L - Q NB - 3 + / + ( L and M ) and NB - 3 − / − ( O and P ) pyramidal neurons were co - cultured with astrocytes that did not ( L and O ) or did ( M and P ) express NB - 3 , and immunostained for Tuj1 , NB - 3 , and GFAP ( L , M , O and P ) . NB - 3 + / + pyramidal neurons ( N ) and NB - 3 − / − pyramidal neurons that overexpressed NB - 3ΔECD - Myc ( Q ) were co - cultured with astrocytes that overexpressed NB - 3ΔECD - Myc , and immunostained for Tuj1 , Myc , and GFAP ( N and Q ) . R The length of neurites on astrocytes relative to total neurite length . n . s . , not significant , and * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified .

L-Q NB-3+/+ (L and M) and NB-3−/− (O and P) pyramidal neurons were co-cultured with astrocytes that did not (L and O) or did (M and P) express NB-3, and immunostained for Tuj1, NB-3, and GFAP (L, M, O and P). NB-3+/+ pyramidal neurons (N) and NB-3−/−pyramidal neurons that overexpressed NB-3ΔECD-Myc (Q) were co-cultured with astrocytes that overexpressed NB-3ΔECD-Myc, and immunostained for Tuj1, Myc, and GFAP (N and Q).R The length of neurites on astrocytes relative to total neurite length. n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The neurite lengths of more than 150 pyramidal neurons from 4 independent experiments in each group were quantified.


A Layer V cortex explants from E18 . 5 NB - 3 + / + and NB - 3 − / − mice were cultured in medium supplemented with either Fc or NB - 3 - Fc ( 50 μg / ml ) . B Quantification of neurite outgrowth in ( A ) . n . s . , not significant , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group .

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

A Layer V cortex explants from E18.5 NB-3+/+ and NB-3−/−mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml).B Quantification of neurite outgrowth in (A). n.s., not significant, **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 50 layer V cortical explants from 5 independent experiments in each group.


C Pyramidal neurons of NB - 3 + / + and NB - 3 − / − mice were cultured in medium supplemented with either Fc or NB - 3 - Fc ( 50 μg / ml ) . The neurons were co - stained for Ctip2 , NB - 3 , and Rhodamine phalloidin . ( C1 - C4 ) High - magnification images of the growth cones ( white arrowheads ) of the neurons in ( C ) , respectively . D Quantification of neurite length in ( C ) , and the percentage of collapsed growth cones in ( C ) . n . s . , not significant , * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group .

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

C Pyramidal neurons of NB-3+/+ and NB-3−/− mice were cultured in medium supplemented with either Fc or NB-3-Fc (50 μg/ml). The neurons were co-stained for Ctip2, NB-3, and Rhodamine phalloidin. (C1-C4) High-magnification images of the growth cones (white arrowheads) of the neurons in (C), respectively.D Quantification of neurite length in (C), and the percentage of collapsed growth cones in (C). n.s., not significant, *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from more than 300 corticospinal neurons from 6 independent experiments in each group.


A Images of coronal sections of layer Vsensorimotor cortex from NB - 3 + / + and NB - 3 − / − mice after SCI . Corticospinal neurons were retrogradely labeled with Fluorogold ( FG ) , and coronal sections were co - stained with p - mTOR ( A ) ( arrowheads ) . B Quantification of fluorescence intensity of p - mTOR in FG - labeled corticospinal neurons in ( A ) . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

A Images of coronal sections of layer Vsensorimotor cortex from NB-3+/+ and NB-3−/−mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-mTOR (A) (arrowheads).B Quantification of fluorescence intensity of p-mTOR in FG-labeled corticospinal neurons in (A). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


C Images of coronal sections of layer Vsensorimotor cortex from NB - 3 + / + and NB - 3 − / − mice after SCI . Corticospinal neurons were retrogradely labeled with Fluorogold ( FG ) , and coronal sections were co - stained with p - S6 ( S240 / 244 ) ( C ) ( arrowheads ) . D Quantification of fluorescence intensity of p - S6 ( S240 / 244 ) in FG - labeled corticospinal neurons in ( C ) . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

C Images of coronal sections of layer Vsensorimotor cortex from NB-3+/+ and NB-3−/−mice after SCI. Corticospinal neurons were retrogradely labeled with Fluorogold (FG), and coronal sections were co-stained with p-S6 (S240/244) (C) (arrowheads).D Quantification of fluorescence intensity of p-S6 (S240/244) in FG-labeled corticospinal neurons in (C). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


E - H Western blot analysis and quantification of the expression of p - Akt / Akt ( E and F ) and p - S6 ( S240 / 244 ) ( G and H ) in sensorimotor cortex from NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from 3 independent experiments each including 3 mice per group .

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

E-H Western blot analysis and quantification of the expression of p-Akt/Akt (E and F) and p-S6 (S240/244) (G and H) in sensorimotor cortex from NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.


I , K , M , O Expression of Akt ( I ) , p - Akt ( K ) , p - mTOR ( M ) , and p - S6 ( S240 / 244 ) ( O ) in NB - 3 + / + pyramidal neuron - astrocyte co - cultures . The astrocytes were transfected either with NB - 3 - Myc or a control vector . The dashed lines outline the astrocytes . The asterisks indicate the NB - 3 - expressing astrocytes . J , L , N , P Quantification of fluorescence intensity of Akt ( J ) , p - Akt ( L ) , p - mTOR ( N ) , and p - S6 ( S240 / 244 ) ( P ) , respectively , in pyramidal neurons of the co - cultures in ( I , K , M , and O ) . n . s . , not significant , and * p < 0 . 05 ; one sample t - test . The intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified .

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

I, K, M, O Expression of Akt (I), p-Akt (K), p-mTOR (M), and p-S6 (S240/244) (O) in NB-3+/+ pyramidal neuron-astrocyte co-cultures. The astrocytes were transfected either with NB-3-Myc or a control vector. The dashed lines outline the astrocytes. The asterisks indicate the NB-3-expressing astrocytes.J, L, N, P Quantification of fluorescence intensity of Akt (J), p-Akt (L), p-mTOR (N), and p-S6 (S240/244) (P), respectively, in pyramidal neurons of the co-cultures in (I, K, M, and O). n.s., not significant, and *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 6 independent experiments in each group were quantified.


A Co - immunostaining of CHL1 ( blue ) , NB - 3 ( green ) , and BDA ( red ) in injured spinal cords of NB - 3 + / + ( A1 ) and NB - 3 − / − ( A5 ) mice . ( A2 - A4 and A6 - A8 ) High - magnification images of boxed areas in ( A1 and A5 ) , respectively . Images in ( A2 and A6 ) are presented as single - channel fluorescence images in ( A3 - A4 and A7 - A8 ) , respectively . B Quantification of fluorescence intensity of CHL1 at the spinal lesion sites of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . n = 11 mice per group .

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

A Co-immunostaining of CHL1 (blue), NB-3 (green), and BDA (red) in injured spinal cords of NB-3+/+ (A1) and NB-3−/− (A5) mice. (A2-A4 and A6-A8) High-magnification images of boxed areas in (A1 and A5), respectively. Images in (A2 and A6) are presented as single-channel fluorescence images in (A3-A4 and A7-A8), respectively.B Quantification of fluorescence intensity of CHL1 at the spinal lesion sites of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. n = 11 mice per group.


C , D Western blot analysis ( C ) and quantification ( D ) of CHL1 expression in spinal lesion areas of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from 3 independent experiments each including 2 mice per group .

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

C, D Western blot analysis (C) and quantification (D) of CHL1 expression in spinal lesion areas of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 2 mice per group.


E Co - immunoprecipitation of NB - 3 and CHL1 from spinal cord lesion areas of NB - 3 + / + and NB - 3 − / − mice .

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

E Co-immunoprecipitation of NB-3 and CHL1 from spinal cord lesion areas of NB-3+/+ and NB-3−/−mice.


F Co - immunostaining of CHL1 and NB - 3 in FG - labeled corticospinal neurons ( arrowheads ) of NB - 3 + / + and NB - 3 − / − mice 14 days post - injury ( dpi ) . G Quantification of the fluorescence intensity of CHL1 in ( F ) . * p < 0 . 05 , and * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

F Co-immunostaining of CHL1 and NB-3 in FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi).G Quantification of the fluorescence intensity of CHL1 in (F). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


H , I Western blot analysis ( H ) and quantification ( I ) of CHL1 expression in sensorimotor cortices of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . Data were analyzed from 3 independent experiments each including 3 mice per group .

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

H, I Western blot analysis (H) and quantification (I) of CHL1 expression in sensorimotor cortices of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.


J CHL1 expression in the somas and axons of NB - 3 + / + and NB - 3 − / − pyramidal neurons when co - culturing with NB - 3 - expressing astrocytes ( dashed lines and asterisks ) or astrocytes that didn ' t express NB - 3 ( dashed lines ) . Co - localization of CHL1 and NB - 3 in astrocytes overexpressed NB - 3 ( asterisks ) . Co - localization of CHL1 and NB - 3 in the somas and axons of co - cultured NB - 3 + / + pyramidal neurons ( arrowheads ) . Left inset , high - magnification view of the soma ( white arrowhead ) . Right inset , high - magnification view of the axon tip ( green arrowhead ) . K Quantification of fluorescence intensity of CHL1 in ( J ) . * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified .

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

J CHL1 expression in the somas and axons of NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3-expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Co-localization of CHL1 and NB-3 in astrocytes overexpressed NB-3 (asterisks). Co-localization of CHL1 and NB-3 in the somas and axons of co-cultured NB-3+/+ pyramidal neurons (arrowheads). Left inset, high-magnification view of the soma (white arrowhead). Right inset, high-magnification view of the axon tip (green arrowhead).K Quantification of fluorescence intensity of CHL1 in (J). *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified.


L - N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( L ) and LV - CHL1 shRNA - GFP ( M ) into both the sensorimotor cortex and lesion sites . High - magnification image ( N ) of the area in ( M ) ( asterisk ) . The arrows indicate the lesion sites ; the asterisk indicates the lesion epicenter . O Quantification of intensity index of BDA - labeled axons at certain distances from the lesion border in ( L and M ) . * p < 0 . 05 and * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 9 mice in ( L ) , and n = 10 mice in ( M ) .

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

L-N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (L) and LV-CHL1 shRNA-GFP (M) into both the sensorimotor cortex and lesion sites. High-magnification image (N) of the area in (M) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.O Quantification of intensity index of BDA-labeled axons at certain distances from the lesion border in (L and M). *p < 0.05 and **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 9 mice in (L), and n= 10 mice in (M).


A Co - immunostaining of PTPσ ( blue ) , NB - 3 ( green ) , and BDA ( red ) at spinal lesion sites of NB - 3 + / + ( A1 ) and NB - 3 − / − ( A6 ) mice . ( A2 - A5 and A7 - A10 ) High - magnification images of boxed areas in ( A1 and A6 ) showing PTPσ expression at the terminations of BDA - labeled corticospinal axons , respectively . Images in ( A2 and A7 ) are presented as single - channel fluorescence images in ( A3 - A5 and A8 - A10 ) , respectively . B Quantification of fluorescence intensity of PTPσ at BDA - labeled axonal terminations of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . n = 12 mice per group .

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

A Co-immunostaining of PTPσ (blue), NB-3 (green), and BDA (red) at spinal lesion sites of NB-3+/+ (A1) and NB-3−/− (A6) mice. (A2-A5 and A7-A10) High-magnification images of boxed areas in (A1 and A6) showing PTPσ expression at the terminations of BDA-labeled corticospinal axons, respectively. Images in (A2 and A7) are presented as single-channel fluorescence images in (A3-A5 and A8-A10), respectively.B Quantification of fluorescence intensity of PTPσ at BDA-labeled axonal terminations of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. n = 12 mice per group.


C Co - immunostaining of PTPσ and NB - 3 in the FG - labeled corticospinal neurons ( arrowheads ) of NB - 3 + / + and NB - 3 − / − mice 14 days post - injury ( dpi ) . Insets , only the signals from PTPσ and FG in NB - 3 + / + corticospinal neurons ( red arrowheads ) were visualized . D Quantification of fluorescence intensity of PTPσ in ( C ) . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

C Co-immunostaining of PTPσ and NB-3 in the FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi). Insets, only the signals from PTPσ and FG in NB-3+/+ corticospinal neurons (red arrowheads) were visualized.D Quantification of fluorescence intensity of PTPσ in (C). *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


E Co - immunoprecipitation of NB - 3 and PTPσ from sensorimotor cortex lysates of NB - 3 + / + mice after SCI . F Quantification of immunoprecipitated PTPσ by NB - 3 in ( E ) . * * p < 0 . 01 ; one sample t - test . Data were analyzed from 3 independent experiments each including 15 mice per group .

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

E Co-immunoprecipitation of NB-3 and PTPσ from sensorimotor cortex lysates of NB-3+/+mice after SCI.F Quantification of immunoprecipitated PTPσ by NB-3 in (E). **p < 0.01; one sample t-test. Data were analyzed from 3 independent experiments each including 15 mice per group.


G PTPσ expression in NB - 3 + / + and NB - 3 − / − pyramidal neurons when co - culturing with NB - 3 - expressing astrocytes ( dashed lines and asterisks ) or astrocytes that didn ' t express NB - 3 ( dashed lines ) . Upper inset , high - magnification view of the soma ( white arrowhead ) . Lower inset , high - magnification view of the axon tip ( green arrowhead ) . H Quantification of fluorescence intensity of PTPσ in ( G ) . n . s . , not significant , and * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test .

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

G PTPσ expression in NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3-expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Upper inset, high-magnification view of the soma (white arrowhead). Lower inset, high-magnification view of the axon tip (green arrowhead).H Quantification of fluorescence intensity of PTPσ in (G). n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test.


I Expression of PTPσ , p - Akt , p - mTOR , and p - S6 ( S240 / 244 ) in pyramidal neurons that expressed PTPσ shRNA - GFP . A control plasmid expressing GFP was transfected as the negative control ( nc ) . J Quantification of fluorescence intensity of PTPσ , p - Akt , p - mTOR , and p - S6 ( S240 / 244 ) in ( I ) . * p < 0 . 05 ; one sample t - test . The intensities of more than 300 pyramidal neurons from 4 independent experiments in each group in ( H ) and ( J ) were quantified , respectively .

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

I Expression of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in pyramidal neurons that expressed PTPσ shRNA-GFP. A control plasmid expressing GFP was transfected as the negative control (nc).J Quantification of fluorescence intensity of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in (I). *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 4 independent experiments in each group in (H) and (J) were quantified, respectively.


K - M Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( K ) and LV - PTPσ shRNA - GFP ( L ) into the sensorimotor cortex . High - magnification image ( M ) of the area in ( L ) ( asterisk ) . The arrows indicate the lesion sites ; the asterisk indicates the lesion epicenter . N Quantification of the intensity index of BDA - labeled axons at certain distances from the lesion border in ( K and L ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 8 mice in ( K ) , and n = 10 mice in ( L ) .

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

K-M Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (K) and LV-PTPσ shRNA-GFP (L) into the sensorimotor cortex. High-magnification image (M) of the area in (L) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.N Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (K and L). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n = 8 mice in (K), and n = 10 mice in (L).


( A ) Sirt5 deficiency leads to higher ROS levels in MEFs . ROS levels were determined in the indicated MEFs as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

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

(A) Sirt5 deficiency leads to higher ROS levels in MEFs. ROS levels were determined in the indicated MEFs as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( B ) Sirt5 deficiency suppresses GSH production in MEFs . The ratio of [ GSH / GSSG ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - tailed unpaired t - test ) .

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

(B) Sirt5 deficiency suppresses GSH production in MEFs. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).


( C ) Sirt5 deficiency inhibits NADPH production in MEFs . The ratio of [ NADPH / NADP + ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(C) Sirt5 deficiency inhibits NADPH production in MEFs. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).


( D ) Sirt5 deficiency decreases cell growth . MEF cells were seeded in 6 - well plates at a density of 50 , 000 cells / well , and cell growth rate was carefully monitored every 1 - 2 days by cell counting for 8 days . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(D) Sirt5 deficiency decreases cell growth. MEF cells were seeded in 6-well plates at a density of 50,000 cells/well, and cell growth rate was carefully monitored every 1-2 days by cell counting for 8 days. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).


( E ) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat . The levels of cleaved PARP and Caspase - 3 were determined by western blot analysis .

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

(E) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat. The levels of cleaved PARP and Caspase-3 were determined by western blot analysis.


( F ) Sirt5 protects MEFs from ROS - induced cell death . Wild - type and Sirt5 KO MEFs were treated with Paraquat ( 500 μM ) for 24 hrs , and then cell viability was determined by counting the remaining adherent cells . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

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

(F) Sirt5 protects MEFs from ROS-induced cell death. Wild-type and Sirt5 KO MEFs were treated with Paraquat (500 μM) for 24 hrs, and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( G - I ) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection . Female Sirt5 KO and WT littermates ( n = 3 ~ 5 per group ) were intraperitoneally injected with saline or paraquat ( 10 mg / kg ) once a week for 3 consecutive weeks . The ratios of [ NADPH / NADP + ] and [ GSH / GSSG ] and the level of lipid peroxide were determined in tissue extracts . The results are average ± SD . * p < 0 . 05 , n . s . : not significant ( two - tailed unpaired t - test ) .

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

(G-I) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection. Female Sirt5 KO and WT littermates (n=3~5 per group) were intraperitoneally injected with saline or paraquat (10 mg/kg) once a week for 3 consecutive weeks. The ratios of [NADPH/NADP+] and [GSH/GSSG] and the level of lipid peroxide were determined in tissue extracts. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( J - K ) Sirt5 KO mice are more sensitive to Paraquat - induced nigrostriatal dopaminergic degeneration . Female Sirt5 KO and WT littermates were treated with paraquat as in ( G - I ) . The brain sections were stained to detect tyrosine hydroxylase ( TH ) positive dopaminergic neurons in the SNc region , as described in ' Materials and Methods ' . Representative immunohistochemistry images ( original magnification , 100 × ; scale bar , 100 μm ) and the corresponding quantifications are shown in ( J ) and ( K ) , respectively . The results are average ± SD . * p < 0 . 05 , n . s . : not significant ( two - tailed unpaired t - test ) .

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

(J-K) Sirt5 KO mice are more sensitive to Paraquat-induced nigrostriatal dopaminergic degeneration. Female Sirt5 KO and WT littermates were treated with paraquat as in (G-I). The brain sections were stained to detect tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNc region, as described in 'Materials and Methods'. Representative immunohistochemistry images (original magnification, 100×; scale bar, 100 μm) and the corresponding quantifications are shown in (J) and (K), respectively. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( A ) Knockdown of SIRT5 inhibits NADPH production in HEK293T cells . The ratio of [ NADPH / NADP + ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(A)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( B ) Knockdown of SIRT5 suppresses GSH production in HEK293T cells . The ratio of [ GSH / GSSG ] was determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

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

(B) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( C ) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells . ROS levels were determined in the indicated stable cells as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).


( D ) Knockdown of SIRT5 leads to higher sensitivity to Paraquat . The expression levels of PARP and Caspase - 3 were determined by western blot analysis .

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

(D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis.


( E ) SIRT5 protects HEK293T cells from ROS - induced cell death . The indicated stable HEK293T cells were treated as mentioned above in ( D ) , and then cell viability was determined by counting the remaining adherent cells . The results are average ± SD of 3 independent experiments * * p < 0 . 01 ( two - tailed unpaired t - test ) .

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

(E) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).


( G ) The activities of G6PD / 6PGD and IDH1 / 2 are reduced in stable HEK293T cells with SIRT5 knockdown . Activities of the indicated endogenous proteins were determined in cell extracts as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . : not significant ( two - tailed unpaired t - test ) .

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

(G) The activities of G6PD/6PGD and IDH1/2 are reduced in stable HEK293T cells with SIRT5 knockdown. Activities of the indicated endogenous proteins were determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01, ***p<0.001, n.s.: not significant (two-tailed unpaired t-test).


( H ) G6PD and IDH2 are activated by SIRT5 in vitro . Upon pre - incubation with SIRT5 in vitro , the activities of the indicated ectopically expressed proteins were determined as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , n . s . : not significant ( two - tailed unpaired t - test ) .

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

(H) G6PD and IDH2 are activated by SIRT5 in vitro. Upon pre-incubation with SIRT5 in vitro, the activities of the indicated ectopically expressed proteins were determined as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, n.s.: not significant (two-tailed unpaired t-test).


( A ) G6PD activity is not regulated by lysine succinylation . Purified Flag - tagged G6PD was incubated with or without succinyl - CoA ( 1 mM ) at 30 ° C for 15 mins , followed by measurement of G6PD enzyme activity as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments . n . s . : not significant ( two - tailed unpaired t - test ) .

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

(A) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( B ) IDH2 activity is suppressed by lysine succinylation . Purified Flag - tagged IDH2 was incubated with or without succinyl - CoA ( 1 mM ) at 30 ° C for 15 mins , followed by measurement of IDH2 enzyme activity as described in ' Materials and Methods ' . The results are average ± SD of 3 independent experiments * p < 0 . 05 ( two - tailed unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).


( C ) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation , respectively , and decreases both enzyme activity . Flag - G6PD or Flag - IDH2 was ectopically expressed in the indicated stable HEK293T cells . G6PD / IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide , followed by determination of their lysine modifications and enzyme activity . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(C) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation, respectively, and decreases both enzyme activity. Flag-G6PD or Flag-IDH2 was ectopically expressed in the indicated stable HEK293T cells. G6PD/IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


( D ) SIRT5 decreases G6PD glutarylation and IDH2 succinylation , and increases both enzyme activity . Flag - G6PD or Flag - IDH2 was co - overexpressed with HA - SIRT5 in HEK293T cells . G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide , followed by determination of their lysine modifications and enzyme activity . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * p < 0 . 01 ( two - tailed unpaired t - test ) .

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

(D) SIRT5 decreases G6PD glutarylation and IDH2 succinylation, and increases both enzyme activity. Flag-G6PD or Flag-IDH2 was co-overexpressed with HA-SIRT5 in HEK293T cells. G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01 (two-tailed unpaired t-test).


( E ) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket ( PDB ID : 1LWD ) [ 33 ] , made by Pymol ( www . pymol . org ) . The protein is colored in light green and isocitrate in rainbow , with Lys 360 and Lys 413 residues in IDH2 being colored in red .

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

(E) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket (PDB ID: 1LWD) [33], made by Pymol (www.pymol.org). The protein is colored in light green and isocitrate in rainbow, with Lys 360 and Lys 413 residues in IDH2 being colored in red.


( F - G ) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5 . The indicated Flag - tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co - overexpression . Wild - type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide , followed by measurement of IDH2 enzyme activity . The results are average ± SD of 3 independent experiments * p < 0 . 05 , n . s . : not significant ( two - tailed unpaired t - test ) .

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

(F-G) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5. The indicated Flag-tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co-overexpression. Wild-type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide, followed by measurement of IDH2 enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, n.s.: not significant (two-tailed unpaired t-test).


( A - B ) SIRT5 expression is not affected by chemical oxidant treatment . HEK293T cells were treated with Paraquat ( A ) or H2O2 ( B ) for the indicated periods . The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real - time PCR and western blot analysis , respectively . The results are average ± SD of 3 independent experiments . n . s . : not significant ( two - tailed unpaired t - test ) .

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

(A-B) SIRT5 expression is not affected by chemical oxidant treatment. HEK293T cells were treated with Paraquat (A) or H2O2 (B) for the indicated periods. The mRNA and protein expression of endogenous SIRT5 were determined by quantitative real-time PCR and western blot analysis, respectively. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( D ) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli . Flag - SIRT5 was ectopically expressed in HEK293T cells . The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods . Flag - SIRT5 was purified by immunoprecipitation with Flag beads , eluted with Flag peptide , and then subjected to the desuccinylase activity assay as described in ' Materials and Methods ' . The SIRT5 desuccinylase activity was normalized against its protein level . The results are average ± SD of 3 independent experiments * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(D) The desuccinylase activity of SIRT5 is stimulated by oxidative stimuli. Flag-SIRT5 was ectopically expressed in HEK293T cells. The transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).


( E ) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5 . Flag - tagged SIRT5 was ectopically expressed in HEK293T cells , and then purified by immunoprecipitation with Flag - beads and eluted with Flag peptide . The purified Flag - SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr , and then subjected to the desuccinylase activity assay as described in ' Materials and Methods ' . The SIRT5 desuccinylase activity was normalized against its protein level . The results are average ± SD of 3 independent experiments . n . s . : not significant ( two - tailed unpaired t - test ) .

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

(E) In vitro incubation with chemical oxidants does not affect the desuccinylase activity of SIRT5. Flag-tagged SIRT5 was ectopically expressed in HEK293T cells, and then purified by immunoprecipitation with Flag-beads and eluted with Flag peptide. The purified Flag-SIRT5 was treated with increased concentrations of Paraquat or H2O2 at room temperature for 1 hr, and then subjected to the desuccinylase activity assay as described in 'Materials and Methods'. The SIRT5 desuccinylase activity was normalized against its protein level. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).


( F ) Chemical oxidants enhance the ability of SIRT5 to activate IDH2 . Flag - SIRT5 was ectopically expressed in HEK293T cells , and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods . Flag - SIRT5 was purified by immunoprecipitation with Flag beads , eluted with Flag peptide , and then incubated with purified Flag - IDH2 in vitro . IDH2 enzyme activity was measured as described in ' Materials and Methods ' , normalized by its protein level . The results are average ± SD of 3 independent experiments * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed unpaired t - test ) .

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

(F) Chemical oxidants enhance the ability of SIRT5 to activate IDH2. Flag-SIRT5 was ectopically expressed in HEK293T cells, and the transfected cells were treated with increased concentrations of Paraquat or H2O2 for the indicated periods. Flag-SIRT5 was purified by immunoprecipitation with Flag beads, eluted with Flag peptide, and then incubated with purified Flag-IDH2 in vitro. IDH2 enzyme activity was measured as described in 'Materials and Methods', normalized by its protein level. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).


C - E KRAS and TP53 alterations in the cancer genome atlas ( TCGA ) pan - cancer MPM dataset ( n = 86 patients ) . Shown are clinical and molecular data plot with alteration frequencies ( C ) and patients reclassified as KRAS - or TP53 - altered ( asterisks ) , copy number variation data summary ( D ) , and segments of the KRAS and TP53 loci ( E ) .

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

C-E KRAS and TP53 alterations in the cancer genome atlas (TCGA) pan-cancer MPM dataset (n = 86 patients). Shown are clinical and molecular data plot with alteration frequencies (C) and patients reclassified as KRAS- or TP53-altered (asterisks), copy number variation data summary (D), and segments of the KRAS and TP53 loci (E).


Molecular and clinical features of the cancer genome atlas ( TCGA ) pan - cancer MPM patients ( n = 87 ) stratified by the presence of KRAS standalone ( n = 10 ) and combined KRAS / TP53 ( n = 7 ) alterations . Shown are unsupervised hierarchical clustering of n = 86 patients ( gene expression data were not available for one patient ) by 40 genes significantly overexpressed in KRAS / TP53 - altered over KRAS - altered over KRAS / TP53 - normal patients ( A ) and data summaries of mononucleotide change signatures ( B ) , of indices of genomic instability and mutation burden ( C ) , of clinical

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

Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients (A) and data summaries of mononucleotide change signatures (B), of indices of genomic instability and mutation burden (C), of clinical features


Molecular and clinical features of the cancer genome atlas ( TCGA ) pan - cancer MPM patients ( n = 87 ) stratified by the presence of KRAS standalone ( n = 10 ) and combined KRAS / TP53 ( n = 7 ) alterations . Shown are unsupervised hierarchical clustering of n = 86 patients ( gene expression data were not available for one patient ) by 40 genes significantly overexpressed in KRAS / TP53 - altered over KRAS - altered over KRAS / TP53 - normal patients overall survival ( F ) .

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

Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients overall survival (F).


H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from ( A - G ) .

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H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from (A-G).


Pleural fluid cell pellets and supernatants from 10 patients ( called CRCINA # ) with pleural effusion and pleural tumor samples from 17 patients ( called TR # ) with MPM were subjected to digital droplet polymerase chain reaction ( ddPCR ) for the detection of mutant ( MUT ) copies of KRAS codon 12 / 13 ( KRASG12 / 13 ) and KRAS codon 61 ( KRASQ61 ) , as well as copies of TP53 and TERT . Diagnoses were lung adenocarcinoma ( LUAD ; n = 4 ) and MPM ( n = 23 ) . The assays were designed for detection of down to 1 : 20000 copies using EKVX ( KRASWTTP53G610T ) , A549 ( KRASG12STP53WT ) , NCI - H460 ( KRASQ61HTP53WT ) , NCI - H3122 ( KRASWTTP53E285V ) , and NCI - H3255 ( KRASWTTP53G560 - 1A ) human LUAD cells as controls . Shown are individual patient ( KRAS plot ) and individual sample ( TP53 plot ) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot ( A ) Any number of KRAS - mutant droplets detected in any sample ( KRAS plot in A ) and any patient that failed to achieve normal TP53 ploidy by any sample ( TP53 plot in A ) was deemed altered .

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Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion and pleural tumor samples from 17 patients (called TR#) with MPM were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A) Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.


F Representative May - Gruenwald - Giemsa - stained pleural fluid cytocentrifugal specimen from a KRASG12D ; Trp53f / f mouse showing macrophages ( MΦ , black arrow ) , lymphocytes ( LΦ , purple arrow ) , and neutrophils ( NΦ , green arrow ) and summary of cellular and biochemical features of effusions of KRASG12D ; Trp53f / f mice ( n = 10 ) .

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F Representative May-Gruenwald-Giemsa-stained pleural fluid cytocentrifugal specimen from a KRASG12D;Trp53f/f mouse showing macrophages (MΦ, black arrow), lymphocytes (LΦ, purple arrow), and neutrophils (NΦ, green arrow) and summary of cellular and biochemical features of effusions of KRASG12D;Trp53f/f mice (n = 10).


G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [ n is given in table in ( E ) ] .

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G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [n is given in table in (E)].


Wild - type ( Wt ) , KRASG12D , and Trp53f / f mice were intercrossed , and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad - Cre and were sacrificed when moribund . In parallel , C57BL / 6 mice received ten consecutive weekly intraperitoneal injections of 1 g / Kg urethane and were sacrificed after six months . Data summary ( heatmap ) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma ( MPM ) and lung adenocarcinoma ( LUAD ) . n = 10 mice / group were analyzed for each marker . Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining . Note the ubiquitous strong expression of Wilms ' tumor 1 ( WT1 ) , patchy moderate expression of vimentin ( VIM ) , ubiquitous moderate expression of mesothelin ( MSLN ) , ubiquitous strong expression of calretinin ( CALB2 ) , podoplanin ( PDPN ) , and osteopontin ( SPP1 ) , patchy moderate expression of cytokeratin 5 / 6 ( CK5 / 6 ) , and the absence of expression of surfactant protein C ( SFTPC ) in murine KRAS - driven mesotheliomas . Note also the ubiquitous strong expression of WT1 , the patchy moderate expression of VIM , the ubiquitous low - level expression of MSLN , the ubiquitous strong expression of CALB2 and SPP1 , the ubiquitous low - level expression of PDPN , the variable moderate expression of CK5 / 6 , and the ubiquitous moderate expression of SFTPC in murine KRASG12D - driven and urethane - induced LUAD .

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Wild-type (Wt), KRASG12D, and Trp53f/f mice were intercrossed, and all possible offspring genotypes received 5 x 108 PFU intrapleural or intratracheal Ad-Cre and were sacrificed when moribund. In parallel, C57BL/6 mice received ten consecutive weekly intraperitoneal injections of 1 g/Kg urethane and were sacrificed after six months. Data summary (heatmap) and representative images of immunoreactivity of tissue sections of pleural and pulmonary tissues and tumors from these mice for different markers of human malignant pleural mesothelioma (MPM) and lung adenocarcinoma (LUAD). n = 10 mice/group were analyzed for each marker. Brown color indicates immunoreactivity and blue color nuclear hematoxylin counterstaining. Note the ubiquitous strong expression of Wilms' tumor 1 (WT1), patchy moderate expression of vimentin (VIM), ubiquitous moderate expression of mesothelin (MSLN), ubiquitous strong expression of calretinin (CALB2), podoplanin (PDPN), and osteopontin (SPP1), patchy moderate expression of cytokeratin 5/6 (CK5/6), and the absence of expression of surfactant protein C (SFTPC) in murine KRAS-driven mesotheliomas. Note also the ubiquitous strong expression of WT1, the patchy moderate expression of VIM, the ubiquitous low-level expression of MSLN, the ubiquitous strong expression of CALB2 and SPP1, the ubiquitous low-level expression of PDPN, the variable moderate expression of CK5/6, and the ubiquitous moderate expression of SFTPC in murine KRASG12D-driven and urethane-induced LUAD.


C Data summaries from in vitro MTT reduction ( top ; 2 x 104 cells / well ; n = 6 independent experiments ) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL / 6 mouse ( bottom ; n = 5 / group ) .

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C Data summaries from in vitro MTT reduction (top; 2 x 104 cells/well; n = 6 independent experiments) and in vivo subcutaneous tumor growth after injection of 106 cells per C57BL/6 mouse (bottom; n = 5/group).


E , F RT - PCR ( E ) and qPCR ( F ) of KPM cells and parental tumors show Trp53f / f allele deletion ( Δ ) and Bap1 and Cdkn2a overexpression compared with PMC .

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E, F RT-PCR (E) and qPCR (F) of KPM cells and parental tumors show Trp53f/f allele deletion (Δ) and Bap1 and Cdkn2a overexpression compared with PMC.


A Kaplan - Meier survival plot with survival table .

A Kaplan-Meier survival plot with survival table.


F , G C57BL / 6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1 % DMSO or 15 mg / Kg deltarasin in 1 % DMSO at day 9 post - tumor cells . Shown are data summaries of MPE volume ( n = 8 and 7 DMSO and deltarasin - treated mice / group , respectively ) and pleural fluid nucleated cells at day 19 post - KPM1 cells ( F ) , as well as representative images of pleural effusions ( dashed lines ) and tumors ( t in ( G ) ) .

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F, G C57BL/6 mice received pleural KPM1 cells followed by a single intrapleural injection of liposomes containing 1% DMSO or 15 mg/Kg deltarasin in 1% DMSO at day 9 post-tumor cells. Shown are data summaries of MPE volume (n = 8 and 7 DMSO and deltarasin-treated mice/group, respectively) and pleural fluid nucleated cells at day 19 post-KPM1 cells (F), as well as representative images of pleural effusions (dashed lines) and tumors (t in (G)).


B Volcano plot showing some top KPM versus PMC differentially expressed genes .

B Volcano plot showing some top KPM versus PMC differentially expressed genes.


C KPM and PMC expression of classic mesothelioma markers ( top ) and top KPM versus PMC overexpressed genes ( bottom ) .

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C KPM and PMC expression of classic mesothelioma markers (top) and top KPM versus PMC overexpressed genes (bottom).


( A ) RT - qPCR measurement of intracellular level of the SARS - CoV - 2 RNA in Huh7 cells after 72 hours and 96 hours of infection . Data information : In A values were normalized to that of the GAPDH and represented as mean ± SD of samples from five independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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(A) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA in Huh7 cells after 72 hours and 96 hours of infection. Data information: In A values were normalized to that of the GAPDH and represented as mean±SD of samples from five independent experiments. Student's t-test was applied to determine the significance between the groups.


( B ) Western blot analysis of the SARS - CoV - 2 N protein ( upper panel ) and GAPDH ( lower panel ) levels in Huh7 cells , as mentioned in ( A ) .

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(B) Western blot analysis of the SARS-CoV-2 N protein (upper panel) and GAPDH (lower panel) levels in Huh7 cells, as mentioned in (A).


( C - J ) RT - qPCR analysis of the intracellular mRNA levels of HERV - T ( C ) , HERV - E ( D ) , HERV - R ( E ) , HERV - V ( F ) , HERV - FRD ( G ) , HERV - MER34 ( H ) , HERV - W ( I ) and HERV - K - HML - 2 ( J ) in the SARS - CoV - 2 infected Huh7 cells , as mentioned in ( A ) . Data information : In C - J , values were normalized to that of the GAPDH and represented as mean ± SD of samples from five independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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(C-J) RT-qPCR analysis of the intracellular mRNA levels of HERV-T (C), HERV-E (D), HERV-R (E), HERV-V (F), HERV-FRD (G), HERV-MER34 (H), HERV-W (I) and HERV-K-HML-2 (J) in the SARS-CoV-2 infected Huh7 cells, as mentioned in (A). Data information: In C-J, values were normalized to that of the GAPDH and represented as mean±SD of samples from five independent experiments. Student's t-test was applied to determine the significance between the groups.


( A ) RT - qPCR measurement of intracellular level of the SARS - CoV - 2 RNA ( normalized to that of the GAPDH ) in Calu 3 cells after 48 hours and 72 hours of infection . Data information : In A values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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(A) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA (normalized to that of the GAPDH) in Calu 3 cells after 48 hours and 72 hours of infection. Data information: In A values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


( B ) Western blot analysis of the SARS - CoV - 2 N protein ( upper panel ) and GAPDH ( lower panel ) levels in Calu 3 cells , as mentioned in ( A ) .

(B) Western blot analysis of the SARS-CoV-2 N protein (upper panel) and GAPDH (lower panel) levels in Calu 3 cells, as mentioned in (A).


( C - J ) RT - qPCR analysis of the intracellular mRNA levels of HERV - T ( C ) , HERV - E ( D ) , HERV - R ( E ) , HERV - V ( F ) , HERV - FRD ( G ) , HERV - MER34 ( H ) , HERV - W ( I ) and HERV - K - HML - 2 ( J ) in the SARS - CoV - 2 infected Calu 3 cells , as mentioned in ( A ) . Data information : In C - J , values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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(C-J) RT-qPCR analysis of the intracellular mRNA levels of HERV-T (C), HERV-E (D), HERV-R (E), HERV-V (F), HERV-FRD (G), HERV-MER34 (H), HERV-W (I) and HERV-K-HML-2 (J) in the SARS-CoV-2 infected Calu 3 cells, as mentioned in (A). Data information: In C-J, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


( B ) RT - qPCR measurement of exon 1 region [ shown in ( A ) ] transcript in Huh7 cells infected with SARS - CoV - 2 for 72hours . ( C ) RT - qPCR measurement of exon 1 region transcript in Calu 3 cells infected with SARS - CoV - 2 for 72hours . Data information : In B and C , values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments .

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(B) RT-qPCR measurement of exon 1 region [shown in (A)] transcript in Huh7 cells infected with SARS-CoV-2 for 72hours. (C) RT-qPCR measurement of exon 1 region transcript in Calu 3 cells infected with SARS-CoV-2 for 72hours. Data information: In B and C, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments.


( E ) RT - qPCR measurement of HERV - R - Env transcript level in PBMCs isolated from 19 healthy individuals and 16 COVID - 19 patients . ( F ) RT - qPCR measurement of Exon1 region transcript level in PBMCs isolated from 19 Healthy individuals and 16 COVID - 19 patients . Data information : values were normalized to that of the GAPDH and represented as mean ± SD of samples from 19 healthy and 16 COVID - 19 patients , assayed in duplicate .

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(E) RT-qPCR measurement of HERV-R-Env transcript level in PBMCs isolated from 19 healthy individuals and 16 COVID-19 patients. (F) RT-qPCR measurement of Exon1 region transcript level in PBMCs isolated from 19 Healthy individuals and 16 COVID-19 patients. Data information: values were normalized to that of the GAPDH and represented as mean±SD of samples from 19 healthy and 16 COVID-19 patients, assayed in duplicate.


( G ) Quantitation of HERV - R - Env RNA level in transcriptome dataset of indicated human lung tissues Data information : In G , HERV - R - Env copy number in two biological replicates ( Healthy lung ) and two technical replicates ( COVID - 19 lung ) of the transcriptome dataset are represented as mean ± SD .

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(G) Quantitation of HERV-R-Env RNA level in transcriptome dataset of indicated human lung tissues Data information: In G, HERV-R-Env copy number in two biological replicates (Healthy lung) and two technical replicates (COVID-19 lung) of the transcriptome dataset are represented as mean±SD.


( H ) Quantitation of HERV - R - Env RNA level in transcriptome dataset of Calu 3 cells , analyzed at 4 , 8 , 12 and 24 hours of SARS - CoV - 2 infection , as indicated . Data information : In H , HERV - R - Env copy number in three biological replicates of the transcriptome dataset are represented as mean ± SD . Student ' s t - test was applied to determine the significance between the groups .

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(H) Quantitation of HERV-R-Env RNA level in transcriptome dataset of Calu 3 cells, analyzed at 4, 8, 12 and 24 hours of SARS-CoV-2 infection, as indicated. Data information: In H, HERV-R-Env copy number in three biological replicates of the transcriptome dataset are represented as mean±SD. Student's t-test was applied to determine the significance between the groups.


( A ) RT - qPCR measurement of intracellular level of the SARS - CoV - 2 delta variant RNA in Huh7 and Calu 3 cells after 24 , 48 and 72 hours of infection . ( B ) RT - qPCR measurement of intracellular level of the SARS - CoV - 2 omicron variant RNA in Huh7 and Calu 3 cells after 24 , 48 and 72 hours of infection . Data information : values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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

(A) RT-qPCR measurement of intracellular level of the SARS-CoV-2 delta variant RNA in Huh7 and Calu 3 cells after 24, 48 and 72 hours of infection. (B) RT-qPCR measurement of intracellular level of the SARS-CoV-2 omicron variant RNA in Huh7 and Calu 3 cells after 24, 48 and 72 hours of infection. Data information: values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


( C ) RT - qPCR analysis of the HERV - R - Env RNA level in the SARS - CoV - 2 delta variant infected Huh7 and Calu 3 cells , as mentioned in ( A ) . ( D ) RT - qPCR analysis of the HERV - R - Env RNA level in the SARS - CoV - 2 omicron variant infected Huh7 and Calu 3 cells , as mentioned in ( B ) . Data information : values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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(C) RT-qPCR analysis of the HERV-R-Env RNA level in the SARS-CoV-2 delta variant infected Huh7 and Calu 3 cells, as mentioned in (A). (D) RT-qPCR analysis of the HERV-R-Env RNA level in the SARS-CoV-2 omicron variant infected Huh7 and Calu 3 cells, as mentioned in (B). Data information: values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


( B ) Western blot analysis of the SARS - CoV - 2 N protein ( upper panel ) , HERV - R - Env protein ( middle panel ) and GAPDH ( lower panel ) levels in Huh7 cells , transiently transfected with HERV - R expression plasmid and infected with the SARS - CoV - 2 for 24 and 48 hrs .

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(B) Western blot analysis of the SARS-CoV-2 N protein (upper panel), HERV-R-Env protein (middle panel) and GAPDH (lower panel) levels in Huh7 cells, transiently transfected with HERV-R expression plasmid and infected with the SARS-CoV-2 for 24 and 48 hrs.


( C ) Densitometry quantification of N - protein band intensities ( normalized to that of GAPDH ) ) from three independent blots , as shown in Fig ( B ) . Values are mean ± SD of 3 independent experiments . Data information : In values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments .

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(C) Densitometry quantification of N-protein band intensities (normalized to that of GAPDH) ) from three independent blots, as shown in Fig (B). Values are mean±SD of 3 independent experiments. Data information: In values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments.


( F ) RT - qPCR measurement of intracellular level of the SARS - CoV - 2 RNA ( normalized to that of the GAPDH ) in Huh7 cells transfected with NT - siRNA ( Non target siRNA ) or HERV - R - Env siRNA and infected with the SARS - CoV - 2 for 24 , 48 or 72 hrs , as indicated . Values are mean ± SD of 3 independent experiments . Data information : In F , values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments .

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(F) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA (normalized to that of the GAPDH) in Huh7 cells transfected with NT-siRNA (Non target siRNA) or HERV-R-Env siRNA and infected with the SARS-CoV-2 for 24, 48 or 72 hrs, as indicated. Values are mean±SD of 3 independent experiments. Data information: In F, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments.


( G ) Dual luciferase assay showing activation of IFNγ , ISRE , ISG56 and NFκB promoters in HEK 293T cells expressing the empty vector ( V ) , constitutively active mutant of RIG - I , R - C , 25 and 50 ng of HERV - R - Env . Activation of these promoters by R - C was taken as 100 % . Data information : In G , FF Luc values were normalized to that of the RL Luc and represented as % mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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(G) Dual luciferase assay showing activation of IFNγ, ISRE, ISG56 and NFκB promoters in HEK 293T cells expressing the empty vector (V), constitutively active mutant of RIG-I, R-C, 25 and 50 ng of HERV-R-Env. Activation of these promoters by R-C was taken as 100%. Data information: In G, FF Luc values were normalized to that of the RL Luc and represented as % mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


( H ) Western blot analysis of the indicated proteins in Huh7 cells , transiently transfected with HERV - R - Env expression plasmid .

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(H) Western blot analysis of the indicated proteins in Huh7 cells, transiently transfected with HERV-R-Env expression plasmid.


( A ) Western blot analysis of the indicated proteins in Huh7 cells , transiently transfected with HERV - R expression plasmid and infected with the SARS - CoV - 2 . ( B ) Densitometry quantification of P - ERK and N - protein band intensities ( normalized to that of total - ERK and GAPDH , respectively ) from three independent blots , representative image shown in panel ( A ) . Data information : In B , P - ERK and N - protein band intensities were normalized to that of total - ERK and GAPDH , respectively [ as shown in panel ( A ) ] and represented as mean ± SD of three independent blots . Student ' s t - test was applied to determine the significance between the groups .

(A) Western blot analysis of the indicated proteins in Huh7 cells, transiently transfected with HERV-R expression plasmid and infected with the SARS-CoV-2. (B) Densitometry quantification of P-ERK and N-protein band intensities (normalized to that of total-ERK and GAPDH, respectively) from three independent blots, representative image shown in panel (A). Data information: In B, P-ERK and N-protein band intensities were normalized to that of total-ERK and GAPDH, respectively [as shown in panel (A)] and represented as mean±SD of three independent blots. Student's t-test was applied to determine the significance between the groups.


( C ) Western blot analysis of the indicated proteins in Huh7 cells , transiently transfected with HERV - R expression plasmid , infected with the SARS - CoV - 2 and treated with U0126 . ( D ) Densitometry quantification of P - ERK and N - protein band intensities ( normalized to that of total - ERK and GAPDH , respectively ) from two independent blots , representative image shown in panel ( C ) . Data information : In D , P - ERK and N - protein band intensities were normalized to that of GAPDH [ as shown in panel ( C ) ] and represented as mean of two independent blots . Individual values are shown as circles .

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

(C) Western blot analysis of the indicated proteins in Huh7 cells, transiently transfected with HERV-R expression plasmid, infected with the SARS-CoV-2 and treated with U0126. (D) Densitometry quantification of P-ERK and N-protein band intensities (normalized to that of total-ERK and GAPDH, respectively) from two independent blots, representative image shown in panel (C). Data information: In D, P-ERK and N-protein band intensities were normalized to that of GAPDH [as shown in panel (C)] and represented as mean of two independent blots. Individual values are shown as circles.


( B ) HERV - R 5 ' - LTR activity in Huh7 cells cotransfected with the pGL3 Fl 5 ' - LTR G - Luc and pGL3 promoter Firefly - Luc ( denoted as Fl 5 ' - LTR G - Luc ) or pGL3 G - Luc and pGL3 promoter Firefly - Luc ( denoted as G - Luc ) plasmids for 48 hours . ( C ) HERV - R 5 ' - LTR activity in Huh7 cells cotransfected with the pGL3 Fl 5 ' - LTR G - Luc and pGL3 promoter Firefly - Luc ( denoted as Fl 5 ' - LTR G - Luc ) or pGL3 ∆ 5 ' - LTR G - Luc and pGL3 promoter Firefly - Luc ( denoted as ∆ 5 ' - LTR G - Luc ) or pGL3 G - Luc and pGL3 promoter Firefly - Luc ( denoted as G - Luc ) plasmids for 48 hours . Data information : G - Luc values were normalized to that of the Firefly - Luc and represented as mean ± SD of samples from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HERV-R 5'-LTR activity in Huh7 cells cotransfected with the pGL3 Fl 5'-LTR G-Luc and pGL3 promoter Firefly-Luc (denoted as Fl 5'-LTR G-Luc) or pGL3 G-Luc and pGL3 promoter Firefly-Luc (denoted as G-Luc) plasmids for 48 hours. (C) HERV-R 5'-LTR activity in Huh7 cells cotransfected with the pGL3 Fl 5'-LTR G-Luc and pGL3 promoter Firefly-Luc (denoted as Fl 5'-LTR G-Luc) or pGL3 ∆ 5'-LTR G-Luc and pGL3 promoter Firefly-Luc (denoted as ∆ 5'-LTR G-Luc) or pGL3 G-Luc and pGL3 promoter Firefly-Luc (denoted as G-Luc) plasmids for 48 hours. Data information: G-Luc values were normalized to that of the Firefly-Luc and represented as mean±SD of samples from three independent experiments.


( I ) RT - qPCR measurement of HERV - R - Env RNA in Huh7 cells infected with the SARS - CoV - 2 and treated with the indicated compounds for 48 hours . Data information : In I values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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

(I) RT-qPCR measurement of HERV-R-Env RNA in Huh7 cells infected with the SARS-CoV-2 and treated with the indicated compounds for 48 hours. Data information: In I values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


( J ) Western blot analysis of the HERV - R - Env ( upper panel ) , SARS - CoV - 2 N ( middle panel ) and GAPDH ( lower panel ) protein levels in Huh7 cells infected with the SARS - CoV - 2 and treated with the indicated compounds for 48 hours .

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

(J) Western blot analysis of the HERV-R-Env (upper panel), SARS-CoV-2 N (middle panel) and GAPDH (lower panel) protein levels in Huh7 cells infected with the SARS-CoV-2 and treated with the indicated compounds for 48 hours.


( K ) RT - qPCR measurement of intracellular level of the SARS - CoV - 2 RNA in Huh7 cells infected with the SARS - CoV - 2 and treated with the indicated compounds for 48 hours . Data information : In K , values were normalized to that of the GAPDH and represented as mean ± SD of samples from three independent experiments . Student ' s t - test was applied to determine the significance between the groups .

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

(K) RT-qPCR measurement of intracellular level of the SARS-CoV-2 RNA in Huh7 cells infected with the SARS-CoV-2 and treated with the indicated compounds for 48 hours. Data information: In K, values were normalized to that of the GAPDH and represented as mean±SD of samples from three independent experiments. Student's t-test was applied to determine the significance between the groups.


B . Representative traces for C - H , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and shRNA - expressing neurons ( green traces ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for C-H, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and shRNA-expressing neurons (green traces).


C , D . Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at − 60 mV ( AMPAR - mediated EPSCs ; C ) or at + 40 mV ( NMDAR - mediated EPSCs ; D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . E . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices expressing shRNA against GSK3α at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and GSK3α shRNA lentivirus infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at − 60 mV ( AMPAR - mediated EPSCs ; C ) or at + 40 mV ( NMDAR - mediated EPSCs ; D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from uninfected and infected slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . F , G . As C , D , for GSK3β . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . H . As E , for GSK3β . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

Scatter plots showing individual recordings of pairs of uninfected and infected neurons in slices at −60 mV (AMPAR-mediated EPSCs; C) or at +40 mV (NMDAR-mediated EPSCs; D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from uninfected and infected slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. F, G. As C, D, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. H. As E, for GSK3β. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C , where baseline responses ( dark trace ) are taken between 10 and 0 minutes before LTD induction , and responses after LTD induction ( light trace ) are taken between 40 and 45 minutes after the end of LTD induction .

B. Representative traces for C, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.


C . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of LTD by LFS ( 300 pulses at 1 Hz ) , in control ( uninfected ) and GSK3α ( blue ) or GSK3β ( orange ) shRNA - expressing neurons . Error bars represent SEM . D . Quantification of average synaptic depression from the last 5 minutes of the time course shown in C . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . E . Cumulative frequency distribution of the ESPC values plotted in D . Statistical significance was calculated according to the Kolmogorov - Smirnov test .

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

C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (uninfected) and GSK3α (blue) or GSK3β (orange) shRNA-expressing neurons. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. E. Cumulative frequency distribution of the ESPC values plotted in D. Statistical significance was calculated according to the Kolmogorov-Smirnov test.


A . Representative traces for B and C , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in neurons from inactive control - , GSK3α or GSK3β inhibitor - treated slices ( all at 10 µM ) . Slices were incubated with each compound for 1 hour before recording , and during whole - cell recording itself . Compounds were also included in the internal solution of the recording pipette ( all at 10 µM ) .

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

A. Representative traces for B and C, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from inactive control-, GSK3α or GSK3β inhibitor-treated slices (all at 10 µM). Slices were incubated with each compound for 1 hour before recording, and during whole-cell recording itself. Compounds were also included in the internal solution of the recording pipette (all at 10 µM).


B . Quantification of individual ( open circles ) and average ( grey bars ) AMPA / NMDA ratios of neurons from inactive control , GSK3α or GSK3β inhibitor - treated slices , where AMPAR - mediated current is taken as the peak response at − 60 mV , and NMDAR - mediated current is measured at + 40 mV at a latency of 60 ms following stimulation . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test . C . Cumulative frequency distribution of the AMPA / NMDA ratios plotted in B . Statistical significance was calculated using the Kolmogorov - Smirnov test .

B. Quantification of individual (open circles) and average (grey bars) AMPA/NMDA ratios of neurons from inactive control, GSK3α or GSK3β inhibitor-treated slices, where AMPAR-mediated current is taken as the peak response at −60 mV, and NMDAR-mediated current is measured at +40 mV at a latency of 60 ms following stimulation. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test. C. Cumulative frequency distribution of the AMPA/NMDA ratios plotted in B. Statistical significance was calculated using the Kolmogorov-Smirnov test.


D . Representative traces for E and F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in neurons from hippocampal slices treated with DMSO ( vehicle control ) , AR - 18 ( 10 µM ) , CHIR ( 1 µM ) or SC79 ( 20 µM ) . E , F . As B , C , for DMSO , AR - 18 , CHIR and SC79 . Error bars represent SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

D. Representative traces for E and F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in neurons from hippocampal slices treated with DMSO (vehicle control), AR-18 (10 µM), CHIR (1 µM) or SC79 (20 µM). E, F. As B, C, for DMSO, AR-18, CHIR and SC79. Error bars represent SEM. Statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C , D , where baseline responses ( dark trace ) are taken between 10 and 0 minutes before LTD induction , and responses after LTD induction ( light trace ) are taken between 40 and 45 minutes after the end of LTD induction .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for C, D, where baseline responses (dark trace) are taken between 10 and 0 minutes before LTD induction, and responses after LTD induction (light trace) are taken between 40 and 45 minutes after the end of LTD induction.


C . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of LTD by LFS ( 300 pulses at 1 Hz ) , in control ( inactive control ) , GSK3α ( blue ) and GSK3β ( orange ) inhibitor , and AR - 18 - treated neurons ( yellow ) . Drug treatment was for 1 hour before recording at 10 µM for all compounds . Error bars represent SEM . D . Quantification of average synaptic depression from the last 5 minutes of the time course shown in C . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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

C. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of LTD by LFS (300 pulses at 1 Hz), in control (inactive control), GSK3α (blue) and GSK3β (orange) inhibitor, and AR-18-treated neurons (yellow). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Error bars represent SEM. D. Quantification of average synaptic depression from the last 5 minutes of the time course shown in C. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


E . As B , for F and G . F . Time course of the peak AMPAR - mediated synaptic response to Schaffer collateral stimulation before , during , and after the induction of mGluR - dependent LTD by PP - LFS ( 900 paired pulses , separated by 50 ms , at 1 Hz ) , in control ( inactive control ) and GSK3α inhibitor - treated neurons ( blue ) . Drug treatment was for 1 hour before recording at 10 µM for all compounds . Representative traces for synaptic responses before ( dark trace ) and after ( light trace ) LTD induction are shown above the time course . Error bars represent SEM . G . Quantification of average synaptic depression from the last 5 minutes of the time course shown in F . Error bars represent SEM . No statistically significant difference was found , according to the Mann - Whitney U test .

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

E. As B, for F and G. F. Time course of the peak AMPAR-mediated synaptic response to Schaffer collateral stimulation before, during, and after the induction of mGluR-dependent LTD by PP-LFS (900 paired pulses, separated by 50 ms, at 1 Hz), in control (inactive control) and GSK3α inhibitor-treated neurons (blue). Drug treatment was for 1 hour before recording at 10 µM for all compounds. Representative traces for synaptic responses before (dark trace) and after (light trace) LTD induction are shown above the time course. Error bars represent SEM. G. Quantification of average synaptic depression from the last 5 minutes of the time course shown in F. Error bars represent SEM. No statistically significant difference was found, according to the Mann-Whitney U test.


B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - or EGFP - GSK3β - expressing neurons ( green traces ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α- or EGFP-GSK3β -expressing neurons (green traces).