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 B-CONTROLLED_VAR 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 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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

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

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

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 .

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

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 of NAD + / NADH 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 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 ) .

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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 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 .

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(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 ) .

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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 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 .

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

(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 ( + ) , IKKβ inhibitors BI605906 ( 5 μM ) , TPCA - 1 ( 5 μM ) or PS1145 ( 10 μM ) , TAK1 inhibitor NG25 ( 2 μM ) or NLRP3 inhibitor MCC950 . were then co - stimulated for 30 min without ( - ) or with ( + ) 100 ng / ml LPS / or 4 mM ATP ( A ) / or 5 μM nigericin ( B ) . culture medium was removed lysed . in culture medium was precipitated ( see Methods ) , dissolved in SDS . subjected to SDS - PAGE , along with lysates . After transfer to PVDF membranes , immunoblotting was performed with antibodies recognizing both full ( FL ) ( CL ) gasdermin D ( GSDMD ) , p20 p10 fragments of caspase - 1 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 ) control WT 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O B-MEASURED_VAR O O O O O O B-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 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

(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 / or 5 μM nigericin or left unstimulated . were lysed in buffer containing 1 % ( v / v ) Triton - X - 100 , Triton - X - 100 - soluble Triton - X - 100 - insoluble fractions were prepared as in Methods . Triton - X - 100 - insoluble fraction was dissolved SDS subjected to SDS - PAGE immunoblotted with antibodies indicated . Histone H3B ( H3B ) was used as a loading control in Tritin X - 100 insoluble fraction . Similar results were obtained in three independent experiments . ( B ) As in A , except that 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 . U2 Small Auxiliary Factor 1 ( U2AF1 ) was used as a loading control . Similar results were obtained in two independent experiments .

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 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

(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 .

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 O O O O

(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 IKKβ inhibitors TPCA - 1 ( 5 μM ) ( C , G ) or BI605906 ( 5 μM ) ( D , H ) . 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 5 μM nigericin ( B , C , D ) or left unstimulated ( control ) ( A ) . were fixed processed for PLA using a rabbit polyclonal antibody against TGN38 a mouse monoclonal antibody against NLRP3 . A PLA signal is only if NLRP3 antibody TGN38 antibody come into close proximity . ( A - H ) panel show PLA signal ( red ) DAPI for nuclei ( blue ) . were acquired by sequential scanning on a confocal microscope . Similar results were obtained in three independent experiments representative are shown . Scale bar = 50 μm .

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 O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O B-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 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 iBMDM from IKKβ - CXCR3 - Cre ( flox / flox ) ( IKKβ ( fl / fl ) ) or WT mice were co - stimulated for 1 h with 100 ng / ml LPS 10 μM nigericin or left unstimulated ( control ) . Scale bar = 50 μm .

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 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

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

(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 10 μM nigericin or left unstimulated . Similar results were obtained in two independent experiments . Data information : In panels , scale bar = 50 μm .

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

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

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

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

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 B-CONTROLLED_VAR 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

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

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

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

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

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

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

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

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 B-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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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 were transfected with Atg7 siRNA or control scrambled siRNA 48 h later were treated with 50 ng / ml FGF19 for 12 h . LC3 actin in extracts determined by IB ( n = 3 culture plates ) .

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

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

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

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 : of TFEB detected by IB in SHP IgG immunoprecipitated samples . Lower panel : of TFEB SHP in input samples detected by IB . Similar results were obtained in a second experiment .

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

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

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

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

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

(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 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 . of indicated determined by IB . LC3 II / I p62 / actin ratios are shown at right ( n = 3 - 4 culture plates ) . B , mean SD are plotted . Statistical significance was determined by ( B ) two - way ANOVA with Tukey post - , P - values are indicated .

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

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

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

(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 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 actin in 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 . fractions were isolated from intestine or liver , indicated 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 ( B ) were re - fed for 6 h after fasting overnight . indicated were detected by IB ( n = 2 mice ) . TBLN indicates tubulin .

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

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 were fasted overnight re - fed for indicated times . ( C ) fraction was isolated of TFEB , SHP , 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 were fasted overnight re - fed for indicated times . ( D ) of LC3 , p62 , actin in 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 were cultured overnight in serum free media treated with vehicle or 50 ng / ml FGF19 for 30 min . indicated 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 were re - fed for 0 , 2 , or 3 h after fasting overnight . indicated in intestinal extracts were detected by IB ( n = 2 mice ) .

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

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

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 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

(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 presence of 35S - methionine mixed with SecA SecYEG proteoliposomes . After incubation with ATP , samples were treated with proteinase K to degrade any non - translocated material . Where indicated , reaction was performed in absence of ATP proteolysis in presence of Triton X - 100 ( TX - 100 ) . Translocation reactions were performed in presence of reducing agent DTT or oxidizing agent NaTT , generating protease - protected full - ( FL ) substrate or a translocation intermediate ( IM ) , respectively . samples were analyzed by SDS PAGE followed by autoradiography . Lane 1 shows 10 % of input sample . gel is representative of three replicates . Molecular 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 presence of different nucleotides ( Nuc . ) for different time periods then treated with proteinase K ( Prot . K ) . One sample received hexokinase glucose ( HKG ) to generate ADP , other samples ADP • BeFx or ATPγS . gel is representative of two replicates . of ( C ) . Points show IM band intensities normalized to 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to different nucleotides as shown in inset box . As in ( C ) , but DTT was added concurrently with HKG or nucleotides . gel is representative of two replicates . of ( E ) as in ( D ) , except intensities were calculated for 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) incubated for indicated times before proteolysis . gel is representative of two replicates . of ( B ) . Points show IM band intensities normalized to 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to WB SecA carrying additional mutations , as shown in inset box .

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

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

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) indicated knockout mice after intranasal infection with 750 plaque forming units ( PFU ) of PR8 .

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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 eosin ( H & amp ; amp ; E ) - stained lung sections from mice infected with PR8 on day 2 . Arrows indicate infiltrates necrotic debris occluding airways . Original magnification , × 40 . ( c ) Disease severity ( ' lung score ' ) extent of ( necrosis score ) on day 2 after infection on of H & amp ; amp ; E - stained lung sections . ( d ) Necrosis score on day 7 , estimated visually from H & amp ; amp ; E - stained lung sections , flow cytometry for annexin V - stained lung 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 of neutrophils occluding airways of PR8 - infected mice . Original magnification , × 20 .

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


( f , g ) Flow cytometry of neutrophil , dendritic ( DC ) macrophage numbers on days 2 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 7 after infection in Ripk2 − / − WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( log - rank ( a ) unpaired two - sided Student ' s t - ( 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 7 after infection of wild - type mice ( WT ) Ripk2 − / − mice with PR8 were analyzed for IL - 6 TNF ( day 2 ; a ) , IL - 18 IFN - γ ( b ) , indicated chemokines ( c ) . * P 0 . 05 , * * P 0 . 01 ( unpaired two - sided Student ' s t - ) . Data were normalized for total ( t . p . ) from lung homogenates . Data are pooled from three experiments ( mean ± s . e . m . ; n = 10 - 12 mice total ) .

O 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) on day 2 after infection with PR8 of wild - type mice ( WT ) or Ripk2 − / − mice injected intraperitoneally with control rabbit serum Ripk2 − / − mice injected intraperitoneally with anti - IL - 18 neutralizing antiserum ( αIL - 18Ab ) 3 h before infection , for IL - 18 , IFN - γ CCL2 amounts in whole lung homogenates ( a ; t . p . , total ) , for neutrophil numbers by immunohistochemistry ( b ) by flow cytometry ( c ) . Original magnification , × 20 .

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(a-c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti-IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20.


(d) Analysis of survival of indicated mice.


( e ) Neutrophil numbers in WT , Ripk2 − / − Ripk2 − / − Il18 − / − mice on day 2 after PR8 infection .

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(e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection.


( f ) Survival of Ripk2 − / − Il18 − / − , Ripk2 − / − WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc ( a , c , e ) log - rank ( d , f ) ) . Data are representative of two independent experiments ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

O O O O O B-CONTROLLED_VAR 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 O O O O O O O O O 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) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6-8 mice per experiment).


( a , b ) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL - 18 production by using IL - 18 immunohistochemistry ( a ) IL - 18 ELISA ( b ) of whole lung homogenates . WT , wild type ; t . p . , total . In a , blue arrows denote IL - 18 + macrophages , red arrows denote IL - 18 + bronchial epithelium . Original magnification , × 200 .

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

(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200.


( c ) Flow cytometry of NK in Ripk2 − / − wild - type mice .

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(c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice.


( d ) of wild - type Ripk2 − / − NK for IFN - γ production on a per basis .

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(d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis.


(e) Accumulation of T and B cells in lungs.


( f ) of CD8 + T for IFN - γ CD44 expression in wild - type Ripk2 − / − mice . * P 0 . 05 , * * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc ( b ) unpaired two - sided Student ' s t - ( d , f ) ) . Data are representative of two experiments ( a , b , f ) or three experiments ( c - e ) ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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

(f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c-e) (mean ± s.e.m.; n = 6-8 mice per experiment).


( a ) Immunoblots showing active caspase - 1 ( casp - 1 ) p20 compared to GAPDH loading controls in wild - type ( WT ) , Ripk2 − / − Nod2 − / − BMDCs after PR8 infection .

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(a) Immunoblots showing active caspase-1 (casp-1) p20 compared to GAPDH loading controls in wild-type (WT), Ripk2−/− and Nod2−/− BMDCs after PR8 infection.


( b ) IL - 18 release in PR8 - infected WT , Ripk2 − / − Nod2 − / − BMDCs .

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(b) IL-18 release in PR8-infected WT, Ripk2−/− and Nod2−/− BMDCs.


( c , d ) Immunoblot showing active casp - 1 p20 ( c ) IL - 18 production ( d ) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10 .

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(c,d) Immunoblot analysis showing active casp-1 p20 (c) and IL-18 production (d) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10.


( e , f ) IL - 18 casp - 1 activation in Ripk2 − / − BMDCs treated with NLRP3 inflammasome - specific inhibitor glyburide ( glyb . ) during PR8 infection .

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(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.


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( g ) Immunoblot of casp - 1 p20 during Listeria infection of WT Aim2 − / − BMDCs . * P 0 . 01 , * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc ) . Data are representative of six experiments ( a , b ) or three experiments ( c - g ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Immunoblot analysis of casp-1 p20 during Listeria infection of WT and Aim2−/− BMDCs. *P 0.01, **P 0.001 (one-way ANOVA with Tukey's post-hoc test). Data are representative of six experiments (a,b) or three experiments (c-g) (mean ± s.d.; n = 2-3 replicates per experiment).


( a ) Immunoblot of wild - type ( WT ) , Ripk2 − / − Nod2 − / − BMDCs for LC3 - II ( marker for autophagy ) after 18 h of PR8 infection .

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(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection.


( b , c ) Numbers of LC3 - II + ( b ) , counted in at least 100 from five random fields from confocal ( c ) of BMDCs after PR8 infection / or chloroquine treatment . Original magnification , × 400 .

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(b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400.


( d , e ) Immunoblots of LC3 - II caspase - 1 ( casp - 1 ; e ) IL - 18 production ( d ) in wild - type Ripk2 − / − BMDCs after treatment with rapamycin ( Rap ) .

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

(d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap).


( f ) Immunoblot for LC3 - II in lung lysates of PR8 - infected wild - type Ripk2 − / − mice ( 1 - 5 indicate mice ) .

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(f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1-5 indicate individual mice).