP - C knockout .

D) All constructs analyzed in (E) were stably expressed in CENP-C knockout cells.


E ) Localization of GFP - fused CENPC - CT , CENPC - CTR656E , CENPC - CTR655E _ R656E ( green ) on mitotic chromosomes in chicken DT40 . was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

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E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) Competitive pull - down assays for CENP - A nucleosome binding of CENPC - CT CENPN - NT . left panel displays a of MBP - CENPN - NT ( MBP - CN - NT ) with CENP - A nucleosome ( CA - nuc ) incubated in absence or presence of phosphorylated CENPC - CT ( CC - CT ) pulled down by MBP affinity . right panel displays a of phosphorylated MBP - CENPC - CT ( MBP - CC - CT ) with CENP - A nucleosome ( CA - nuc ) incubated in absence or presence of CENPN - NT ( CN - NT ) pulled down by MBP affinity . signal intensities of histones in CENP - A nucleosomes , which were precipitated with MBP in indicated lanes ( * ) , were quantified . signal intensities were normalized to MBP - CN - NT or MBP - CC - CT signals . Bar graph indicates mean with SD ( n = 3 ) .

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A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3).


Immunoprecipitation of SATB2 from mouse neonatal cortical lysates . LEMD2 was detected by Western blotting in SATB2 - immunoprecipitate from control but not SATB2 - deficient cortical lysate . equal input of total from control SATB2 - deficient cortical lysates was controlled by ERK2 detection . Representative of immunoblots are shown ; I ( Input ) , F ( Flow - through ) , B ( Beads ) .

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Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2-immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads).


Reverse immunoprecipitation with LEMD2 antibody control IgG antibody , followed by WB detection of LEMD2 SATB2 . Lysates from cortical culture lysates were used .

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Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used.


GST pull - down assays . Representative of n = 3 independent experiments are shown . GST pull - down of transiently overexpressed V5 - tagged LEMD2 ( i ) , SATB2 ( ii ) , LEMD2ΔLEM ( iii ) from HeLa lysates using GST , GST - SATB2 , GST - LEMD2 ( 413 - 503 ) hybrid .

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GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins.


Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa transfected with expression plasmids encoding V5 - tagged full - SATB2 ( Satb21 - 733 ) EGFP as control . equal input of total was controlled by GAPDH detection . Representative of immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular marker ) .

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Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21-733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


CUT - like domain of SATB2 is required for interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa transfected with V5 - tagged Satb21 - 247 Satb21 - 157 deletion mutants . LEMD2 BAF were detected only in immunoprecipitates from HeLa transfected with deletion mutant containing CUT - like domain ( Satb21 - 247 ) . equal input of total was controlled by GAPDH detection . Representative of immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular marker ) .

The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21-247 and Satb21-157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21-247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


CUT1 CUT2 domains are required for interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from HeLa transfected with V5 - tagged Satb2346 - 733 Satb2616 - 733 deletion mutants . LEMD2 BAF were detected only in immunoprecipitates from HeLa expressing CUT1 CUT2 containing deletion mutant ( Satb2346 - 733 ) .

CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).


Representative of z - axis projected stacks of confocal of hippocampal neuronal nuclei immunostained for Lamin B2 . Examples of spherical infolded nuclei are shown . Scale bars : 3 μm .

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Representative images of z-axis projected stacks of confocal images of hippocampal neuronal nuclei immunostained for Lamin B2. Examples of spherical and infolded nuclei are shown. Scale bars: 3 μm.


Activity - induced formation of infoldings is abolished in SATB2 - defficient hippocampal neurons . Bic - induced AP bursting for 1 h caused a significant increase in of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx / flx mice but not from Satb2flx / flx : : Nes - Cre mice ( Satb2NesCre ) ( n = 6 independent cultures , two - way ANOVA , F1 , 20 = 9 . 51 , significant interaction p = 0 . 0058 , simple main effects , Satb2flx / flx cultures , Bic - treated vs untreated p = 0 . 0000043 , Satb2NesCre cultures , Bic - treated vs untreated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , untreated - 680 , Satb2flx / flx cultures , Bic - treated - 711 , Satb2NesCre , untreated - 755 , Satb2NesCre , Bic - treated - 720 ) . Data are presented as mean ± , * * * p < 0 . 001 .

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Activity-induced formation of nuclear infoldings is abolished in SATB2-defficient hippocampal neurons. Bic-induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx/flx mice but not from Satb2flx/flx::Nes-Cre mice (Satb2NesCre) (n = 6 independent primary cultures, two-way ANOVA, F1,20 = 9.51, significant interaction p = 0.0058, simple main effects analysis, Satb2flx/flx cultures, Bic-treated vs untreated p = 0.0000043, Satb2NesCre cultures, Bic-treated vs untreated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, untreated - 680, Satb2flx/flx cultures, Bic-treated - 711, Satb2NesCre, untreated - 755, Satb2NesCre, Bic-treated - 720). Data are presented as mean ± SEM, ***p < 0.001.


Activity - induced formation of infoldings is impaired in SATB2 - deficient cortical neurons . DIV14 cortical cultures form Satb2flx / flx Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h . of infolded nuclei following AP bursting was increased in control Satb2flx / flx cultures but not in SATB2 - deficient cultures ( n = 3 - 4 independent cultures , two - way ANOVA , F1 , 10 = 39 . 124 , significant interaction p = 0 . 000094 , simple main effects , Satb2flx / flx cultures : Bic - treated vs NBQX - treated p = 7 . 66E - 07 , Satb2NesCre cultures : Bic - treated vs NBQX - treated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , NBQX - 606 , Satb2flx / flx cultures , Bic - 910 ; Satb2NesCre cultures , NBQX - 604 , Satb2NesCre cultures , Bic - 835 ) . Data are presented as mean ± , * * * p < 0 . 001 .

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Activity-induced formation of nuclear infoldings is impaired in SATB2-deficient cortical neurons. DIV14 cortical cultures form Satb2flx/flx and Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h. The percentage of infolded nuclei following AP bursting was increased in control Satb2flx/flx cultures but not in SATB2-deficient cultures (n = 3-4 independent primary cultures, two-way ANOVA, F1,10 = 39.124, significant interaction p = 0.000094, simple main effects analysis, Satb2flx/flx cultures: Bic-treated vs NBQX-treated p = 7.66E-07, Satb2NesCre cultures: Bic-treated vs NBQX-treated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, NBQX - 606, Satb2flx/flx cultures, Bic - 910; Satb2NesCre cultures, NBQX -604, Satb2NesCre cultures, Bic - 835). Data are presented as mean ± SEM, ***p < 0.001.


Confocal ( z - axis - projected stacks ) of Lamin B2 stained neuronal nuclei from CA1 pyramidal layer of adult Satb2flx / flx Satb2flx / flx : : CamK2a - Cre ( Satb2CamkCre ) mice . Scale bars : 10 μm .

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Confocal images (z-axis-projected stacks) of Lamin B2 stained neuronal nuclei from the CA1 pyramidal cell layer of adult Satb2flx/flx and Satb2flx/flx::CamK2a-Cre (Satb2CamkCre) mice. Scale bars: 10 μm.


of of infolded nuclei in hippocampus of Satb2flx / flx mice vs Satb2CamkCre mice . In Satb2flx / flx mice , number of infolded nuclei was significantly higher in CA1 compared to CA3 ( p = 0 . 000001 ) DG ( p = 1 . 8 E - 12 ) . In Satb2CamkCre mice , number of infolded nuclei in CA1 was significantly reduced compared to corresponding number in littermate controls ( p = 1 . 9 E - 09 ) . No significant differences were observed in this number in CA3 DG between Satb2CamkCre mice floxed controls ( n = 4 mice , two - way ANOVA , significant interaction , F2 , 18 = 47 . 81 , p = 0 . 00001 , simple main effects , CA1 , Satb2flx / flx vs CKO mice p = 1 . 9 E - 09 , CA3 , Satb2flx / flx vs CKO mice p = 0 . 909 , DG , Satb2flx / flx vs CKO mice p = 0 . 262 , adjustment for multiple comparisons : Bonferroni ) . Number of analyzed nuclei : Satb2flx / flx mice : 469 ( CA1 ) , 225 ( CA3 ) , 665 ( DG ) ; Satb2CamkCre : 508 ( CA1 ) , 224 ( CA3 ) , 575 ( DG ) . Data are presented as mean ± , * * * p < 0 . 001 .

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

Analysis of the percentage of infolded nuclei in the hippocampus of Satb2flx/flx mice vs Satb2CamkCre mice. In Satb2flx/flx mice, the number of infolded nuclei was significantly higher in the CA1 area compared to the CA3 area (p = 0.000001) and DG (p = 1.8 E-12). In Satb2CamkCre mice, the number of infolded nuclei in the CA1 area was significantly reduced compared to the corresponding number in littermate controls (p = 1.9 E-09). No significant differences were observed in this number in the CA3 and DG between Satb2CamkCre mice and floxed controls (n = 4 mice, two-way ANOVA, significant interaction, F2,18 = 47.81, p = 0.00001, simple main effects analysis, CA1 area, Satb2flx/flx vs CKO mice p = 1.9 E-09, CA3 area, Satb2flx/flx vs CKO mice p = 0.909, DG, Satb2flx/flx vs CKO mice p = 0.262, adjustment for multiple comparisons: Bonferroni). Number of analyzed nuclei: Satb2flx/flx mice: 469 (CA1), 225 (CA3), 665 (DG); Satb2CamkCre: 508 (CA1), 224 (CA3), 575 (DG). Data are presented as mean ± SEM, ***p < 0.001.


of of infolded nuclei in CA1 pyramidal layer of Satb2CamkCre mice vs . littermate controls after stereotaxic injections of AAV - SATB2 - V5 or AAV8 - EGFP . Viral delivery of SATB2 resulted in restoration of number of infolded nuclei in CA1 pyramidal layer of Satb2CamkCre mice ( n = 3 - 5 mice , one - way ANOVA followed by Hochberg post hoc , F2 , 9 = 33 . 1 , Satb2CamkCre : : AAV - SATB2 vs . Satb2flx / flx : : AAV - EGFP , p = 0 . 0012 ; Satb2flx / flx : : AAV - EGFP vs . Satb2CamkCre : : AAV - EGFP , p = 0 . 00008 , Satb2flx / flx : : AAV - EGFP vs . Satb2CamkCre : : AAV - SATB2 , p = 0 . 072 ) . Number of analyzed nuclei : 388 ( Satb2flx / flx : : AAV - EGFP ) , 560 ( Satb2CamkCre : : AAV - EGFP ) , 546 ( Satb2CamkCre : : AAV - SATB2 ) . Data are presented as mean ± , * * * p < 0 . 001 compared to Satb2flx / flx : : AAV - EGFP , # # p < 0 . 01 , compared to Satb2CamkCre : : AAV - EGFP .

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

Analysis of the percentage of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice vs. littermate controls after stereotaxic injections of AAV-SATB2-V5 or AAV8-EGFP. Viral delivery of SATB2 resulted in restoration of the number of infolded nuclei in the CA1 pyramidal cell layer of Satb2CamkCre mice (n = 3-5 mice, one-way ANOVA followed by Hochberg post hoc test, F2,9 = 33.1, Satb2CamkCre::AAV-SATB2 vs. Satb2flx/flx::AAV-EGFP, p = 0.0012; Satb2flx/flx::AAV-EGFP vs. Satb2CamkCre::AAV-EGFP, p = 0.00008, Satb2flx/flx::AAV-EGFP vs. Satb2CamkCre::AAV-SATB2, p = 0.072). Number of analyzed nuclei: 388 (Satb2flx/flx::AAV-EGFP), 560 (Satb2CamkCre::AAV-EGFP), 546 (Satb2CamkCre::AAV-SATB2). Data are presented as mean ± SEM, ***p < 0.001 compared to Satb2flx/flx::AAV-EGFP, ##p < 0.01, compared to Satb2CamkCre::AAV-EGFP.


infolding triggered by SATB2 overexpression requires LEMD2 . of of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression Lemd2 knockdown . AAV - SATB2 - transduced neurons ( both non - transfected scrambled siRNA - transfected , Scr ) exhibited higher of infolded nuclei compared to AAV - EGFP - transduced neurons . In contrast , LEMD2 - depleted AAV - SATB2 - transduced cultures showed similar of infolded nuclei to AAV - EGFP - transduced neurons ( n = 3 - 4 independent cultures ; one - way ANOVA F3 , 11 = 30 . 982 , p = 0 . 000011 ; Hochberg post hoc , AAV - EGFP vs . AAV - SATB2 p = 0 . 0001 , AAV - EGFP vs . AAV - SATB2 + Scr p = 0 . 0001 , AAV - EGFP vs . AAV - SATB2 + siLemd2 p = 0 . 9999 , AAV - SATB2 + Scr vs . AAV - SATB2 + siLemd2 p = 0 . 0001 ) . Number of nuclei analyzed : 460 ( AAV - EGFP ) , 466 ( AAV - SATB2 ) , 537 ( AAV - SATB2 + Scr ) , 597 ( AAV - SATB2 + siLem2 ) . Data are presented as mean ± , * * * p < 0 . 001 compared to AAV - EGFP ; # # # p < 0 . 001 compared to AAV - SATB2 + Scr .

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

Nuclear infolding triggered by SATB2 overexpression requires LEMD2. Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression and Lemd2 knockdown. AAV-SATB2-transduced neurons (both non-transfected and scrambled siRNA-transfected, Scr) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, LEMD2-depleted AAV-SATB2-transduced cultures showed similar percentage of infolded nuclei to AAV-EGFP-transduced neurons (n = 3-4 independent primary cultures; one-way ANOVA F3,11 = 30.982, p = 0.000011; Hochberg post hoc test, AAV-EGFP vs. AAV-SATB2 p = 0.0001, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0001, AAV-EGFP vs. AAV-SATB2 + siLemd2 p= 0.9999, AAV-SATB2 + Scr vs. AAV-SATB2 + siLemd2 p = 0.0001). Number of nuclei analyzed: 460 (AAV-EGFP), 466 (AAV-SATB2), 537 (AAV-SATB2 + Scr), 597 (AAV-SATB2+siLem2). Data are presented as mean ± SEM, ***p < 0.001 compared to AAV-EGFP; ###p < 0.001 compared to AAV-SATB2 + Scr.


Activity - induced formation of infoldings is impaired in LEMD2 - depleted cortical neurons . Bic - triggered AP bursting for 1 h caused a significant increase in of infolded nuclei in cortical neurons transduced with AAV - scrambled but not with AAV - shLemd2 ( n = 5 independent cultures , two - way ANOVA , F1 , 16 = 8 . 35 , significant interaction p = 0 . 0107 , simple main effects : AAV - scrambled - transduced cultures , Bic - treated vs untreated p = 0 . 0049 , AAV - shLemd2 - transduced cultures , Bic - treated vs untreated p > 0 . 99 , adjustment for multiple comparisons : Bonferroni ) . Data are presented as mean ± , * * p < 0 . 01 compared to NBQX - treated cultures .

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

Activity-induced formation of nuclear infoldings is impaired in LEMD2-depleted cortical neurons. Bic-triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in cortical neurons transduced with AAV-scrambled but not with AAV-shLemd2 virus (n = 5 independent primary cultures, two-way ANOVA, F1,16 = 8.35, significant interaction p = 0.0107, simple main effects analysis: AAV-scrambled-transduced cultures, Bic-treated vs untreated p = 0.0049, AAV-shLemd2-transduced cultures, Bic-treated vs untreated p > 0.99, adjustment for multiple comparisons: Bonferroni). Data are presented as mean ± SEM, **p < 0.01 compared to NBQX-treated cultures.


LEMD2 is required for envelope infoldings of CA1 pyramidal neurons in vivo . Top panel : representative confocal ( z - axis - projected stack ) of Lamin B2 / DAPI - stained coronal brain sections from wild - type mice after stereotaxic injection of AAV - shRNA ( AAV - shLemd2 - mCherry or AAV - scrambled - mCherry ) into dorsal hippocampus . Merged , colocalization of three signals . Scale bars : 50 μm . Middle panel : higher magnification of AAV - shLemd2 - transduced CA1 pyramidal neurons . Arrows show examples of mCherry - positive nuclei that are devoid of infoldings , whereas stars denote non - transduced infolded nuclei . Lower panel : higher magnification of AAV - scrambled siRNA - transduced CA1 pyramidal neurons . Arrows show examples of infolded , mCherry - positive nuclei ; stars denote infolded , non - transduced neurons . Scale bars ( middle lower panels ) : 10 μm .

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

LEMD2 is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo. Top panel: representative confocal images (z-axis-projected stack) of Lamin B2/DAPI-stained coronal brain sections from wild-type mice after stereotaxic injection of AAV-shRNA viruses (AAV-shLemd2-mCherry or AAV-scrambled-mCherry) into the dorsal hippocampus. Merged, colocalization of the three signals. Scale bars: 50 μm. Middle panel: higher magnification images of AAV-shLemd2-transduced CA1 pyramidal neurons. Arrows show examples of mCherry-positive nuclei that are devoid of nuclear infoldings, whereas stars denote non-transduced infolded nuclei. Lower panel: higher magnification images of AAV-scrambled siRNA-transduced CA1 pyramidal neurons. Arrows show examples of infolded, mCherry-positive nuclei; stars denote infolded, non-transduced neurons. Scale bars (middle and lower panels): 10 μm.


of of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression Vps4a / Vps4b knockdown . AAV - SATB2 - transduced neurons ( non - transfected or scrambled siRNA ( Scr ) - transfected ) exhibited higher of infolded nuclei compared to AAV - EGFP - transduced neurons . In contrast , VPS4 - depleted AAV - SATB2 - transduced cultures showed similar number of infolded nuclei to AAV - EGFP - transduced neurons ( n = 4 independent cultures , one - way ANOVA followed by Tuckey post hoc , F3 , 11 = 15 . 866 , p = 0 . 00026 , AAV - EGFP vs . AAV - SATB2 p = 0 . 0104 , AAV - EGFP vs . AAV - SATB2 + Scr p = 0 . 0028 , AAV - EGFP vs . AAV - SATB2 + siVps4 p = 0 . 605 , AAV - SATB2 + Scr vs . AAV - SATB2 + siVps4 p = 0 . 0007 ) . Number of nuclei analyzed : 594 ( AAV - EGFP ) , 657 ( AAV - SATB2 ) , 612 ( AAV - SATB2 + Scr ) , 428 ( AAV - SATB2 + siVps4 ) . Data are presented as mean ± , * * p < 0 . 01 compared with AAV - EGFP ; # # # p < 0 . 001 compared with AAV - SATB2 + Scr .

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

Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression and Vps4a/ Vps4b knockdown. AAV-SATB2-transduced neurons (non-transfected or scrambled siRNA (Scr)-transfected) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, VPS4-depleted AAV-SATB2-transduced cultures showed similar number of infolded nuclei to AAV-EGFP-transduced neurons (n = 4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,11 =15.866, p = 0.00026, AAV-EGFP vs. AAV-SATB2 p = 0.0104, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0028, AAV-EGFP vs. AAV-SATB2 + siVps4 p = 0.605, AAV-SATB2 + Scr vs. AAV-SATB2 + siVps4 p = 0.0007). Number of nuclei analyzed: 594 (AAV-EGFP), 657 (AAV-SATB2), 612 (AAV-SATB2 + Scr), 428 (AAV-SATB2 + siVps4). Data are presented as mean ± SEM, **p < 0.01 compared with AAV-EGFP; ###p < 0.001 compared with AAV-SATB2 + Scr.


Activity - induced formation of infoldings is impaired in VPS4 - depleted cortical neurons . Upon AP bursting , of infolded nuclei was increased in non - transfected cultures in cultures transfected with scramble siRNA ( Scr ) but not with siVps4 ( n = 3 - 4 independent cultures , one - way ANOVA followed by Tuckey post hoc , F3 , 8 = 90 . 525 , p = 1 . 63E - 06 , Bic - treated vs NBQX - treated p = 0 . 000012 , Scr Bic - treated vs NBQX - treated p = 0 . 000052 , siVps4 Bic - treated vs NBQX - treated p = 0 . 396 , siVps4 Bic - treated vs Scr Bic - treated p = 0 . 000015 ) . Number of analyzed nuclei : 474 ( NBQX ) , 456 ( Bic ) , 409 ( Bic + Scr ) , 383 ( Bic + siVps4 ) . Data are presented as mean ± , * * * p < 0 . 001 compared to NBQX - treated , # # # p < 0 . 001 compared to Scr Bic - treated .

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

Activity-induced formation of nuclear infoldings is impaired in VPS4-depleted cortical neurons. Upon AP bursting, the percentage of infolded nuclei was increased in non-transfected cultures and in cultures transfected with scramble siRNA (Scr) but not with siVps4 (n = 3-4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,8 = 90.525, p = 1.63E-06, Bic-treated vs NBQX-treated p = 0.000012, Scr Bic-treated vs NBQX-treated p = 0.000052, siVps4 Bic-treated vs NBQX-treated p = 0.396, siVps4 Bic-treated vs Scr Bic-treated p = 0.000015). Number of analyzed nuclei: 474 (NBQX), 456 (Bic), 409 (Bic + Scr), 383 (Bic + siVps4). Data are presented as mean ± SEM, ***p < 0.001 compared to NBQX-treated, ###p < 0.001 compared to Scr Bic-treated.


Representative confocal ( Z - axis projection ) of CA1 pyramidal neurons of : ( top panel ) Satb2 floxed mice ( n = 3 ) maintained in their home cages ( HC ) , ( middle panel ) Satb2 floxed mice ( n = 4 ) subjected to NEE for 1 . 5 h , ( bottom panel ) Satb2CamkCre mice ( n = 5 ) exposed to NE for 1 . 5 h . Brain sections were stained for cFos Lamin B2 . Scale bars : 10 μm . Higher magnification views of boxed areas in A ( middle panel ) . Arrows show examples of infolded , cFos - positive nuclei of CA1 field of Satb2 floxed mice subjected to NEE . Scale bars : 5 μm .

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

Representative confocal images (Z-axis projection) of CA1 pyramidal neurons of: (top panel) Satb2 floxed mice (n = 3) maintained in their home cages (HC), (middle panel) Satb2 floxed mice (n = 4) subjected to NEE for 1.5 h, (bottom panel) Satb2CamkCre mice (n = 5) exposed to NE for 1.5 h. Brain sections were stained for cFos and Lamin B2. Scale bars: 10 μm. Higher magnification views of boxed areas in A (middle panel). Arrows show examples of infolded, cFos-positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE. Scale bars: 5 μm.


( B ) Quantitative PCR of post - natal lungs for mitochondrial ( mtDNA ) . Actin was used as a normalization control . ( mean ± SD , : p < 0 . 05 ; : p < 0 . 01 ; N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative PCR analysis of post-natal lungs for mitochondrial DNA (mtDNA). Actin was used as a normalization control. (mean ± SD, : p<0.05;: p<0.01; N=3).


( C ) Quantitative of oxidative stress in post - natal pups of indicated ( in days ) using peroxide linked oxidative stress marker DHR - 123 , as described in methods ( mean ± SD , : p < 0 . 01 ; N = 8 ) .

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

(C) Quantitative analysis of oxidative stress in post-natal pups of indicated age (in days) using the peroxide linked oxidative stress marker DHR-123, as described in methods (mean ± SD, : p<0.01; N=8).


( E ) Immunofluorescence of oxidative damage ( left panel ) double - strand breaks sensor kinase marker pATM ( right panel ) in post - natal murine lungs . Here anti - 8 - oxo - 7 , 8 - dihydroguanine was used for detection of oxidative modification ( 8 - oxoG , left panel ) , anti - pATM Serine 1981 was used for of activation of DDR ( pATM , right panel ) . Green represents α - Tubulin ; blue DAPI represents nucleus ( scale 10μm ) . No antibody control served as a negative control ( P14 ) of .

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

(E) Immunofluorescence analysis of oxidative DNA damage (left panel) and the DNA double-strand breaks sensor kinase marker pATM (right panel) in post-natal murine lungs. Here anti-8-oxo-7, 8-dihydroguanine was used for the detection of oxidative DNA modification (8-oxoG, left panel), and anti-pATM Serine 1981 was used for the analysis of the activation of the DDR (pATM, right panel). Green cytoplasmic staining represents α-Tubulin; blue DAPI staining represents nucleus (scale 10μm). No primary antibody control served as a negative control (P14) of the staining.


( F ) Mean of nuclei positive for oxidative damage marker 8 - oxoG in post - natal murine lungs , as determined by immunofluorescence ( mean ± , : p < 0 . 01 ; : p < 0 . 001 ; N = 8 ) . ( G ) Mean of nuclei positive for damage marker pATM in post - natal murine lungs , as determined by immunofluorescence ( mean ± , : p < 0 . 01 ; N = 8 ) .

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

(F) Mean percentage of nuclei positive for oxidative DNA damage marker 8-oxoG in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; : p<0.001; N=8). (G) Mean percentage of nuclei positive for DNA damage marker pATM in post-natal murine lungs, as determined by immunofluorescence analysis (mean ± SEM, : p<0.01; N=8).


( H ) Quantitative of NAD + / NADH in post - natal lungs after homogenization in extraction buffer ( mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ) .

O O O O O B-MEASURED_VAR I-MEASURED_VAR 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

(H) Quantitative analysis of the NAD+/NADH ratio in post-natal lungs after homogenization in extraction buffer (mean ± SD, : p<0.01; : p<0.001).


( A ) Immunofluorescence of DSBs associated foci as marked by γH2AX in lung adenocarcinoma ( A549 ) , cultured either in low glucose ( 5 . 5mM , left panel ) or high glucose ( 30mM , right panel ) for 5 days , then treated with etoposide ( 5µM for 60 minutes ) . resolution of - DSBs foci , as marked by γH2AX was monitored over 24 hours after drug treatment , CML ( marked in green ) served as induction control ( scale 10µm ) . ( B ) Mean of DSBs positive nuclei as evidenced by γH2AX positivity after pre - treatment of different reducing carbohydrates at 0 , 3 or 24 hours . More than 400 were analyzed for each bar ( mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ) .

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 O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence analysis of DSBs associated foci as marked by γH2AX in lung adenocarcinoma (A549) cells, cultured either in low glucose (5.5mM, left panel) or high glucose (30mM, right panel) for 5 days, then treated with etoposide (5µM for 60 minutes). The resolution of DNA-DSBs foci, as marked by γH2AX was monitored over 24 hours after drug treatment, CML (marked in green) served as induction control (scale 10µm). (B) Mean percentage of DSBs positive nuclei as evidenced by γH2AX positivity after pre-treatment of different reducing carbohydrates at 0, 3 or 24 hours. More than 400 cells were analyzed for each bar (mean ± SD, : p<0.01;: p<0.001).


( C ) Still of A549 showing live recruitment of hPARP - mCherry hXRCC4 - GFP , at site of - induced - DSBs from pre - treated with indicated reducing sugars as described in methods .

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

(C) Still images of A549 cells showing the live recruitment of hPARP-mCherry and hXRCC4-GFP, at the site of laser-induced DNA-DSBs from the cells pre-treated with the indicated reducing sugars as described in methods.


( D ) Quantitative of efficiency of NHEJ - repair in a reporter , as described in Methods . were cultured in low glucose ( 5 . 5mM ) or high glucose ( 30mM ) for 5 days , fructose ( 30mM ) , ribose ( 20mM ) , for 3 days ( graph bar 4 ) . I - SceI , untransfected served as a negative control . Shown is average from three independent experiments ( mean ± , : p < 0 . 01 : p < 0 . 001 ) .

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 O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O 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 O O O O O 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) Quantitative analysis of the efficiency of NHEJ-repair in a reporter cell line, as described in Methods. Cells were cultured in low glucose (5.5mM) or high glucose (30mM) for 5 days, fructose (30mM), ribose (20mM), for 3 days (graph bar 4). I-SceI, untransfected cells served as a negative control. Shown is the average from three independent experiments (mean ± SEM, : p<0.01 : p<0.001).


( E ) Quantitative of NAD + / NADH in A549 , cultured in either low ( 5 . 5mM , LG ) , or high glucose ( 30mM , HG ) for 5 days , fructose ( 30mM , FR ) , ribose ( 20mM , RI ) , for 3 days ( graph bar 4 ) . was performed after homogenization in extraction buffer ( Data represent mean ± SD , : p < 0 . 01 ; : p < 0 . 001 ; N = 3 ) .

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(E) Quantitative analysis of the NAD+/NADH ratio in A549 cells, cultured in either low (5.5mM, LG), or high glucose (30mM, HG) for 5 days, fructose (30mM, FR), ribose (20mM, RI), for 3 days (graph bar 4). Quantification was performed after homogenization in extraction buffer (Data represent mean ± SD, : p<0.01;: p<0.001; N=3).


( F ) A549 were cultured in either low ( 5 . 5mM ; lane 1 ) or high glucose ( 30mM ; lane 2 ) for 5 days , fructose ( 30mM ; lane 3 ) , ribose ( 20mM ; lane 4 ) , for 3 days . extracts were then for immunoprecipitation by using anti - PARP , or a non - specific species control antibody . PARP , or its interacting partner DBC1 was then detected using PARP , or a DBC1 specific antibody .

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(F) A549 cells were cultured in either low (5.5mM; lane 1) or high glucose (30mM; lane 2) for 5 days, fructose (30mM; lane 3), ribose (20mM; lane 4), for 3 days. The cell extracts were then for immunoprecipitation by using anti-PARP, or a non-specific species control antibody. The PARP, or its interacting partner DBC1 was then detected using PARP, or a DBC1 specific antibody.


( A ) Representative of lungs for nuclei positive for - DSBs marker γH2AX , versus DAPI , in - matched 3 - , or 6 - months control , versus STZ induced diabetic mice , as determined by immunofluorescence ( scale 10μm ) . ( B ) Mean of nuclei positive for - DSBs marker γH2AX in lungs of - matched control versus 6 - month STZ induced diabetic mice , as determined by immunofluorescence of lungs ( mean ± , : p < 0 . 05 ; : p < 0 . 01 ; : p < 0 . 001 ; N = 6 ) .

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(A) Representative images of lungs for nuclei positive for the DNA-DSBs marker γH2AX, versus DAPI, in age-matched 3-, or 6- months control, versus STZ induced diabetic mice, as determined by immunofluorescence analysis (scale 10μm). (B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in lungs of age-matched control versus 6-month STZ induced diabetic mice, as determined by immunofluorescence analysis of lungs (mean ± SEM, :p<0.05;: p<0.01; : p<0.001; N=6).


( C ) Representative of lungs from - matched control versus 3 or 6 - month STZ mice , stained for peroxide linked oxidative stress marker DHR - 123 ( in red ) , as determined by mean . Blue represents DAPI ( Scale 10µm ) . ( D ) Quantitative of respectively - matched control versus 3 or 6 - month STZ induced diabetic lungs for persistent damage signaling associated inflammatory marker IL - 6 , as determined by mean of respective group lungs ( mean ± SD ; : p < 0 . 01 : p < 0 . 001 ; N = 6 ) .

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(C) Representative images of lungs from age-matched control versus 3 or 6-month STZ mice, stained for the peroxide linked oxidative stress marker DHR-123(in red), as determined by mean fluorescence intensity. Blue nuclear staining represents DAPI (Scale 10µm). (D) Quantitative analysis of respectively age-matched control versus 3 or 6-month STZ induced diabetic lungs for persistent DNA damage signaling associated inflammatory marker IL-6, as determined by mean fluorescence intensity of respective group lungs (mean ± SD; : p<0.01: p<0.001; N=6).


( E ) Representative of lungs from - matched controls , versus 3 or 6 - month STZ induced diabetic mice . Sections stained for senescence - associated β - galactosidase [ β - Gal ] as described in Methods visualized by bright field polarized , senescent areas are recognized by its bluish - green ( Scale 40µm ) .

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(E) Representative images of lungs from age-matched controls, versus 3 or 6-month STZ induced diabetic mice. Sections stained for cellular senescence-associated β-galactosidase [β-Gal] as described in Methods and visualized by bright field and polarized light, the senescent areas are recognized by its bluish-green staining (Scale 40µm).


( A ) Pressure - volume curves were determined using FlexiVent system in - matched control versus 3 - month STZ induced diabetic mice . curves represent group averages ( mean ± SD ; : p < 0 . 05 ; N = 5 ) .

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(A) Pressure-volume curves were determined using the FlexiVent system in age-matched control versus 3-month STZ induced diabetic mice. The curves represent group averages (mean ± SD; : p<0.05; N=5).


( B ) Quantitative of static pulmonary compliance in - matched control versus 3 months STZ induced diabetic mice , as described in Figure 3a . curves represent group averages ( mean ± SD ; : P < 0 . 05 , : p < 0 . 01 ; N = 5 ) .

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(B) Quantitative analysis of static pulmonary compliance in age-matched control versus 3 months STZ induced diabetic mice, as described in Figure 3a. The curves represent group averages (mean ± SD; : P<0.05, : p<0.01; N=5).


( C ) Representative of lungs from ( 3 - or 6 - month ) matched control versus STZ induced diabetic mice , stained for Masson ' s Trichrome stain , as described in Methods visualized by bright field polarized , accumulated ECM is recognized by its blue ( Scale 40µm ) .

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(C) Representative images of lungs from age (3- or 6- month) matched control versus STZ induced diabetic mice, stained for Masson's Trichrome stain, as described in Methods and visualized by bright field and polarized light, the accumulated ECM is recognized by its blue staining (Scale 40µm).


( D ) total lung capacity of controls versus with diabetes . dotted represents cut - off decided as per guidelines of European Respiratory Society ( ERS ) American Thoracic Society ( ATS ) , ( mean ± SD , : p < 0 . 05 ; triplicates from each Ncontrol = 44 , Ntype - 1 = 35 , Ntype - 2 = 110 ) . ( E ) forced vital capacity of lungs in controls versus with diabetes . dotted represents cut - off decided as per guidelines of ERS ATS , ( mean ± SD ; : p < 0 . 05 ; triplicates from each Ncontrol = 44 , Ntype - 1 = 35 , Ntype - 2 = 110 ) .

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(D) Percentage total lung capacity of controls versus patients with diabetes. The dotted line represents the cut-off decided as per the guidelines of the European Respiratory Society (ERS) and American Thoracic Society (ATS), (mean ± SD, : p<0.05; triplicates from each Ncontrol=44, Ntype-1=35, Ntype-2=110). (E) Percentage forced vital capacity of lungs in controls versus patients with diabetes. The dotted line represents the cut-off decided as per the guidelines of ERS and ATS, (mean ± SD; : p<0.05; triplicates from each Ncontrol=44, Ntype-1=35, Ntype-2=110).


( F ) Quantitative of NAD + / NADH in lungs harvested from - matched 3 , or 6 months control , or STZ induced diabetic mice , after homogenization in extraction buffer ( mean ± SD ; : p < 0 . 01 , : p < 0 . 001 ; N = 5 ) .

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(F) Quantitative analysis of the NAD+/NADH ratio in lungs harvested from age-matched 3, or 6 months control, or STZ induced diabetic mice, after homogenization in extraction buffer (mean ± SD; : p<0.01, : p<0.001; N=5).


( G ) Quantitative of NAD + / NADH determined in serum from with diabetes , as described in methods ( N = 3 ; mean ± SD , : p < 0 . 01 , : p < 0 . 001 ) .

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(G) Quantitative analysis of the NAD+/NADH ratio determined in serum from patients with diabetes, as described in methods (N=3; mean ± SD, : p<0.01, : p<0.001).


( A ) Representative of γH2AX positive nuclei in lungs of 6 - month STZ induced diabetic mice , transduced with respective RAGE virions as described in Methods . lungs were harvested 6 - after viral transduction . empty vector served as control . Red represents γH2AX foci green represents α - tubulin ; blue represents DAPI ( Scale 10µm ) . No antibody control served as a negative control ( from control lung ) of .

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(A) Representative images of γH2AX positive nuclei in lungs of 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. The lungs were harvested 6-weeks after viral transduction. The empty vector served as control. Red represents γH2AX foci green cytoplasmic staining represents α-tubulin; blue nuclear staining represents DAPI (Scale 10µm). No primary antibody control served as a negative control (from control lung) of the staining.


( B ) Mean of nuclei positive for - DSBs marker γH2AX in transduced lungs of 6 - month STZ diabetic mice as described in Figure 5a ( mean ± ; : p < 0 . 01 ; N = 8 ) .

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(B) Mean percentage of nuclei positive for the DNA-DSBs marker γH2AX in transduced lungs of 6-month STZ diabetic mice as described in Figure 5a (mean ± SEM; : p<0.01; N=8).


( C ) Representative of lungs from 6 - month STZ induced diabetic mice , transduced with respective RAGE virions as described in Methods . Lungs were stained for senescence - associated β - galactosidase as described in Figure 5a visualized by bright field polarized , where accumulated senescent areas are recognized by its blue ( Scale 40µm ) .

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(C) Representative images of lungs from 6-month STZ induced diabetic mice, transduced with respective RAGE virions as described in Methods. Lungs were stained for cellular senescence-associated β-galactosidase as described in Figure 5a and visualized by bright field and polarized light, where the accumulated senescent areas are recognized by its blue staining (Scale 40µm).


( D ) Quantitative of persistent damage associated inflammatory marker IL - 6 , in transduced lungs from 6 - month STZ - diabetic mice , as determined by mean of respective group lungs ( mean ± SD , : p < 0 . 05 , : p < 0 . 001 ; N = 8 ) .

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(D) Quantitative analysis of persistent DNA damage associated inflammatory marker IL-6, in transduced lungs from 6-month STZ-diabetic mice, as determined by mean fluorescence intensity of respective group lungs (mean ± SD,: p<0.05, : p<0.001; N=8).


( E ) Quantitative of Masson ' s Trichrome stain for lungs of 6 - month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F ( mean ± SD , : p < 0 . 001 ; N = 8 ) .

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(E) Quantitative analysis of Masson's Trichrome stain for lungs of 6-month STZ induced diabetic mice with respective RAGE virions as described in Figure 5F (mean ± SD, : p<0.001; N=8).


( F ) Representative of Masson ' s Trichrome stain for lungs transduced with respective RAGE expressing virions as described in Figure 5a visualized by bright field polarized , where accumulated ECM areas are recognized by its blue ( Scale 40µm ) . dotted lines represent respective zoomed window .

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(F) Representative images of Masson's Trichrome stain for lungs transduced with the respective RAGE expressing virions as described in Figure 5a and visualized by bright field and polarized light, where the accumulated ECM areas are recognized by its blue staining (Scale 40µm). The dotted lines represent the respective zoomed window.


( G ) Pressure - volume curves of diabetic non - diabetic mice were determined using FlexiVent system 6 after transduction using respective RAGE virions , as described in Figure 5a . curves represent group averages ( N = 8 ) . lungs studied in Figure 5a , b , c d are identical ( mean ± SD ; Vector alone vs . RAGE - EE ; : p < 0 . 05 ; N = 8 ) .

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(G) Pressure-volume curves of diabetic and non-diabetic mice were determined using the FlexiVent system 6 weeks after transduction using respective the RAGE virions, as described in Figure 5a. The curves represent group averages (N=8). The lungs studied in Figure 5a, b, c and d are identical (mean ± SD; Vector alone vs. RAGE-EE; : p<0.05; N=8).


A ) M . florum doubling time at different incubation temperatures measured by colorimetric assays . bars represent mean standard deviation values obtained from three technical replicates . asterisk indicates absence of significant growth , preventing calculation of a doubling time .

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A) M. florum doubling time at different incubation temperatures measured by colorimetric assays. The bars represent the mean and standard deviation values obtained from three technical replicates. The asterisk indicates the absence of significant growth, preventing the calculation of a doubling time.


D ) Representative of SYTO 9 propidium iodide ( PI ) double stained M . florum , harvested from an exponential or - phase culture , observed by widefield microscopy . brightness of each channel was adjusted equally between conditions . Scale bar : 5 µm .

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D) Representative images of SYTO 9 and propidium iodide (PI) double stained M. florum cells, harvested from an exponential or death-phase culture, observed by widefield fluorescence microscopy. The brightness of each channel was adjusted equally between conditions. Scale bar: 5 µm.


A ) Representative image of M . florum observed by transmission electronic microscopy ( ) . Scale : 100 nm .

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A) Representative image of M. florum cells observed by transmission electronic microscopy (TEM). Scale: 100 nm.


D ) Boxplots showing median interquartile range of average diameter calculated from 389 169 analyzed by STED , respectively . Whiskers indicate 10 - 90 percentile range .

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D) Boxplots showing the median and interquartile range of the average cell diameter calculated from 389 and 169 individual cells analyzed by TEM and STED, respectively. Whiskers indicate the 10-90 percentile range.


A ) Circular representation of M . florum L1 chromosome enhanced with 5 ' - RACE data generated in this study . Outer to inner circle : genomic coordinates ( kbp ) ; genes encoded on positive ( blue for coding sequences , turquoise for RNAs ) negative ( red for coding sequences , fuchsia for RNAs ) strands ; raw 5 ' - RACE signal ( 0 - 1 , 000 read starts scale ) observed at each genomic position for positive ( black ) negative ( gray ) strands ; putative transcription start sites ( TSSs ) identified on positive ( green ) negative ( orange ) strands from significant 5 ' - RACE peaks .

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A) Circular representation of the M. florum L1 chromosome enhanced with 5'-RACE data generated in this study. Outer to inner circle: genomic coordinates (kbp); genes encoded on the positive (blue for coding sequences, turquoise for RNAs) and negative (red for coding sequences, fuchsia for RNAs) DNA strands; raw 5'-RACE signal (0-1,000 read starts scale) observed at each genomic position for the positive (black) and negative (gray) DNA strands; putative transcription start sites (TSSs) identified on the positive (green) and negative (orange) DNA strands from significant 5'-RACE peaks.


A ) Transcription of M . florum coding sequences ( CDS ) quantified by - seq . Transcription were calculated according to number of fragments per kilobase per million of mapped reads ( FPKM ) observed over six replicates . corresponding numbers of mRNA copies per , estimated from measured M . florum mass , are also indicated . CDS were sorted from least to most transcribed . transcription level of selected genes of importance are presented . LDH , L - lactate dehydrogenase ( peg . 600 / mfl596 ) ; G3PDH , glyceraldehyde - 3 - phosphate dehydrogenase ( peg . 583 / mfl578 ) ; PGK , phosphoglycerate kinase ( peg . 582 / mfl577 ) ; L24p L7 / L12 , large subunit ribosomal L24p ( peg . 133 / mfl134 ) L7 / L12 ( peg . 605 / mfl601 ) ; S7p , small subunit ribosomal S7p ( peg . 626 / mfl623 ) ; RNAPβ , RNAPβ ' , RNAPα , polymerase subunits β , β ' , α ( peg . 601 / mfl597 , peg . 602 / mfl598 , peg . 149 / mfl150 ) ; HPr , phosphotransferase system phosphocarrier HPr ( peg . 570 / mfl565 ) ; σ70 , polymerase sigma factor RpoD ( peg . 269 / mfl270 ) .

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A) Transcription levels of all M. florum coding sequences (CDS) quantified by RNA-seq. Transcription levels were calculated according to the number of fragments per kilobase per million of mapped reads (FPKM) observed over six replicates. The corresponding numbers of mRNA copies per cell, estimated from the measured M. florum RNA mass, are also indicated. CDS were sorted from least to most transcribed. The transcription level of selected genes of importance are presented. LDH, L-lactate dehydrogenase (peg.600/mfl596); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (peg.583/mfl578); PGK, phosphoglycerate kinase (peg.582/mfl577); L24p and L7/L12, large subunit ribosomal proteins L24p (peg.133/mfl134) and L7/L12 (peg.605/mfl601); S7p, small subunit ribosomal protein S7p (peg.626/mfl623); RNAPβ, RNAPβ', and RNAPα, RNA polymerase subunits β, β', and α (peg.601/mfl597, peg.602/mfl598, and peg.149/mfl150); HPr, phosphotransferase system phosphocarrier protein HPr (peg.570/mfl565); σ70, RNA polymerase sigma factor RpoD (peg.269/mfl270).


C ) Expression of M . florum reference quantified by two - dimensional liquid chromatography - tandem mass spectrometry ( 2D LC - MS / MS ) . Abundance was estimated according to normalized spectral abundance factor ( NSAF ) calculated for each . A NSAF value of 0 was assigned to undetected . corresponding number of molecules per ( derived from biomass data ) are indicated . were sorted from least to most abundant . selected genes of importance presented in panel A are also highlighted .

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C) Expression levels of all M. florum reference proteins quantified by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS). Abundance was estimated according to the normalized spectral abundance factor (NSAF) calculated for each protein. A NSAF value of 0 was assigned to undetected proteins. The corresponding number of protein molecules per cell (derived from the biomass data) are indicated. Proteins were sorted from least to most abundant. The selected genes of importance presented in panel A are also highlighted.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) TAK1 inhibitor NG25 ( 2 μM ) , IKKβ inhibitor BI605906 ( 5 μM ) or IKKβ inhibitor PS1145 ( 10 μM ) . were then co - stimulated for 30 min with 100 ng / ml LPS 4 mM ATP . lysates ( 10 μg ) were denatured in SDS , subjected to SDS - PAGE immunoblotted with antibodies indicated . ( B ) As in A , except that were co - stimulated for 1 h with 100 ng / ml LPS 5 μM nigericin . Similar results were obtained in three independent experiments in A B .

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(A) WT BMDM were incubated for 1 h without (-) or with (+) the TAK1 inhibitor NG25 (2 μM), the IKKβ inhibitor BI605906 (5 μM) or the IKKβ inhibitor PS1145 (10 μM). The cells were then co-stimulated for 30 min with 100 ng/ml LPS and 4 mM ATP. Cell lysates (10 μg protein) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. (B) As in A, except that the cells were co-stimulated for 1 h with 100 ng/ml LPS and 5 μM nigericin. Similar results were obtained in three independent experiments in A and B.


( C , D ) As in A , B , except that human monocyte - derived macrophages were used instead of mouse BMDM . Similar results were obtained in two independent experiments .

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(C, D) As in A, B, except that primary human monocyte-derived macrophages were used instead of mouse BMDM. Similar results were obtained in two independent experiments.


( A , B ) Mouse macrophage J774A . 1 was transfected with control siRNA or siRNA against IKKβ ( A ) or IKKα ( B ) . 72 h post - transfection , were co - stimulated for 60 min without ( - ) or with ( + ) 100 ng / ml LPS / or 5 μM nigericin . lysates ( 10 μg ) were denatured in SDS , subjected to SDS - PAGE immunoblotted with antibodies indicated . Similar results were obtained in three ( A ) or two ( B ) independent experiments .

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(A, B) The Mouse macrophage J774A.1 cell line was transfected with control siRNA or siRNA against IKKβ (A) or IKKα (B). 72 h post-transfection, the cells were co-stimulated for 60 min without (-) or with (+) 100 ng/ml LPS and/or 5 μM nigericin. Cell lysates (10 μg) were denatured in SDS, subjected to SDS-PAGE and immunoblotted with the antibodies indicated. Similar results were obtained in three (A) or two (B) independent experiments.


( C , D ) As in ( A , B ) except that BMDM from IKKβ - LysM - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT control mice were co - stimulated without ( - ) or with ( + ) 100 ng / ml LPS / or 4 mM ATP ( C ) or with 100 ng / ml LPS / or 5 μM nigericin ( D ) . Similar results were obtained in two independent experiments .

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(C, D) As in (A, B) except that BMDM from IKKβ-LysM-Cre (flox/flox) (IKKβ (fl/fl)) or WT control mice were co-stimulated without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (C) or with 100 ng/ml LPS and/or 5 μM nigericin (D). Similar results were obtained in two independent experiments.