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 ) .

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

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 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 .

O O O O O O O O O O O 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 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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(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 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 ) .

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(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 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 .

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(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 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 ) .

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

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(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 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 ) .

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(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 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 .

(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 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 ) .

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(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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 ) 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 . 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 .

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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 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 .

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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 cells observed by transmission electronic microscopy ( TEM ) . 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 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 .

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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 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 .

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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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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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 ( + ) 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 .

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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 primary 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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, 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 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 .

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

(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.


( A , B ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) , the IKKβ inhibitors BI605906 ( 5 μM ) , TPCA - 1 ( 5 μM ) or PS1145 ( 10 μM ) , the TAK1 inhibitor NG25 ( 2 μM ) or the NLRP3 inhibitor MCC950 . The cells were then co - stimulated for 30 min without ( - ) or with ( + ) 100 ng / ml LPS and / or 4 mM ATP ( A ) and / or 5 μM nigericin ( B ) . The cell culture medium was removed and the cells lysed . Protein in the culture medium was precipitated ( see Methods ) , dissolved in SDS . and subjected to SDS - PAGE , along with cell lysates . After transfer to PVDF membranes , immunoblotting was performed with antibodies recognizing both full length ( FL ) and cleaved ( CL ) gasdermin D ( GSDMD ) , the p20 and p10 fragments of caspase - 1 and IL - 18 . Similar results were obtained in two independent experiments . ( C ) As in A , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) and control WT cells were used . Similar results were obtained in three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 O O O 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, B) WT BMDM were incubated for 1 h without (-) or with (+), the IKKβ inhibitors BI605906 (5 μM), TPCA-1 (5 μM) or PS1145 (10 μM), the TAK1 inhibitor NG25 (2 μM) or the NLRP3 inhibitor MCC950. The cells were then co-stimulated for 30 min without (-) or with (+) 100 ng/ml LPS and/or 4 mM ATP (A) and/or 5 μM nigericin (B). The cell culture medium was removed and the cells lysed. Protein in the culture medium was precipitated (see Methods), dissolved in SDS. and subjected to SDS-PAGE, along with cell lysates. After transfer to PVDF membranes, immunoblotting was performed with antibodies recognizing both full length (FL) and cleaved (CL) gasdermin D (GSDMD), the p20 and p10 fragments of caspase-1 and IL-18. Similar results were obtained in two independent experiments. (C) As in A, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) and control WT cells were used. Similar results were obtained in three independent experiments.


( A ) WT BMDM were incubated for 1 h without ( - ) or with ( + ) 5 μM BI605906 , then stimulated for 1 h with ( + ) 100 ng / ml LPS and / or 5 μM nigericin or left unstimulated . The cells were lysed in buffer containing 1 % ( v / v ) Triton - X - 100 , and the Triton - X - 100 - soluble and Triton - X - 100 - insoluble fractions were prepared as in Methods . The Triton - X - 100 - insoluble fraction was dissolved SDS subjected to SDS - PAGE and immunoblotted with the antibodies indicated . The nuclear protein Histone H3B ( H3B ) was used as a loading control in the Tritin X - 100 insoluble fraction . Similar results were obtained in three independent experiments . ( B ) As in A , except that the Triton - X - 100 - insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2 . 0 mM DSS .

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

(A) WT BMDM were incubated for 1 h without (-) or with (+) 5 μM BI605906, then stimulated for 1 h with (+) 100 ng/ml LPS and/or 5 μM nigericin or left unstimulated. The cells were lysed in buffer containing 1% (v/v) Triton-X-100, and the Triton-X-100-soluble and Triton-X-100-insoluble fractions were prepared as in Methods. The Triton-X-100-insoluble fraction was dissolved SDS subjected to SDS-PAGE and immunoblotted with the antibodies indicated. The nuclear protein Histone H3B (H3B) was used as a loading control in the Tritin X-100 insoluble fraction. Similar results were obtained in three independent experiments. (B) As in A, except that the Triton-X-100-insoluble fraction was first subjected to crosslinking for 45 min at 37oC with 2.0 mM DSS.


( C , As in A , B except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) mice ( IKKβ ( fl / fl ) ) were used . The U2 Small Nuclear RNA Auxiliary Factor 1 ( U2AF1 ) was used as a loading control . Similar results were obtained in two independent experiments .

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(C, As in A, B except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) mice (IKKβ (fl/fl)) were used. The U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) was used as a loading control. Similar results were obtained in two independent experiments.


( E , F ) As in A , B except that BMDM from caspase - 1 KO mice were used . Similar results were obtained in two independent experiments .

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(E, F) As in A, B except that BMDM from caspase-1 KO mice were used. Similar results were obtained in two independent experiments.


( A - H ) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA - 1 ( 5 μM ) ( C , G ) or BI605906 ( 5 μM ) ( D , H ) . The cells were then stimulated for 1 h with 100 ng / ml LPS ( E ) or 5 μM nigericin ( F , G , H ) , or co - stimulated with 100 ng / ml LPS and 5 μM nigericin ( B , C , D ) or left unstimulated ( control ) ( A ) . The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3 . A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity . ( A - H ) the panel images show the PLA signal ( red ) and DAPI staining for nuclei ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were obtained in three independent experiments and representative images are shown . Scale bar = 50 μm .

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 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 B-CONTROLLED_VAR 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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) WT BMDM were incubated for 1 h without or with the IKKβ inhibitors TPCA-1 (5 μM) (C, G) or BI605906 (5 μM) (D, H). The cells were then stimulated for 1 h with 100 ng/ml LPS (E) or 5 μM nigericin (F, G, H), or co-stimulated with 100 ng/ml LPS and 5 μM nigericin (B, C, D) or left unstimulated (control) (A). The cells were fixed and processed for PLA using a rabbit polyclonal antibody against TGN38 and a mouse monoclonal antibody against NLRP3. A PLA signal is only if the NLRP3 antibody and the TGN38 antibody come into close proximity. (A-H) the panel images show the PLA signal (red) and DAPI staining for nuclei (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were obtained in three independent experiments and representative images are shown. Scale bar = 50 μm.


( I - L ) As in A - H , except that IKKβ inhibitors were omitted and iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated ( control ) . Scale bar = 50 μm .

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(I-L) As in A-H, except that IKKβ inhibitors were omitted and iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl)) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated (control). Scale bar = 50 μm.


( A - H ) The experiments were performed as in Figs 5A - 5H , except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody , which was visualized using a secondary antibody ( red ) . Nuclei were counterstained with DAPI ( blue ) . Images were acquired by sequential laser scanning on a confocal microscope . Similar results were observed in many fields and obtained in three independent experiments . Representative images are shown Data information : In all panels , scale bar = 50 μm .

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(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown Data information: In all panels, scale bar = 50 μm.


( I - L ) As in A - H , except that iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS and 10 μM nigericin or left unstimulated . Similar results were obtained in two independent experiments . Data information : In all panels , scale bar = 50 μm .

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(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments. Data information: In all panels, scale bar = 50 μm.


C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice were fasted overnight and then re - fed for 6 ( A - C ) Levels of the indicated proteins detected by IB in ( A ) liver and intestinal extracts from C57BL / 6 mice and in intestinal extracts from ( B ) C57BL / 6 or SHP - KO mice or ( C ) FGF15 - KO mice or littermates . Band quantitation was done using Image J , and the LC3 II / I ratio and p62 / actin and lamp1 / actin ratios are shown at the right . ( A - C ) The mean and standard deviation ( SD ) are plotted . Statistical significance was determined by ( A ) two - tailed Student ' s t - tests or ( B , C ) two - way ANOVA with the Tukey post - test . P - values are indicated ( n = 3 mice ) .

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C57BL/6, SHP-KO, and FGF15-KO or littermate mice were fasted overnight and then re-fed for 6 (A-C) Levels of the indicated proteins detected by IB in (A) liver and intestinal extracts from C57BL/6 mice and in intestinal extracts from (B) C57BL/6 or SHP-KO mice or (C) FGF15-KO mice or littermates. Band quantitation was done using Image J, and the LC3 II/I ratio and p62/actin and lamp1/actin ratios are shown at the right. (A-C) The mean and standard deviation (SD) are plotted. Statistical significance was determined by (A) two-tailed Student's t-tests or (B, C) two-way ANOVA with the Tukey post-test. P-values are indicated (n=3 mice).


C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice as indicated were fasted for 12 h and then , re - fed for 6 h . levels of the indicated proteins in intestinal extracts from ( B ) C57BL / 6 and SHP - KO mice or ( D ) FGF15 - KO mice and littermates with quantitation of the bands at right . , ( B , D ) n = 3 mice . The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are

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C57BL/6, SHP-KO, and FGF15-KO or littermate mice as indicated were fasted for 12 h and then, re-fed for 6 h. levels of the indicated proteins in intestinal extracts from (B) C57BL/6 and SHP-KO mice or (D) FGF15-KO mice and littermates with quantitation of the bands at right. , (B, D) n=3 mice. The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated


( G ) HT29 cells were transfected with GFP - LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 , control plasmid DNA , and scrambled siRNA . The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 10 μm . G , The mean and SD are plotted . Statistical significance was determined by ( G ) one - way ANOVA with the Tukey post - test . P - values are

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(G) HT29 cells were transfected with GFP-LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19, control plasmid DNA, and scrambled siRNA. The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 10 μm. G, The mean and SD are plotted. Statistical significance was determined by (G) one-way ANOVA with the Tukey post-test. P-values are indicated


( H ) HT29 cells were treated with 50 ng / ml FGF19 with or without 100 nM bafilomycin A1 for 12 h . LC3 and actin levels in cell extracts were determined by IB . Quantification of bands is shown at the right ( n = 3 culture plates ) . H ) The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are

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(H) HT29 cells were treated with 50 ng/ml FGF19 with or without 100 nM bafilomycin A1 for 12 h. LC3 and actin levels in cell extracts were determined by IB. Quantification of bands is shown at the right (n=3 culture plates). H) The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated


( I ) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for the indicated times . ApoB48 and actin levels were determined by IB ( n = 2 culture plates ) .

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(I) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for the indicated times. ApoB48 and actin levels were determined by IB (n=2 culture plates).


( A ) HT29 cells were transfected with GFP - LC3 and with Atg7 siRNA or control scrambled RNA ( siCtl ) and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and control siRNA . The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 20 μm . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with GFP-LC3 and with Atg7 siRNA or control scrambled RNA (siCtl) and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and control siRNA. The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 20 μm. The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


( B ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for 12 h . LC3 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

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(B) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for 12 h. LC3 and actin levels in cell extracts determined by IB (n=3 culture plates).


D ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA ( siCtl ) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 12 h . ( D ) ApoB48 and actin levels in cell extracts determined by IB . Bands were quantified and the LC3 - II / LC3 - I ratio is shown at the right ( n = 3 culture plates ) . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

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D) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA (siCtl) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 12 h. (D) ApoB48 and actin levels in cell extracts determined by IB. Bands were quantified and the LC3-II/LC3-I ratio is shown at the right (n=3 culture plates). The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


Mice were re - fed for 2 h ( B ) after fasting overnight . ( B ) CoIP of TFEB with SHP . Upper panel : Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples . Lower panel : levels of TFEB and SHP in input samples detected by IB . Similar results were obtained in a second experiment .

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Mice were re-fed for 2 h (B) after fasting overnight. (B) CoIP of TFEB with SHP. Upper panel: Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples. Lower panel: levels of TFEB and SHP in input samples detected by IB. Similar results were obtained in a second experiment.


( F , G ) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from ( F ) C57BL / 6 and SHP - KO mice or ( G ) FGF15 - KO mice or littermates re - fed for 6 h after fasting overnight ( n = 3 mice ) .

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(F, G) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from (F) C57BL/6 and SHP-KO mice or (G) FGF15-KO mice or littermates re-fed for 6 h after fasting overnight (n=3 mice).


( A ) HT29 cells were transfected with GFP - LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA . The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 cells ) . Scale bar , 10 μm . ( A , The mean and SD are plotted . Statistical significance was determined by ( A ) one - way ANOVA with the Tukey post - test , P - values are indicated .

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(A) HT29 cells were transfected with GFP-LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA. The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20 cells). Scale bar, 10 μm. (A, The mean and SD are plotted. Statistical significance was determined by (A) one-way ANOVA with the Tukey post-test, P-values are indicated.


( B ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or control scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 ( BAF ) for 12 h . Levels of the indicated proteins determined by IB . LC3 II / I and p62 / actin ratios are shown at the right ( n = 3 - 4 culture plates ) . B , The mean and SD are plotted . Statistical significance was determined by ( B ) two - way ANOVA with the Tukey post - test , P - values are indicated .

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(B) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or control scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 (BAF) for 12 h. Levels of the indicated proteins determined by IB. LC3 II/I and p62/actin ratios are shown at the right (n=3-4 culture plates). B, The mean and SD are plotted. Statistical significance was determined by (B) two-way ANOVA with the Tukey post-test, P-values are indicated.


( C ) HT29 cells were transfected with siRNA for TFEB ( siTFEB ) or control RNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . Protein levels detected by IB of ULK1 and ATGL ( n = 3 culture plates ) .

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(C) HT29 cells were transfected with siRNA for TFEB (siTFEB) or control RNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. Protein levels detected by IB of ULK1 and ATGL (n=3 culture plates).


( E ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . ApoB48 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

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(E) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. ApoB48 and actin levels in cell extracts determined by IB (n=3 culture plates).


( A , C57BL / 6 ( A ) were re - fed for 6 h after fasting overnight . The cytosolic and nuclear fractions were isolated from intestine or liver , and the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

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(A, C57BL/6 (A) were re-fed for 6 h after fasting overnight. The cytosolic and nuclear fractions were isolated from intestine or liver, and the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.


B ) FGF15 - KO mice or littermates ( B ) were re - fed for 6 h after fasting overnight . the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

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B) FGF15-KO mice or littermates (B) were re-fed for 6 h after fasting overnight. the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.


FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( C ) The nuclear fraction was isolated and the nuclear levels of TFEB , SHP , and LAMIN were determined by IB .

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FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (C) The nuclear fraction was isolated and the nuclear levels of TFEB, SHP, and LAMIN were determined by IB.


FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( D ) Levels of LC3 , p62 , and actin in cell extracts were determined by IB .

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FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (D) Levels of LC3, p62, and actin in cell extracts were determined by IB.


( E ) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng / ml FGF19 for 30 min . The indicated proteins were detected by IB ( n = 3 culture plates ) .

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(E) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng/ml FGF19 for 30 min. The indicated proteins were detected by IB (n=3 culture plates).


( F ) FGF15 - KO mice or littermates were re - fed for 0 , 2 , or 3 h after fasting overnight . The indicated proteins in intestinal extracts were detected by IB ( n = 2 mice ) .

(F) FGF15-KO mice or littermates were re-fed for 0, 2, or 3 h after fasting overnight. The indicated proteins in intestinal extracts were detected by IB (n=2 mice).


( G ) HT29 cells were transfected with expression plasmids for flag - tagged TFEB or its mutants as indicated and 24 h later , cells were treated with FGF19 ( 50 ng / ml ) for 12 h . ∆ CT indicates the TFEB mutant with a C - terminal deletion of 15 amino acids . The indicated proteins were detected by IB ( n = 3 culture plates ) .

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(G) HT29 cells were transfected with expression plasmids for flag-tagged TFEB or its mutants as indicated and 24 h later, cells were treated with FGF19 (50 ng/ml) for 12 h. ∆CT indicates the TFEB mutant with a C-terminal deletion of 15 amino acids. The indicated proteins were detected by IB (n=3 culture plates).


Substrate was synthesized in reticulocyte lysate in the presence of 35S - methionine and mixed with SecA and SecYEG proteoliposomes . After incubation with ATP , the samples were treated with proteinase K to degrade any non - translocated material . Where indicated , the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X - 100 ( TX - 100 ) . Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT , generating protease - protected full - length ( FL ) substrate or a translocation intermediate ( IM ) , respectively . All samples were analyzed by SDS PAGE followed by autoradiography . Lane 1 shows 10 % of the input sample . The gel is representative of three replicates . Molecular weight markers are estimated from Coomassie - stained gels run in parallel .

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

Substrate was synthesized in reticulocyte lysate in the presence of 35S-methionine and mixed with SecA and SecYEG proteoliposomes. After incubation with ATP, the samples were treated with proteinase K to degrade any non-translocated material. Where indicated, the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X-100 (TX-100). Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT, generating protease-protected full-length (FL) substrate or a translocation intermediate (IM), respectively. All samples were analyzed by SDS PAGE followed by autoradiography. Lane 1 shows 10% of the input sample. The gel is representative of three replicates. Molecular weight markers are estimated from Coomassie-stained gels run in parallel.


Translocation intermediates ( IM ) were incubated in the presence of different nucleotides ( Nuc . ) for different time periods and then treated with proteinase K ( Prot . K ) . One sample received hexokinase and glucose ( HKG ) to generate ADP , other samples ADP • BeFx or ATPγS . The gel is representative of two replicates . Quantification of ( C ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show the average of replicates . Colors correspond to different nucleotides as shown in the inset box . As in ( C ) , but DTT was added concurrently with HKG or nucleotides . The gel is representative of two replicates . Quantification of ( E ) as in ( D ) , except intensities were calculated for the FL bands .

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

Translocation intermediates (IM) were incubated in the presence of different nucleotides (Nuc.) for different time periods and then treated with proteinase K (Prot. K). One sample received hexokinase and glucose (HKG) to generate ADP, other samples ADP•BeFx or ATPγS. The gel is representative of two replicates.   Quantification of (C). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show the average of replicates. Colors correspond to different nucleotides as shown in the inset box.   As in (C), but DTT was added concurrently with HKG or nucleotides. The gel is representative of two replicates.   Quantification of (E) as in (D), except intensities were calculated for the FL bands.


WB SecA with additional mutations ( WT , Y794G , R342E ) was added to preformed intermediates ( IM ) and incubated for the indicated times before proteolysis . The gel is representative of two replicates . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to WB SecA carrying additional mutations , as shown in the inset box .

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WB SecA with additional mutations (WT, Y794G, R342E) was added to preformed intermediates (IM) and incubated for the indicated times before proteolysis. The gel is representative of two replicates.   Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to WB SecA carrying additional mutations, as shown in the inset box.


Imidazole ( imi ) and either WT or mutant SecA were added to translocation intermediates ( IM ) pre - formed with SecA dN20 : : H6 . The samples were incubated for the indicated times before proteolysis with proteinase K ( Prot . K ) . The gel is representative of two replicates , except for the experiment with the R792G / K797G mutant and the 30 - min time points . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to different SecA mutants as shown in the inset box . As in ( B ) , but ATPγS was added together with imidazole . The gel is representative of two replicates . Quantification of ( E ) as in ( C ) . As in ( D ) , but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA . The gel is representative of two replicates . Quantification of ( G ) as in ( F ) , except that both the intensities of the IM ( darker colors ) and FL ( lighter colors ) bands were measured .

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

Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points. Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box. As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates. Quantification of (E) as in (C). As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.   Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.


( a ) Survival rate of wild - type mice ( WT ) and indicated knockout mice after intranasal infection with 750 plaque forming units ( PFU ) of the PR8 virus .

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(a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus.


( b ) Hematoxylin and eosin ( H & amp ; amp ; E ) - stained lung sections from mice infected with PR8 virus on day 2 . Arrows indicate cellular infiltrates and necrotic debris occluding the airways . Original magnification , × 40 . ( c ) Disease severity ( ' lung score ' ) and extent of cell death ( necrosis score ) on day 2 after infection based on analysis of H & amp ; amp ; E - stained lung sections . ( d ) Necrosis score on day 7 , estimated visually from H & amp ; amp ; E - stained lung sections , and flow cytometry analysis for annexin V - stained lung cell populations .

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(b) Hematoxylin and eosin (H&amp;E)-stained lung sections from mice infected with PR8 virus on day 2. Arrows indicate cellular infiltrates and necrotic debris occluding the airways. Original magnification, ×40. (c) Disease severity ('lung score') and extent of cell death (necrosis score) on day 2 after infection based on analysis of H&amp;E-stained lung sections. (d) Necrosis score on day 7, estimated visually from H&amp;E-stained lung sections, and flow cytometry analysis for annexin V-stained lung cell populations.


( e ) Immunohistochemistry staining of neutrophils occluding the airways of PR8 - infected mice . Original magnification , × 20 .

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(e) Immunohistochemistry staining of neutrophils occluding the airways of PR8-infected mice. Original magnification, ×20.


( f , g ) Flow cytometry analysis of neutrophil , dendritic cell ( DC ) and macrophage cell numbers on days 2 and 7 .

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(f,g) Flow cytometry analysis of neutrophil, dendritic cell (DC) and macrophage cell numbers on days 2 and 7.


( h ) Pulmonary viral titers on days 2 and 7 after infection in Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( log - rank test ( a ) and unpaired two - sided Student ' s t - test ( c , d , f , g ) . Data are pooled from three experiments ( a , f - h ; mean ± s . e . m . in f - h ; n = 10 - 12 mice total ) or are representative of three experiments ( b - e ; mean ± s . e . m . ; n = 3 - 5 mice per experiment ) .

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(h) Pulmonary viral titers on days 2 and 7 after infection in Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (log-rank test (a) and unpaired two-sided Student's t-test (c,d,f,g). Data are pooled from three experiments (a,f-h; mean ± s.e.m. in f-h; n = 10-12 mice total) or are representative of three experiments (b-e; mean ± s.e.m.; n = 3-5 mice per experiment).


( a - c ) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild - type mice ( WT ) and Ripk2 − / − mice with the PR8 virus were analyzed for IL - 6 and TNF ( day 2 ; a ) , IL - 18 and IFN - γ ( b ) , and indicated chemokines ( c ) . * P 0 . 05 , * * P 0 . 01 ( unpaired two - sided Student ' s t - test ) . Data were normalized for total protein ( t . p . ) from lung homogenates . Data are pooled from three experiments ( mean ± s . e . m . ; n = 10 - 12 mice total ) .

(a-c) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild-type mice (WT) and Ripk2 −/− mice with the PR8 virus were analyzed for IL-6 and TNF (day 2; a), IL-18 and IFN-γ (b), and indicated chemokines (c). *P 0.05, **P 0.01 (unpaired two-sided Student's t-test). Data were normalized for total protein (t.p.) from lung homogenates. Data are pooled from three experiments (mean ± s.e.m.; n = 10-12 mice total).