Figure 7Splenic infectious centers measured at 12 dpi ( A , left ) , 14 dpi ( B , left ) , 21 dpi ( C , left ) , 28 dpi ( D , up ) , 35 dpi ( E , up ) , or 42 dpi ( F , up ) and viral genome load measured by real - time PCR at 12 dpi ( A , right ) , 14 dpi ( B , right ) , 21 dpi ( C , right ) , 28 dpi ( D , down ) , 35 dpi ( E , down ) , or 42 dpi ( F , down ) , in the BALB / cmice intranasally infected with the WT or recombinant γHV68 mutants , as indicated ( Mean ± SEM of five mice per group / time point / experiment ) . Data of 14 dpi and 28 dpi is pooled from two and three separate experiments , respectively . Preformed infectious virus was negligible in all spleen samples . No significant difference was detected at 12 dpi ( A ) , 14 dpi ( B ) , and 21 dpi ( C ) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [ for Δα1 , P = 0 . 58 ( day 12 ) ; P = 0 . 75 ( day 14 ) ; P = 0 . 18 ( day 21 ) ; for ΔBH2 , P = 0 . 64 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 81 ( day 21 ) ; unpaired t - tests ] and in viral genome loads [ for Δα1 , P = 0 . 85 ( day 12 ) ; P = 0 . 76 ( day 14 ) ; P = 0 . 96 ( day 21 ) ; for ΔBH2 , P = 0 . 56 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 25 ( day 21 ) ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased infectious center titers ( top ) of vBcl - 2 mutant viruses , as compared to WT γHV68 , were statistically significant [ vBcl - 2Δα1 versus vBcl - 2AAA ( 42 dpi ) , P = 0 . 46 ; vBcl - 2Δα1 versus WT γHV68 ( 42 dpi ) , P = 0 . 08 ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased viral DNA loads ( bottom ) of the vBcl - 2Δα1 , vBcl - 2AAA , and vBcl - 2 null mutants , as compared to WT γHV68 and the vBcl - 2ΔBH2 mutant , were statistically significant as follows ( unpaired t - tests ) : at day 28 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 01 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 01 ; vBcl - 2 - null versus WT γHV68 , P < 0 . 01 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2 - null versus vBcl - 2ΔBH2 , P < 0 . 001 ; at day 35 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 05 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 05 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 01 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 05 ; at day 42 , vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2ΔBH2 versus WT γHV68 , P = 0 . 33 . I . C . , infectious center . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/cmice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.


Figure 1A . Retinal whole mount immunostaining for Isolectin - B4 ( red ) and Desmin ( green ) in WT ( top panel ) and Glut1iECKO ( bottom panel ) mice showing pericyte coverage in the vasculature . Left and right panels show representative images with 20x or 63x magnification , respectively . B . Quantification of the pericytes coverage in the retinal vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 9 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) C . Immunostaining for CD31 ( green ) , CD13 ( white ; pericyte marker ) and αSMA ( cyan ) using paraffin section from WT ( top panel ) and Glut1iECKO ( bottom panel ) mice . D . Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 7 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) E . Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 13 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) J . Representative confocal images of fibrin ( green ) , CD31 ( red ) and DAPI ( blue ) immunostaining in the brain vasculature of WT ( top panel ) and Glut1iECKO ( bottom panel ) mice K . Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 13 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) L . Quantification of extravascular fibrin deposits in WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 9 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) M . Quantification of lactate in the cerebrospinal fluid of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 3 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Retinal whole mount immunostaining for Isolectin-B4 (red) and Desmin (green) in WT (top panel) and Glut1iECKO (bottom panel) mice showing pericyte coverage in the vasculature. Left and right panels show representative images with 20x or 63x magnification, respectively. B. Quantification of the pericytes coverage in the retinal vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)C. Immunostaining for CD31 (green), CD13 (white; pericyte marker) and αSMA (cyan) using paraffin section from WT (top panel) and Glut1iECKO (bottom panel) mice.D. Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=7 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) E. Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)J. Representative confocal images of fibrin (green), CD31 (red) and DAPI (blue) immunostaining in the brain vasculature of WT (top panel) and Glut1iECKO (bottom panel) mice K. Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) L. Quantification of extravascular fibrin deposits in WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) M. Quantification of lactate in the cerebrospinal fluid of WT (blue) and Glut1iECKO (orange) mice. (n=3 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


Figure 2A . Quantification of the mTFP / Venus fluorescence ratio of laconic ( lactate sensor ) showing concentration - dependent lactate uptake in HBVPs . ( n = 8 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) ) B . Representative images showing mTFP ( blue ) and Venus ( yellow ) fluorescence in control and lactate ( 20mM ) treated HBVPs . F . Representative images ( high magnification ; 60x ) showing mTFP ( cyan ) , Venus ( yellow ) and VECAD ( purple ) fluorescence from BMECs and laconic - infected HBVPs on a 2 well chamber . The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact . G . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs with endothelial contact ( EC - PC contact ) or HBVPs alone ( PC only ) . ( n = 16 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) H . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or GLUT1 ( lower panel ) siRNA treated BMECs . VECAD immunostaining ( purple ) was used for endothelial staining . I . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or GLUT1 ( siGLUT1 ) siRNA - treated BMECs . ( n = 12 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Quantification of the mTFP/Venus fluorescence ratio of laconic (lactate sensor) showing concentration-dependent lactate uptake in HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM)) B. Representative images showing mTFP (blue) and Venus (yellow) fluorescence in control and lactate (20mM) treated HBVPs.F. Representative images (high magnification; 60x) showing mTFP (cyan), Venus (yellow) and VECAD (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact. G. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs with endothelial contact (EC-PC contact) or HBVPs alone (PC only). (n=16 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)H. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or GLUT1 (lower panel) siRNA treated BMECs. VECAD immunostaining (purple) was used for endothelial staining. I. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or GLUT1 (siGLUT1) siRNA-treated BMECs. (n=12 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


Figure 6E . Western blot analysis for siRNA efficiency of MCT1 . G . Western blot analysis for siRNA efficiency of MCT12 . I . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or MCT1 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial staining . J . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or MCT1 ( siMCT1 ) siRNA - treated BMECs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) K . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or MCT5 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial staining . L . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or MCT5 ( siMCT5 ) siRNA - treated BMECs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6E. Western blot analysis for siRNA efficiency of MCT1.G. Western blot analysis for siRNA efficiency of MCT12.I. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT1 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. J. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT1 (siMCT1) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)K. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT5 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. L. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT5 (siMCT5) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


Figure 7D . Western blot analysis showing expression level of MCT12 and β - actin in HBVPs and BMECs . E . Western blot analysis for siRNA efficiency of MCT12 . G . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in control ( upper panel ) or MCT12 ( lower panel ) siRNA treated HBVPs in a 2 well chamber assay system with BMECs . VECAD ( purple ) immunostaining was used for endothelial staining . H . Quantification of the mTFP / Venus fluorescence ratio of Laconic in control ( blue ) or MCT12 ( orange ) siRNA treated HBVPs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7D. Western blot analysis showing expression level of MCT12 and β-actin in HBVPs and BMECs. E. Western blot analysis for siRNA efficiency of MCT12.G. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in control (upper panel) or MCT12 (lower panel) siRNA treated HBVPs in a 2 well chamber assay system with BMECs. VECAD (purple) immunostaining was used for endothelial staining. H. Quantification of the mTFP/Venus fluorescence ratio of Laconic in control (blue) or MCT12 (orange) siRNA treated HBVPs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.


Figure 8A . Permeability assay using vibratome section showing the cadaverine 555 dye ( red ) , CD31 ( green ) and CD13 ( purple ) in the brain vasculature of Glut1iECKO mice with ( bottom ) or without ( top ) lactate administration . Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice , but not in lactate supplemented Glut1iECKO mice . B . Quantification of the CD13 - positive pericytes coverage in the brain vasculature of Glut1iECKO mice with ( orange ) or without ( blue ) lactate administration . ( n = 16 from 5 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from 1 - way ANOVA . ) C . Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with ( orange ) or without ( blue ) lactate administration . ( n = 24 from 5 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from 1 - way ANOVA . ) D . Quantification of CSF lactate level in WT and Glut1iECKO mice with / without lactate supplementation . ( n = 3 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from 1 - way ANOVA . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice.B. Quantification of the CD13-positive pericytes coverage in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=16 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) C. Quantification of the number of cadaverine 555 positive brain parenchyma cells in the brain vasculature of Glut1iECKO mice with (orange) or without (blue) lactate administration. (n=24 from 5 independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.) D. Quantification of CSF lactate level in WT and Glut1iECKO mice with/without lactate supplementation. (n=3 from independent experiments. Error bars indicate the standard error of the mean (SEM) from 1-way ANOVA.)


Figure 1 ( A ) Immunoblot analysis of BAG1 , BAG3 , Hsc / Hsp70 and Hsp90 in young and old I90 cells . For detection of BAG proteins , an antibody directed against the conserved BAG domain was used . ( B ) Real ‐ time PCR analysis of human BAG family members and Hsc70 and Hsp90 in young and old I90 cells . Depicted is the expression ratio ( log2 ) of target genes in old cells relative to young cells . * P0 . 05 and * * P0 . 01 versus young , n = 3 . ( C ) Co ‐ IP analysis of Hsc / Hsp70 interaction with BAG1 and BAG3 in young ( Y ) and old ( O ) I90 cells . Upper panel shows relative amounts of proteins in cell lysates used for Co ‐ IP ( Input ) . Middle panel shows levels of BAG1 and BAG3 found in Hsc / Hsp70 immunoprecipitates . Lower panel shows levels of Hsc / Hsp70 co ‐ sedimented upon immunoprecipitation of BAG3 . ( D , E ) 293 cells were treated with the indicated agents for 8 h followed by immunoblot analysis of indicated proteins .

O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Immunoblot analysis of BAG1, BAG3, Hsc/Hsp70 and Hsp90 in young and old I90 cells. For detection of BAG proteins, an antibody directed against the conserved BAG domain was used.(B) Real‐time PCR analysis of human BAG family members and Hsc70 and Hsp90 in young and old I90 cells. Depicted is the expression ratio (log2) of target genes in old cells relative to young cells. *P0.05 and **P0.01 versus young, n=3.(C) Co‐IP analysis of Hsc/Hsp70 interaction with BAG1 and BAG3 in young (Y) and old (O) I90 cells. Upper panel shows relative amounts of proteins in cell lysates used for Co‐IP (Input). Middle panel shows levels of BAG1 and BAG3 found in Hsc/Hsp70 immunoprecipitates. Lower panel shows levels of Hsc/Hsp70 co‐sedimented upon immunoprecipitation of BAG3.(D, E) 293 cells were treated with the indicated agents for 8 h followed by immunoblot analysis of indicated proteins.


Figure 2 ( A ) 293 cells were transfected with bag1 , bag3 or nonsense ( nons ) siRNAs , as indicated . After 48 h , cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs . After additional 24 h , levels of indicated proteins were detected by immunoblot analysis . ( B ) 293 cells were transfected with indicated siRNAs for 48 h followed by real ‐ time PCR analysis of BAG1 and BAG3 mRNA levels . Depicted is the mean relative expression ratio ( log2 ) ± s . e . m . * P0 . 05 and * * * P0 . 001 versus nons , n = 3 . ( C ) BAG1 knockdown was performed in 293 cells for 48 h . Thereafter , cells were transfected with GFP or GFP ‐ based UPS reporter genes as indicated . After additional 24 h , levels of GFP ‐ positive proteins were detected by western ‐ blot analysis using a GFP antibody . ( D , E ) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h ( D ) or transfected with indicated amounts of BAG1S plasmid for 24 h ( E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated. After 48 h, cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs. After additional 24 h, levels of indicated proteins were detected by immunoblot analysis.(B) 293 cells were transfected with indicated siRNAs for 48 h followed by real‐time PCR analysis of BAG1 and BAG3 mRNA levels. Depicted is the mean relative expression ratio (log2) ±s.e.m. *P0.05 and ***P0.001 versus nons, n=3.(C) BAG1 knockdown was performed in 293 cells for 48 h. Thereafter, cells were transfected with GFP or GFP‐based UPS reporter genes as indicated. After additional 24 h, levels of GFP‐positive proteins were detected by western‐blot analysis using a GFP antibody.(D, E) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h (D) or transfected with indicated amounts of BAG1S plasmid for 24 h (E).


Figure 3 ( A ) 293 cells were transfected with nonsense ( nons ) , bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 ( both 10 μg / ml ; Pep . A / E64 ) or DMSO as control , followed by immunoblot analysis of the indicated proteins . ( B ) 293 cells were transfected for 48 h with the indicated siRNAs and BAG3 expression plasmid ( BAG3 ‐ N1 ) or vector control ( N1 ) followed by the same analysis as in ( A ) . ( C ) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in ( A ) and ( B ) . Autophagic flux was determined by the strength of LC3 ‐ II accumulation in a 2 ‐ h treatment period with Pep . A / E64 . Therefore , normalised LC3 ‐ II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep . A / E64 . Values are expressed as mean ± s . e . m . * P0 . 05 versus control ‐ transfected cells , n = 3 . ( D ) I90 cells were transfected with GFP fused to LC3 ( pGFP . LC3 ) and co ‐ transfected either with BAG3 ‐ N1 or N1 . After transfection for 48 h , cells were treated as in ( A ) and levels of indicated proteins were detected by immunoblot analysis . ( E ) I90 cells , transfected as in ( D ) for 24 h , were microscopically analysed for GFP ‐ LC3 fluorescence . Representative pictures are shown . Bar : 20 μm . Values expressed in diagram are mean ± s . e . m . from three independent experiments ( 50 cells were counted per experiment ) . * P0 . 05 versus N1 , n = 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.A/E64) or DMSO as control, followed by immunoblot analysis of the indicated proteins.(B) 293 cells were transfected for 48 h with the indicated siRNAs and BAG3 expression plasmid (BAG3‐N1) or vector control (N1) followed by the same analysis as in (A).(C) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in (A) and (B). Autophagic flux was determined by the strength of LC3‐II accumulation in a 2‐h treatment period with Pep.A/E64. Therefore, normalised LC3‐II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64. Values are expressed as mean±s.e.m. *P0.05 versus control‐transfected cells, n=3.(D) I90 cells were transfected with GFP fused to LC3 (pGFP.LC3) and co‐transfected either with BAG3‐N1 or N1. After transfection for 48 h, cells were treated as in (A) and levels of indicated proteins were detected by immunoblot analysis.(E) I90 cells, transfected as in (D) for 24 h, were microscopically analysed for GFP‐LC3 fluorescence. Representative pictures are shown. Bar: 20 μm. Values expressed in diagram are mean±s.e.m. from three independent experiments (50 cells were counted per experiment). *P0.05 versus N1, n=3.


Figure 4 ( A ) I90 cells were transfected with BAG3 ( BAG3 ‐ N1 ) or vector control . After transfection for 48 h , levels of indicated proteins were detected by western ‐ blot analysis . ( B ) Transcript levels of SQSTM1 in I90 cells transfected as in ( A ) were compared by real ‐ time PCR analysis . Depicted is the fold ‐ increase of SQSTM1 mRNA levels in BAG3 ‐ N1 ‐ transfected cells relative to control ( N1 ) ‐ transfected cells . Values are expressed as mean ± s . e . m . * * P0 . 01 versus N1 cells , n = 3 . ( C ) I90 cells were transfected with a BAG3 ‐ GFP fusion plasmid for 48 h followed by indirect immunofluorescence staining of endogenous SQSTM1 . ( a ) Direct fluorescence of BAG3 ‐ GFP ( green ) , ( b ) indirect immunofluorescence of SQSTM1 ( red ) and ( c ) the stainings of ( a ) and ( b ) overlapped . DAPI ( blue ) was used to stain DNA . Representative pictures are shown . Bar : 10 μm . ( D ) Co ‐ IP studies testing the interaction of SQSTM1 and BAG3 . I90 cells were transfected as in ( A ) . After 24 h , SQSTM1 and BAG3 were immunoprecipitated followed by analysis of co ‐ sedimented proteins , as indicated . As negative control purified rabbit ( rb ) and mouse ( ms ) IgG was used . Left panel shows relative amounts of proteins in lysates used for Co ‐ IP ( Input ) .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) I90 cells were transfected with BAG3 (BAG3‐N1) or vector control. After transfection for 48 h, levels of indicated proteins were detected by western‐blot analysis.(B) Transcript levels of SQSTM1 in I90 cells transfected as in (A) were compared by real‐time PCR analysis. Depicted is the fold‐increase of SQSTM1 mRNA levels in BAG3‐N1‐transfected cells relative to control (N1)‐transfected cells. Values are expressed as mean±s.e.m. **P0.01 versus N1 cells, n=3.(C) I90 cells were transfected with a BAG3‐GFP fusion plasmid for 48 h followed by indirect immunofluorescence staining of endogenous SQSTM1. (a) Direct fluorescence of BAG3‐GFP (green), (b) indirect immunofluorescence of SQSTM1 (red) and (c) the stainings of (a) and (b) overlapped. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 10 μm.(D) Co‐IP studies testing the interaction of SQSTM1 and BAG3. I90 cells were transfected as in (A). After 24 h, SQSTM1 and BAG3 were immunoprecipitated followed by analysis of co‐sedimented proteins, as indicated. As negative control purified rabbit (rb) and mouse (ms) IgG was used. Left panel shows relative amounts of proteins in lysates used for Co‐IP (Input).


Figure 5 ( A ) Proteasomal chymotrypsin and cathepsin activity in lysates from young ( Y ) and old ( O ) I90 cells was determined using specific fluorescence probes , as described in the Material and methods section . Values are expressed are mean ± s . e . m . * P0 . 05 and * * P0 . 01 versus young , n = 3 . ( B ) Real ‐ time PCR analysis of LC3 , WIPI1 and SQSTM1 mRNA levels in young and old I90 cells . Depicted is the mean expression ratio ( log2 ) ± s . e . m . of target genes in old cells relative to young cells . * * P0 . 01 and * * * P0 . 001 versus young , n = 3 . ( C ) Immunoblot analysis of LC3 and SQSTM1 in young and old I90 cells . ( D ) Young and old I90 cells were treated for 2 h with bafilomycin A1 ( BafA1 , 2 μM ) or DMSO as control followed by immunoblot analysis of indicated proteins . ( E ) Immunoblot analysis of WIPI1 expression in young and old I90 cells . ( F ) Indirect immunofluorescence staining of endogenous LC3 ( green ) , SQSTM1 ( red ) and WIPI1 ( white ) in I90 cells of young and old age . DAPI ( blue ) was used to stain DNA . Representative pictures are shown . Bar : 20 μm . Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E . ( G ) Old and young I90 cells were treated for 1 h with DMSO as control ( C ) , lactacystin ( L , 2 μM ) or NH4Cl ( 20 mM ) plus leupeptin ( Leu , 5 μM ) ( N , NH4Cl / Leu ) . Western ‐ blot analyses were performed for detection of indicated proteins . In the diagram ( right panel ) , levels of polyUb ‐ proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels . Values are expressed as mean ± s . e . m . * P0 . 05 versus old control , # P0 . 05 versus young control , n = 3 . ( H ) I90 cells of old ( upper panel ) and young ( lower panel ) age were transfected with atg7 or nonsense ( nons ) siRNA . After transfection for 4 days , the cells were treated with NH4Cl / Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX ‐ 100 ( TX ‐ 100 ) soluble and insoluble material . Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins . Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions , respectively . ( I ) Transmission electron microscopic analysis of young ( a ) and old ( b ) I90 cells . Magnifications of marked areas in ( a ) and ( b ) are shown in ( c ) and ( d ) , respectively . An arrow indicates an autolysosome , and arrow heads indicate autophagosomes . M , mitochondrion . N , nucleus .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Proteasomal chymotrypsin and cathepsin activity in lysates from young (Y) and old (O) I90 cells was determined using specific fluorescence probes, as described in the Material and methods section. Values are expressed are mean±s.e.m. *P0.05 and **P0.01 versus young, n=3.(B) Real‐time PCR analysis of LC3, WIPI1 and SQSTM1 mRNA levels in young and old I90 cells. Depicted is the mean expression ratio (log2) ±s.e.m. of target genes in old cells relative to young cells. **P0.01 and ***P0.001 versus young, n=3.(C) Immunoblot analysis of LC3 and SQSTM1 in young and old I90 cells.(D) Young and old I90 cells were treated for 2 h with bafilomycin A1 (BafA1, 2 μM) or DMSO as control followed by immunoblot analysis of indicated proteins.(E) Immunoblot analysis of WIPI1 expression in young and old I90 cells.(F) Indirect immunofluorescence staining of endogenous LC3 (green), SQSTM1 (red) and WIPI1 (white) in I90 cells of young and old age. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E.(G) Old and young I90 cells were treated for 1 h with DMSO as control (C), lactacystin (L, 2 μM) or NH4Cl (20 mM) plus leupeptin (Leu, 5 μM) (N, NH4Cl/Leu). Western‐blot analyses were performed for detection of indicated proteins. In the diagram (right panel), levels of polyUb‐proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels. Values are expressed as mean±s.e.m. *P0.05 versus old control, #P0.05 versus young control, n=3.(H) I90 cells of old (upper panel) and young (lower panel) age were transfected with atg7 or nonsense (nons) siRNA. After transfection for 4 days , the cells were treated with NH4Cl/Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX‐100 (TX‐100) soluble and insoluble material. Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins. Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions, respectively.(I) Transmission electron microscopic analysis of young (a) and old (b) I90 cells. Magnifications of marked areas in (a) and (b) are shown in (c) and (d), respectively. An arrow indicates an autolysosome, and arrow heads indicate autophagosomes. M, mitochondrion. N, nucleus.


Figure 6 ( A ) Immunoblot analysis of indicated proteins of young ( Y ) and old ( O ) I90 cells upon fractionation as described in Figure 5H . ( B , C ) After transfection for 96 h of old I90 cells with bag3 or nonsense ( nons ) siRNA , BAG1 and BAG3 protein and mRNA levels were analysed by immunoblot ( B ) and real ‐ time PCR ( C ) analysis , respectively . Transcript levels in bag3 siRNA cells are depicted as the mean log2 expression ratio ± s . e . m . relative to nons siRNA cells . * P0 . 05 and * * * P0 . 001 versus nons , n = 3 . ( D ) Indirect immunofluorescence analysis of endogenous LC3 ( green ) and WIPI1 ( white ) in old I90 cells transfected with nons ( a , b ) and bag3 siRNA ( c , d ) for 96 h . DAPI ( blue ) was used as a nuclear marker . Representative pictures are shown . Bar : 20 μm . Diagrams show percentage of cells counted as in Figure 3E . ( E ) Same analysis as in Figure 5D but old cells transfected as in ( A ) were used . Values expressed in the diagram are mean ± s . e . m . * P0 . 05 versus nons , n = 3 . ( F ) Same analysis as in Figure 5G but old I90 cells transfected as in ( A ) were used . C , control ; L , lactacystin , N , NH4Cl / Leu . Values are expressed as mean ± s . e . m . * P0 . 05 versus nons control , or as indicated , n = 3 . ( G , H ) Same analysis as in ( F ) but young I90 cells 48 h after transfection with a BAG3 expression plasmid ( BAG3 ‐ N1 ) or vector control ( N1 ) together with nons or sqstm1 siRNA ( G ) and nons or atg7 siRNA ( H ) , as indicated , were used . * , # , ∼ and + , P0 . 05 versus C N1 , C BAG3 ‐ N1 , C BAG3 ‐ N1 sqstm1 or atg7 and C N1 sqstm1 or atg7 , n = 3 .

O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Immunoblot analysis of indicated proteins of young (Y) and old (O) I90 cells upon fractionation as described in Figure 5H.(B, C) After transfection for 96 h of old I90 cells with bag3 or nonsense (nons) siRNA, BAG1 and BAG3 protein and mRNA levels were analysed by immunoblot (B) and real‐time PCR (C) analysis, respectively. Transcript levels in bag3 siRNA cells are depicted as the mean log2 expression ratio ±s.e.m. relative to nons siRNA cells. *P0.05 and ***P0.001 versus nons, n=3.(D) Indirect immunofluorescence analysis of endogenous LC3 (green) and WIPI1 (white) in old I90 cells transfected with nons (a, b) and bag3 siRNA (c, d) for 96 h. DAPI (blue) was used as a nuclear marker. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells counted as in Figure 3E.(E) Same analysis as in Figure 5D but old cells transfected as in (A) were used. Values expressed in the diagram are mean±s.e.m. *P0.05 versus nons, n=3.(F) Same analysis as in Figure 5G but old I90 cells transfected as in (A) were used. C, control; L, lactacystin, N, NH4Cl/Leu. Values are expressed as mean±s.e.m. *P0.05 versus nons control, or as indicated, n=3.(G, H) Same analysis as in (F) but young I90 cells 48 h after transfection with a BAG3 expression plasmid (BAG3‐N1) or vector control (N1) together with nons or sqstm1 siRNA (G) and nons or atg7 siRNA (H), as indicated, were used. *, #, ∼ and +, P0.05 versus C N1, C BAG3‐N1, C BAG3‐N1 sqstm1 or atg7 and C N1 sqstm1 or atg7, n=3.


Figure 7 ( A ) Protein extracts from cerebellum ( CER ) of young ( Y , 3 months ) and old ( O , 24 months ) mice were analysed for BAG1 and BAG3 expression by immunoblot analysis . ( B ) Real ‐ time PCR analysis of indicated mRNA levels in cerebellum of young and old mice . Depicted is the log2 expression ratio of target genes in old mice relative to young mice . Values are expressed as mean ± s . e . m . * P0 . 05 and * * P0 . 01 versus young , n = 3 . ( C ) Immunoblot analysis as in ( A ) but in cortex ( CTX ) , hippocampus ( HIP ) and mid ‐ brain ( MB ) . ( D ) Western ‐ blot analysis of indicated proteins in different brain regions of young and old mice . ( E ) Total cathepsin and specific cathepsin B activity in brain extracts from young and old mice was determined using fluorescence probes Z ‐ FR ‐ AMC and Z ‐ RR ‐ AMC , respectively , as described in the Material and methods section . Values are expressed as mean ± s . e . m . * P0 . 05 versus young , n = 3 . ( F ) Expression analysis of indicated proteins in primary hippocampal astrocytic and neuronal cell cultures from young ( 2 months ) and old ( 24 months ) rats . Detection of GLT1 and NeuN served as astrocyte and neuron markers , respectively . Note the NeuN signals in astrocytic cultures . We attributed these signals to the presence of resting nuclei of dead neurons , which we observed under the microscope .

O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Protein extracts from cerebellum (CER) of young (Y, 3 months) and old (O, 24 months) mice were analysed for BAG1 and BAG3 expression by immunoblot analysis.(B) Real‐time PCR analysis of indicated mRNA levels in cerebellum of young and old mice. Depicted is the log2 expression ratio of target genes in old mice relative to young mice. Values are expressed as mean±s.e.m. *P0.05 and **P0.01 versus young, n=3.(C) Immunoblot analysis as in (A) but in cortex (CTX), hippocampus (HIP) and mid‐brain (MB).(D) Western‐blot analysis of indicated proteins in different brain regions of young and old mice.(E) Total cathepsin and specific cathepsin B activity in brain extracts from young and old mice was determined using fluorescence probes Z‐FR‐AMC and Z‐RR‐AMC, respectively, as described in the Material and methods section. Values are expressed as mean±s.e.m. *P0.05 versus young, n=3.(F) Expression analysis of indicated proteins in primary hippocampal astrocytic and neuronal cell cultures from young (2 months) and old (24 months) rats. Detection of GLT1 and NeuN served as astrocyte and neuron markers, respectively. Note the NeuN signals in astrocytic cultures. We attributed these signals to the presence of resting nuclei of dead neurons, which we observed under the microscope.


f1a , Percentage of ciliated cells in control ( Ctrl ) and IFT20 − MEFs ( * P = 0 . 030 , n = 3 ) and wild - type ( WT ) and Ift88 − / − KECs ( * * P = 0 . 003 , 25 cells each , n = 3 ) after 24 h of serum removal . Arrows , cilia . b , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ctrl and IFT20 − MEFs ( top , * * P = 0 . 0003 , n = 7 ) , and in WT and Ift88 − / − KECs ( bottom , * P = 0 . 035 , n = 9 ) . PI , protease inhibitors ; S , serum . cc , Autophagic flux in WT and Ift88 − / − KECs by mCherry - GFP - LC3 puncta quantification . Puncta : yellow , autophagosomes ; red only , autophagolysosomes ; total , both together ( * * P = 0 . 008 , * P = 0 . 025 ) . . d , LC3 immunoblot ( left ) for quantification of autophagosome formation ( right ) . ( * P = 0 . 048 , n = 4 ) . e , Electron microscopy images ( left ) and morphometric quantification of autophagic vacuoles ( AV , right ) in Ctrl and IFT20 − MEFs ( top , * * P = 0 . 001 , 10 fields in 2 experiments ) and KECs WT and Ift88 − / − ( bottom , * P = 0 . 037 , 5 fields ) . Arrows , autophagic vacuoles ( AV , black ) and endosomal compartments ( yellow ) . f , Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml − 1 platelet - derived growth factor ( PDGF ) treatment . ( * * P = 0 . 001 , * * * P = 0 . 0009 , 121 , 230 and 162 cells , n = 5 ) . g , LC3immunoblot ( top ) for autophagic flux quantification ( bottom ) upon PDGF treatment ( * P = 0 . 005 , n = 3 ) . Scale bars in a , 10 µm . n . s . , statistically non - significant . Mean ± s . d . in b , d and mean ± s . e . m . in other panels .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.030, n = 3) and wild-type (WT) and Ift88−/− KECs (**P = 0.003, 25 cells each, n = 3) after 24 h of serum removal. Arrows, cilia.b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. cc, Autophagic flux in WT and Ift88−/−KECs by mCherry-GFP-LC3 puncta quantification. Puncta: yellow, autophagosomes; red only, autophagolysosomes; total, both together (**P = 0.008, *P = 0.025).. d, LC3 immunoblot (left) for quantification of autophagosome formation (right). (*P = 0.048, n = 4).e, Electron microscopy images (left) and morphometric quantification of autophagic vacuoles (AV, right) in Ctrl and IFT20− MEFs (top, **P = 0.001, 10 fields in 2 experiments) and KECs WT and Ift88−/− (bottom, *P = 0.037, 5 fields). Arrows, autophagic vacuoles (AV, black) and endosomal compartments (yellow).f, Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml−1 platelet-derived growth factor (PDGF) treatment. (**P = 0.001, ***P = 0.0009, 121, 230 and 162 cells, n = 5).g, LC3immunoblot (top) for autophagic flux quantification (bottom) upon PDGF treatment (*P = 0.005, n = 3). Scale bars in a, 10 µm. n.s., statistically non-significant. Mean ± s.d. in b, d and mean ± s.e.m. in other panels.


f2a , GFP - LC3 puncta pattern ( left ) and quantification ( right ) in MEFs treated with purmorphamine ( Purmo ; top , * P = 0 . 028 , n = 4 ) , in Ptc − / − MEFs ( middle , * P = 0 . 015 , n = 4 ) and after myc - GLI1 overexpression ( bottom , * P = 0 . 012 , n = 3 ) . b , Autophagic flux by mCherry - GFP - LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum . Yellow , autophagosomes ; red only , autophagolysosomes ; total , both compartments . ( * P = 0 . 0323 , 40 fields ) . c , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ctrl ( * * P = 0 . 001 ) and MEFs knocked down for SMO ( SMO − ) ( * * P = 0 . 0001 ) upon purmorphamine treatment ( * P = 0 . 009 , n = 4 ) . d , Autophagic flux by mCherry - GFP - LC3 puncta quantification in WT and Ift88 − / − KECs upon purmorphamine treatment ( * P = 0 . 046 , * * P = 0 . 008 , 40 fields ) . . e , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ift88 − / − KECs overexpressing myc - GLI1 ( * * P = 0 . 00006 , n = 3 ) . f , Autophagic flux by mCherry - GFP - LC3puncta quantification in MEFs treated with cyclopamine upon serum removal ( * P = 0 . 011 , * * P = 0 . 013 , 25 fields ) . Scale bars , 10 µm . Mean ± s . d . in c , f and mean ± s . e . m . in other panels .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2a, GFP-LC3 puncta pattern (left) and quantification (right) in MEFs treated with purmorphamine (Purmo; top, *P = 0.028, n = 4), in Ptc−/− MEFs (middle, *P = 0.015, n = 4) and after myc-GLI1 overexpression (bottom, *P = 0.012, n = 3).b, Autophagic flux by mCherry-GFP-LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum. Yellow, autophagosomes; red only, autophagolysosomes; total, both compartments. (*P = 0.0323, 40 fields).c, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl (**P = 0.001) and MEFs knocked down for SMO (SMO−) (**P = 0.0001) upon purmorphamine treatment (*P = 0.009, n = 4).d, Autophagic flux by mCherry-GFP-LC3 puncta quantification in WT and Ift88−/− KECs upon purmorphamine treatment (*P = 0.046, **P = 0.008, 40 fields).. e, LC3 immunoblot (left) for autophagic flux quantification (right) in Ift88−/− KECs overexpressing myc-GLI1 (**P = 0.00006, n = 3).f, Autophagic flux by mCherry-GFP-LC3puncta quantification in MEFs treated with cyclopamine upon serum removal (*P = 0.011, **P = 0.013, 25 fields). Scale bars, 10 µm. Mean ± s.d. in c, f and mean ± s.e.m. in other panels.


f3a , Co - immunostaining ( top ) and 3D reconstruction ( middle and bottom ) for the indicated autophagy - related proteins ( ATGs , green ) and acetylated tubulin ( red , ciliary axoneme marker ) in KECs maintained in the absence of serum for 24 h . Yellow arrows , colocalization . b , Schematic representation of the ATGs associated with the axoneme ( top left ) . Co - immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off . Individual channels and merged images are shown . c , Co - immunostaining ( bottom ) and 3D reconstruction ( top ) for ATGs ( green ) and gamma - tubulin ( red , basal body marker ) in the same cells and conditions . Arrows , colocalization ( yellow ) , no colocalization ( white ) . d - f , Percentage of cells with colocalizing ATGs in the basal body ( BB ) in WT and Ift88 − / − KECs in the presence or absence of serum . d , Serum - and IFT - dependent association of ATGs with the BB ( VPS34 , † P = 0 . 04 , * * P = 0 . 002 , n = 4 ; ATG16L , † † P = 0 . 0003 , * * P = 0 . 0009 , 15 cells each per experiment , n = 7 ) . e , IFT - dependent but serum - independent association of ATGs with the BB ( * * P = 0 . 001 , * * * P = 9 . 33768 × 10 − 6 , n = 4 ; * P = 0 . 018 , 15 cells each per experiment , n = 5 ) . f , BB association of ATGs independent on serum or IFT ( ATG5 , n = 5 ; LC3 , n = 8 ; 15 cells each per experiment ) . Scale bars , 10 & amp ; amp ; mgr ; m . Mean ± s . e . m .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3a, Co-immunostaining (top) and 3D reconstruction (middle and bottom) for the indicated autophagy-related proteins (ATGs, green) and acetylated tubulin (red, ciliary axoneme marker) in KECs maintained in the absence of serum for 24 h. Yellow arrows, colocalization.b, Schematic representation of the ATGs associated with the axoneme (top left). Co-immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off. Individual channels and merged images are shown.c, Co-immunostaining (bottom) and 3D reconstruction (top) for ATGs (green) and gamma-tubulin (red, basal body marker) in the same cells and conditions. Arrows, colocalization (yellow), no colocalization (white).d-f, Percentage of cells with colocalizing ATGs in the basal body (BB) in WT and Ift88−/− KECs in the presence or absence of serum. d, Serum- and IFT-dependent association of ATGs with the BB (VPS34, †P = 0.04, **P = 0.002, n = 4; ATG16L, ††P = 0.0003, **P = 0.0009, 15 cells each per experiment, n = 7). e, IFT-dependent but serum-independent association of ATGs with the BB (**P = 0.001, ***P = 9.33768 × 10−6, n = 4; *P = 0.018, 15 cells each per experiment, n = 5). f, BB association of ATGs independent on serum or IFT (ATG5, n = 5; LC3, n = 8; 15 cells each per experiment). Scale bars, 10 &amp;mgr;m. Mean ± s.e.m.


f4a , b , Immunoblot for the indicated proteins of WT and Ift88 − / − KECs subjected to co - immunoprecipitation of IFT20 ( a ) and ATG16L ( b ) . Inp , 1 / 10 input ; IP , immunoprecipitate ; FT , 1 / 10 flow - through . c , Immunoblot for ATG16L and clathrin in MEF homogenate ( Hom ) , and the pellets from 1 h centrifugation at 100 , 000g ( organelles ( Org ) ) , 300 , 000g ( 300k ) and 500 , 000g ( vesicles , 500k ) . d , Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl , IFT20 − and IFT88 − MEFs in the presence ( top ) and absence ( bottom ) of serum . e , Immunogold electron microscopy for ATG16L ( 15 - nm particles ) and IFT20 ( 10 - nm particles ) in vesicles from MEFs isolated by the same method as in c . Right , quantification of IFT20 and ATG16L presence in the vesicles ( 11 fields ) . Mean ± s . e . m .

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f4a, b, Immunoblot for the indicated proteins of WT and Ift88−/− KECs subjected to co-immunoprecipitation of IFT20 (a) and ATG16L (b). Inp, 1/10 input; IP, immunoprecipitate; FT, 1/10 flow-through.c, Immunoblot for ATG16L and clathrin in MEF homogenate (Hom), and the pellets from 1 h centrifugation at 100,000g (organelles (Org)), 300,000g (300k) and 500,000g (vesicles, 500k).d, Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl, IFT20− and IFT88− MEFs in the presence (top) and absence (bottom) of serum.e, Immunogold electron microscopy for ATG16L (15-nm particles) and IFT20 (10-nm particles) in vesicles from MEFs isolated by the same method as in c. Right, quantification of IFT20 and ATG16L presence in the vesicles (11 fields). Mean ± s.e.m.


f5a , b , Ciliated Atg5 − / − MEFs in serum + ( * P = 0 . 038 , 25 cells each per experiment , n = 3 ) ( a ) or serum − ( * P = 0 . 006 , nonlinear fit regression , 25 cells each per experiment , n = 3 ) ( b ) . c , Scanning electron microscopy ( SEM ) of primary cilia . Arrows , cilia - associated vesicles ; arrowheads , ciliary pocket . . d , Cilia length quantification from SEM ( * P = 0 . 027 n = 28 ; * * P = 0 . 001 , † † P = 0 . 0001 , n = 23 ) . e , f , GLI1 ( * P = 0 . 04 , n = 3 ) ( e ) and GLI2 ( * * * P = 0 . 0005 , * * P = 0 . 0046 , † P = 0 . 0132 , n = 3 ) ( f ) messenger RNA expression in Atg5 − / − MEFs . g , h , IFT20 immunoblot in Atg5 − / − ( g ) and WT MEFs with lysosomal inhibitors ( NL ) ( h ) . i , IFT20 immunoblot in cytosol ( Cyt ) , homogenate ( Hom ) and autophagosomes ( APG ) . j , IFT20 protein levels in Atg5 − / − MEFs ( * P = 0 . 038 , n = 6 ) . k , IFT20 immunoblot in Atg5 − / − MEFs . n . s . , statistically non - significant . Mean ± s . d . in d and j and mean ± s . e . m . in other panels .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5a, b, Ciliated Atg5−/− MEFs in serum+ (*P = 0.038, 25 cells each per experiment, n = 3) (a) or serum− (*P = 0.006, nonlinear fit regression, 25 cells each per experiment, n = 3) (b).c, Scanning electron microscopy (SEM) of primary cilia. Arrows, cilia-associated vesicles; arrowheads, ciliary pocket.. d, Cilia length quantification from SEM (*P = 0.027 n = 28; **P = 0.001, ††P = 0.0001, n = 23).e, f, GLI1 (*P = 0.04, n = 3) (e) and GLI2 (***P = 0.0005, **P = 0.0046, †P = 0.0132, n = 3) (f) messenger RNA expression in Atg5−/− MEFs.g, h, IFT20 immunoblot in Atg5−/− (g) and WT MEFs with lysosomal inhibitors (NL) (h).i, IFT20 immunoblot in cytosol (Cyt), homogenate (Hom) and autophagosomes (APG). j, IFT20 protein levels in Atg5−/− MEFs (*P = 0.038, n = 6). k, IFT20 immunoblot in Atg5−/− MEFs. n.s., statistically non-significant. Mean ± s.d. in d and j and mean ± s.e.m. in other panels.


Figure 1RT - PCR analysis ( top ) and representative direct sequencing ( bottom ) of Ttn exon 326 transcripts from HL - 1 cardiomyocytes transiently transfected with different 2OMePS AONs . Only 2OMePS - AON1 and 2OMePS - AON1 + 3 ( * ) lead to a correct excision of exon 326 as confirmed by direct sequencing . Immunofluorescence images of sarcomeric α - actinin in untransfected and 2OMePS - AON1 + 3 - transfected HL - 1 cardiomyocytes . Scale bars , 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1RT-PCR analysis (top) and representative direct sequencing (bottom) of Ttn exon 326 transcripts from HL-1 cardiomyocytes transiently transfected with different 2OMePS AONs. Only 2OMePS-AON1 and 2OMePS-AON1 + 3 (*) lead to a correct excision of exon 326 as confirmed by direct sequencing.Immunofluorescence images of sarcomeric α-actinin in untransfected and 2OMePS-AON1 + 3-transfected HL-1 cardiomyocytes. Scale bars, 10 μm.


Figure 2RT - PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS - AON1 , 2OMePS - AON3 , and 2OMePS - AON1 + 3 . RT - PCR analysis ( left ) and representative direct sequencing ( right ) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA - TTNAONs - IRES - GFP lentiviral vector carrying the AON1 and 3 sequences ( TTN - AON ) or with a control vector ( U7snRNA - ScrAONs - IRES - GFP ) . Mass spectrometry - based analysis of titin peptides in cells infected with the U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) and U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) vectors . Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down - regulated in DCM Scr - AON cardiomyocytes compared to CTR Scr - AON cardiomyocytes ( n = 3 , P = 9 . 03E − 8 , Fisher ' s exact test , FDR = 0 . 04 , top ) . Down - regulation of exon 326 was also detected in DCM TTN - AON cells when compared to DCM Scr - AON cells ( n = 3 , P = 0 . 02 , Fisher ' s exact test , bottom ) .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2RT-PCR analysis of TTN exon 326 transcripts from DCM cardiomyocytes transiently transfected with 2OMePS-AON1, 2OMePS-AON3, and 2OMePS-AON1 + 3.RT-PCR analysis (left) and representative direct sequencing (right) of TTN exon 326 transcripts from DCM cardiomyocytes infected with the U7snRNA-TTNAONs-IRES-GFP lentiviral vector carrying the AON1 and 3 sequences (TTN-AON) or with a control vector (U7snRNA-ScrAONs-IRES-GFP).Mass spectrometry-based analysis of titin peptides in cells infected with the U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and U7snRNA-TTNAONs-IRES-GFP (TTN-AON) vectors. Unsupervised hierarchical clustering identified a cluster significantly enriched in peptides mapping to exon 326 that was down-regulated in DCM Scr-AON cardiomyocytes compared to CTR Scr-AON cardiomyocytes (n = 3, P = 9.03E−8, Fisher's exact test, FDR = 0.04, top). Down-regulation of exon 326 was also detected in DCM TTN-AON cells when compared to DCM Scr-AON cells (n = 3, P = 0.02, Fisher's exact test, bottom).


Figure 3Percentage of iPSC - derived cardiomyocytes with cross - striated myofibrils occupying a fraction ( partially organized , dark gray ) or the whole cytoplasm ( fully organized , light gray ) from two control and two patient clones . Statistical difference was tested using the two - sided chi - squared test ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 ; * * * P = 1 . 93E − 16 ) . Immunofluorescence images of titin ( Z - disk portion ) and cardiac troponin T ( cTNT , A - band ) in well - organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions . Scale bars , 5 μm . Immunofluorescence images ( left ) of α - actinin and cTNT in CTR and DCM single cardiomyocytes , illustrating 3 different levels of sarcomeric organization ( perinuclear , fully and peripherally organized ) . Percentage of cells with different levels of sarcomeric organization ( right ) under basal and stress conditions . Statistical difference was tested using the chi - squared test ( CTR1 basal : n = 283 , CTR2 basal : n = 250 , DCM1 basal : n = 255 , DCM2 basal : n = 236 , CTR1 Iso : n = 245 , CTR2 Iso : n = 230 ; DCM1 Iso : n = 242 and DCM2 Iso : n = 269 ; * * * P = 1 . 93E − 16 CTR basal versus DCM basal ; * * * P = 9 . 61E − 34 CTR Iso versus DCM Iso , * * P = 0 . 001 DCM basal versus DCM Iso ) . No significant differences were observed comparing CTR basal and CTR Iso groups . Scale bars , 25 μm . Percentage of perinuclear , fully , and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA - ScrAONs - IRES - GFP and U7snRNA - TTNAONs - IRES - GFP lentiviruses . Statistical difference was tested using the two - sided chi - squared test ( CTR1 Scr - AON : n = 190 , CTR2 Scr - AON : n = 200 , DCM1 Scr - AON : n = 221 , DCM2 Scr - AON : n = 115 , CTR1 TTN - AON : n = 223 , CTR2 TTN - AON : n = 187 , DCM1 TTN - AON : n = 171 , DCM2 TTN - AON : n = 243 ; * * * P = 4 . 22E − 15 CTR Scr - AON versus DCM Scr - AON ; * * * P = 4 . 61E − 02 DCM Scr - AON versus DCM TTN - AON ) . No significant differences were observed comparing CTR Scr - AON and CTR TTN - AON groups .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.


Figure 4qRT - PCR analysis of SRF target genes ( MYH6 , MYH7 and ACTC1 ) in CTR and DCM single cardiomyocytes under basal condition ( no infection , NI ) and after infection with control U7snRNA - ScrAONs - IRES - GFP ( Scr - AON ) and the U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses . Statistical difference was tested using the two - sided Student ' s t - test ( * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MYH6 ; * * P = 0 . 002 , CTR Scr - AON versus DCM Scr - AON ; * * P = 0 . 002 , DCM Scr - AON versus DCM TTN - AON for MYH7 ; * * P = 0 . 004 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 02 , DCM Scr - AON versus DCM TTN - AON for ACTC1 ) . No significant differences were observed comparing the CTR NI , CTR Scr - AON and CTR TTN - AON groups and comparing the DCM NI and DCM Scr - AON groups . Expression values were relative to CTR Scr - AON , normalized to GAPDH , and presented as mean ± SEM , n = 3 . Immunofluorescence images showing normal ( a , b , e , f , i , l , o , p ) and altered ( c , d , g , h , m , n , q , r ) intracellular distribution of SRF ( a and b , nuclear ; c and d , cytoplasmic ) , MURF2 ( e and f , sarcomeric ; g and h , nuclear ) , Nbr1 ( i and l , sarcomeric ; m and n , diffused ) , and SQSTM1 / p62 ( o and p , sarcomeric ; q and r , diffused ) in representative single cardiomyocytes ( left ) . Sarcomeres are marked by α - actinin . On the right , percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF , nuclear accumulation of MURF2 , and diffused expression of Nbr1 and of SQSTM1 / p62 after infection with control U7snRNA - ScrambleAONs - IRES - GFP ( Scr - AON ) and the U7snRNA - TTNAONs - IRES - GFP ( TTN - AON ) lentiviruses ( right ) . Data represent mean values ± SEM from two control and two DCM clones . Statistical difference was tested using the two - sided chi - squared test ( CTR Scr - AON : n = 874 , n = 874 , n = 882 and n = 890 , CTR TTN - AON : n = 880 , n = 990 , n = 878 and n = 890 , DCM Scr - AON : n = 890 , n = 887 , n = 884 and n = 886 , DCM TTN - AON : n = 900 , n = 875 , n = 899 and n = 891 for SRF , MURF2 , Nbr1 and SQSTM1 / p62 , respectively ; * P = 0 . 01 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for SRF ; * P = 0 . 02 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 04 , DCM Scr - AON versus DCM TTN - AON for MURF2 ; * P = 0 . 03 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 03 , DCM Scr - AON versus DCM TTN - AON for Nbr1 ; * * P = 0 . 009 , CTR Scr - AON versus DCM Scr - AON ; * P = 0 . 01 , DCM Scr - AON versus DCM TTN - AON for SQSTM1 / p62 ) . No significant differences were observed comparing CTR Scr - AON and CTR TTN - AON groups . Scale bars , 50 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4qRT-PCR analysis of SRF target genes (MYH6, MYH7 and ACTC1) in CTR and DCM single cardiomyocytes under basal condition (no infection, NI) and after infection with control U7snRNA-ScrAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses. Statistical difference was tested using the two-sided Student's t-test (**P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MYH6; **P = 0.002, CTR Scr-AON versus DCM Scr-AON; **P = 0.002, DCM Scr-AON versus DCM TTN-AON for MYH7; **P = 0.004, CTR Scr-AON versus DCM Scr-AON; *P = 0.02, DCM Scr-AON versus DCM TTN-AON for ACTC1). No significant differences were observed comparing the CTR NI, CTR Scr-AON and CTR TTN-AON groups and comparing the DCM NI and DCM Scr-AON groups. Expression values were relative to CTR Scr-AON, normalized to GAPDH, and presented as mean ± SEM, n = 3.Immunofluorescence images showing normal (a, b, e, f, i, l, o, p) and altered (c, d, g, h, m, n, q, r) intracellular distribution of SRF (a and b, nuclear; c and d, cytoplasmic), MURF2 (e and f, sarcomeric; g and h, nuclear), Nbr1 (i and l, sarcomeric; m and n, diffused), and SQSTM1/p62 (o and p, sarcomeric; q and r, diffused) in representative single cardiomyocytes (left). Sarcomeres are marked by α-actinin. On the right, percentage of CTR and DCM cardiomyocytes showing cytoplasmic expression of SRF, nuclear accumulation of MURF2, and diffused expression of Nbr1 and of SQSTM1/p62 after infection with control U7snRNA-ScrambleAONs-IRES-GFP (Scr-AON) and the U7snRNA-TTNAONs-IRES-GFP (TTN-AON) lentiviruses (right). Data represent mean values ± SEM from two control and two DCM clones. Statistical difference was tested using the two-sided chi-squared test (CTR Scr-AON: n = 874, n = 874, n = 882 and n = 890, CTR TTN-AON: n = 880, n = 990, n = 878 and n = 890, DCM Scr-AON: n = 890, n = 887, n = 884 and n = 886, DCM TTN-AON: n = 900, n = 875, n = 899 and n = 891 for SRF, MURF2, Nbr1 and SQSTM1/p62, respectively; *P = 0.01, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for SRF; *P = 0.02, CTR Scr-AON versus DCM Scr-AON; *P = 0.04, DCM Scr-AON versus DCM TTN-AON for MURF2; *P = 0.03, CTR Scr-AON versus DCM Scr-AON; *P = 0.03, DCM Scr-AON versus DCM TTN-AON for Nbr1; **P = 0.009, CTR Scr-AON versus DCM Scr-AON; *P = 0.01, DCM Scr-AON versus DCM TTN-AON for SQSTM1/p62). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups. Scale bars, 50 μm.


Figure 5B , C Immunofluorescence images of sarcomeric α - actinin and myosin ( B ) and assessment of cardiac function ( C ) in homozygous Ttn knock - in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection ( n = 3 per group ) or lentivirus delivery ( n = 7 per group ) . Scale bars , 10 μm . D Electron microscopy analysis in Ttn knock - in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection . mTtnAON treatment of homozygous embryos rescued myofibril formation ( left , black arrows ) , resulting in thicker filaments ( right ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B, C Immunofluorescence images of sarcomeric α-actinin and myosin (B) and assessment of cardiac function (C) in homozygous Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection (n = 3 per group) or lentivirus delivery (n = 7 per group). Scale bars, 10 μm.D Electron microscopy analysis in Ttn knock-in embryos cultured for 24 h after mScrAON and mTtnAON transient transfection. mTtnAON treatment of homozygous embryos rescued myofibril formation (left, black arrows), resulting in thicker filaments (right).


Figure 6Echocardiographic analysis of adult knock - in mice . Statistical difference was tested using the two - sided Student ' s t - test ( WT : n = 6 , HET : n = 6 ; HET + vPMO - mScrAON : n = 10 and HET + vPMO - mTtnAON : n = 14 ; * P = 0 . 013 , HET versus HET + vPMO - mTtnAON and * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LVEDD differences ; * * P = 0 . 006 , HET versus HET + vPMO - mTtnAON and * P = 0 . 02 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for LV - EF differences ; * P = 0 . 03 , HET versus HET + vPMO - mTtnAON and * P = 0 . 04 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON for IVSd differences ; * * P = 0 . 001 , HET versus HET + vPMO - mTtnAON and * P = 0 . 03 , HET + vPMO - mScrAON versus HET + vPMO - mTtnAON and for PWd differences ) . No significant differences were observed among groups at day 0 and day 7 . Data represent mean values ± SEM . Masson ' s trichrome staining of heart sections from HET animals injected with vPMO - mScrAONs and vPMO - mTtnAON . Scale bars , 1 mm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Echocardiographic analysis of adult knock-in mice. Statistical difference was tested using the two-sided Student's t-test (WT: n = 6, HET: n = 6; HET + vPMO-mScrAON: n = 10 and HET + vPMO-mTtnAON: n = 14; *P = 0.013, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LVEDD differences; **P = 0.006, HET versus HET + vPMO-mTtnAON and *P = 0.02, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for LV-EF differences; *P = 0.03, HET versus HET + vPMO-mTtnAON and *P = 0.04, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON for IVSd differences; **P = 0.001, HET versus HET + vPMO-mTtnAON and *P = 0.03, HET + vPMO-mScrAON versus HET + vPMO-mTtnAON and for PWd differences). No significant differences were observed among groups at day 0 and day 7. Data represent mean values ± SEM.Masson's trichrome staining of heart sections from HET animals injected with vPMO-mScrAONs and vPMO-mTtnAON. Scale bars, 1 mm.


Figure 7Fluorescence in situ hybridization ( FISH ) of heart muscle tissue from adult knock - in mice with a probe complementary to vPMO - mTtnAON . Scale bars , 250 and 50 μm ( magnification ) . RT - PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO - mScrAON - and vPMO - mTtnAON - treated mice ( left ) . Representative direct sequencing of Ttn exon 326 transcripts from vPMO - mTtnAON - treated HET heart tissue ( right ) . Mass spectrometry - based analysis of titin peptides in adult knock - in mice injected with vPMO - mScrAONs and vPMO - mTtnAONs . Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down - regulated in vPMO - mTtnAON - treated animals compared to vPMO - mScrAON - treated littermates . Another cluster enriched in C - terminal peptides was up - regulated in the vPMO - mTtnAON group compared to the vPMO - mScrAON group ( n = 3 , P = 0 . 02 , Fisher ' s exact test , FDR = 0 . 04 ) .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Fluorescence in situ hybridization (FISH) of heart muscle tissue from adult knock-in mice with a probe complementary to vPMO-mTtnAON. Scale bars, 250 and 50 μm (magnification).RT-PCR analysis of Ttn exon 326 transcripts from heart tissue of untreated WT and HET animals and vPMO-mScrAON- and vPMO-mTtnAON-treated mice (left). Representative direct sequencing of Ttn exon 326 transcripts from vPMO-mTtnAON-treated HET heart tissue (right).Mass spectrometry-based analysis of titin peptides in adult knock-in mice injected with vPMO-mScrAONs and vPMO-mTtnAONs. Unsupervised hierarchical clustering identified a cluster enriched in exon 326 peptides that was down-regulated in vPMO-mTtnAON-treated animals compared to vPMO-mScrAON-treated littermates. Another cluster enriched in C-terminal peptides was up-regulated in the vPMO-mTtnAON group compared to the vPMO-mScrAON group (n = 3, P = 0.02, Fisher's exact test, FDR = 0.04).


Figure 1E . Double immunofluorescent staining of Fn12230 , fadA - deletion mutant US1 ( ∆ fadA ) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1 : 800 dilution and anti - human β - amyloid polyclonal antibody A11 at 1 : 500 dilution , followed by incubation with Alexa Fluor 680 - conjugated donkey anti - rabbit and Alexa Fluor 555 - conjugated goat anti - mouse , both at 1 : 1 , 000 dilution . Co - staining of FadA and A11 was observed in Fn in stationary phase , not in log phase , or in US1 or lam . The imagines were taken under 60X objective . Scale bar equals 5 μm . F . Scanning electron microscopy of Fn 12230 , US1 ( ∆ fadA ) and lam in log and stationary phase at 10 , 000x magnification . Note the fibrous structure coating Fn in stationary phase , pointed by the clear arrows , but not in log phase . Scale bar equals 1 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1E. Double immunofluorescent staining of Fn12230, fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1:800 dilution and anti-human β-amyloid polyclonal antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in stationary phase, not in log phase, or in US1 or lam. The imagines were taken under 60X objective. Scale bar equals 5 μm.F. Scanning electron microscopy of Fn 12230, US1 (∆fadA) and lam in log and stationary phase at 10,000x magnification. Note the fibrous structure coating Fn in stationary phase, pointed by the clear arrows, but not in log phase. Scale bar equals 1 μm.


Figure 2E . Thioflavin - T binding assay in the presence of 0 . 1 % SDS . FadA proteins ( 0 . 8 mg / ml ) were incubated with 10 μM Thioflavin - T at room temperature for 10 min . The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm . The experiment was performed in duplicate and repeated three times . The error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 ( compared to BSA , t - test ) . G . Analysis of amyloid - like FadA produced by Fn 23726 and its ∆ fap2 mutant . An aliquot of 50 μg detergent - resistant pellets was loaded onto each lane , followed by SDD - AGE and Western blot analysis as described above . The mutant produced significantly less amyloid - like polymers than the wild type .

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Figure 2E. Thioflavin-T binding assay in the presence of 0.1% SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01 (compared to BSA, t-test).G. Analysis of amyloid-like FadA produced by Fn 23726 and its ∆fap2 mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less amyloid-like polymers than the wild type.


Figure 3A . Detection of FadA in subgingival plaque samples by IHC . Three patients with periodontitis are shown here who provided plaque samples from their healthy sites ( probing depth ≤ 3 mm , left panels ) as well as periodontal diseased sites ( probing depth ≥ 7 mm , middle panels ) . IHC was performed as described using mAb 7H7 at 1 : 800 dilution . Anti - mouse IgG isotype control of the diseased site is shown ( right panels ) . The images were taken using 100X objective . Scale bar equals 50 μm . B . Double immunofluorescence staining of paired normal and carcinoma tissues from two CRC patients . The frozen tissue sections were incubated with mAb 7H7 at 1 : 50 dilution and anti - amyloid antibody A11 at 1 : 25 dilution , or mouse and rabbit IgG control , followed by incubation with Alexa Fluor 555 - conjugated goat anti - mouse and Alexa Fluor 680 - conjugated donkey anti - rabbit , both at 1 : 1000 dilution . Co - staining of FadA ( red ) and amyloid oligomers ( green ) was observed in the carcinoma but not normal tissues . The images were taken under 60X objective . Scale bar equals 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Detection of FadA in subgingival plaque samples by IHC. Three patients with periodontitis are shown here who provided plaque samples from their healthy sites (probing depth ≤ 3 mm, left panels) as well as periodontal diseased sites (probing depth ≥ 7 mm, middle panels). IHC was performed as described using mAb 7H7 at 1:800 dilution. Anti-mouse IgG isotype control of the diseased site is shown (right panels). The images were taken using 100X objective. Scale bar equals 50 μm.B. Double immunofluorescence staining of paired normal and carcinoma tissues from two CRC patients. The frozen tissue sections were incubated with mAb 7H7 at 1:50 dilution and anti-amyloid antibody A11 at 1:25 dilution, or mouse and rabbit IgG control, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse and Alexa Fluor 680-conjugated donkey anti-rabbit, both at 1:1000 dilution. Co-staining of FadA (red) and amyloid oligomers (green) was observed in the carcinoma but not normal tissues. The images were taken under 60X objective. Scale bar equals 10 μm.


Figure 4B . Inhibition of biofilm formation mediated by detergent - resistant Fn pellets by Congo Red . An aliquot of 10 μg detergent - resistant pellets prepared from Fn 12230 ( pellet ) was added in 96 - well plates in the presence of 0 , 10 or 50 µg / ml Congo Red ( CR ) , followed by addition of Fn 12230 and incubated as described above . The results shown are the average of four experiments each performed in duplicate . The error bars indicate SD . A representative image following 72 hours of incubation is shown in the top panel . * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) . D . Effects of Congo Red on Fn survival in acidic environment . Wild type Fn 12230 ( Fn ) or its fadA deletion mutant US1 ( ∆ fadA ) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg / ml Congo Red ( CR ) for 1 or 2 hours . The live bacterial counts at time 0 were designated as 100 % , and those after 1 or 2 hrs of incubation were expressed relative to time 0 . The results shown are the average of 5 independent experiments , each performed in duplicate . The error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( One - way ANOVA ) . F . Inhibition of Fn attachment by FadAc in the presence or absence of anti - amyloid antibody OC . An aliquot of 50 μg purified recombinant FadAc was pre - incubated with DLD1 for 45 min either alone , or mixed with 20 μl OC or rabbit IgG control , before Fn was added and the attachment assay performed as above . The attachment value by Fn alone was designated as 100 % , with all other values expressed relative to it . FadA mutant L14A was included as a negative control . Data shown are mean values ± SEM . The experiment was performed in duplicate and repeated three times . * * p < 0 . 01 ( One - way ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Inhibition of biofilm formation mediated by detergent-resistant Fn pellets by Congo Red. An aliquot of 10 μg detergent-resistant pellets prepared from Fn 12230 (pellet) was added in 96-well plates in the presence of 0, 10 or 50 µg/ml Congo Red (CR), followed by addition of Fn 12230 and incubated as described above. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01, ***p<0.001 (t-test).D. Effects of Congo Red on Fn survival in acidic environment. Wild type Fn 12230 (Fn) or its fadA deletion mutant US1 (∆fadA) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg/ml Congo Red (CR) for 1 or 2 hours. The live bacterial counts at time 0 were designated as 100%, and those after 1 or 2 hrs of incubation were expressed relative to time 0. The results shown are the average of 5 independent experiments, each performed in duplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (One-way ANOVA).F. Inhibition of Fn attachment by FadAc in the presence or absence of anti-amyloid antibody OC. An aliquot of 50 μg purified recombinant FadAc was pre-incubated with DLD1 for 45 min either alone, or mixed with 20 μl OC or rabbit IgG control, before Fn was added and the attachment assay performed as above. The attachment value by Fn alone was designated as 100%, with all other values expressed relative to it. FadA mutant L14A was included as a negative control. Data shown are mean values ± SEM. The experiment was performed in duplicate and repeated three times. **p< 0.01 (One-way ANOVA).


Figure 5D . Effect of detergent - resistant pellets prepared from Fn 12230 ( Fn ) and US1 ( ∆ fadA ) on tumor growth . HCT116 cells were inoculated into the nude mice as described above . In one group of mice ( n = 3 ) , sarkosyl - resistant pellets prepared from Fn 12230 and US1 ( ∆ fadA ) were injected into the tumors on opposite sides . In another group ( n = 5 ) , sarkosyl - resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control ( rIgG ) before injecting into the tumors on opposite sides . The tumor volumes were measured described above . The vertical lines represent the standard deviations . The individual tumor pairs are shown ( right panel ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ compared to US1 ( ∆ fadA ) , t - test ] .

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Figure 5D. Effect of detergent-resistant pellets prepared from Fn 12230 (Fn) and US1 (∆fadA) on tumor growth. HCT116 cells were inoculated into the nude mice as described above. In one group of mice (n=3), sarkosyl-resistant pellets prepared from Fn 12230 and US1 (∆fadA) were injected into the tumors on opposite sides. In another group (n=5), sarkosyl-resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control (rIgG) before injecting into the tumors on opposite sides. The tumor volumes were measured described above. The vertical lines represent the standard deviations. The individual tumor pairs are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 [compared to US1 (∆fadA), t-test].