C Representative immunofluorescence showing Phalloidin - labelled actin ( red ) Hoechst ( blue ) in Latrunculin B ( LatB ) ( 0 . 5µM ) or Cytochalasin D ( CytD ) ( 2µM ) - treated HEK293A compared to control across Hippo pathway genome - edited as shown . Scale bar = 20μm . D Average change in volume obtained by DHM in response to hydrostatic pressure with 2µM Cytochalasin D ( CytD ) treatment in WT , Y / T DKO LATS1 / 2 DKO . Each dot represents a single error bars represent mean ± 95 % CI . Data pooled from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . * * p = 0 . 0044 ( WT con vs CytD ) , p * = 0 . 0409 ( Y / T DKO con vs CytD ) , p > 0 . 9999 ( LATS1 / 2 DKO con vs CytD ) , * * * p = 0 . 0003 ( WT con vs Y / T DKO con ) , * * * p < 0 . 001 ( WT con vs Y / T DKO CytD ) , * * * p < 0 . 001 ( Y / T DKO CytD vs LATS1 / 2 DKO CytD ) . E Fold difference in average change in volume in response to dynamic hydrostatic pressure from ( H ) normalised against untreated . Each dot represents a single error bars are 95 % CI . Mann - Whitney U . * * p = 0 . 0069 ( WT vs . Y / T DKO ) , * * p = 0 . 0031 ( WT vs LATS1 / 2 DKO ) . F Average change in volume in response to hydrostatic pressure with 0 . 5µM Latrunculin B ( Lat B ) treatment in WT , Y / T DKO LATS1 / 2 DKO . Each dot represents a single error bars represent mean ± 95 % CI . Data pooled from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . * * * p < 0 . 001 ( comparisons ) , p = 0 . 0972 ( LATS / 12 DKO con vs LatB ) , * p = 0 . 0216 ( Y / T DKO LatB vs LATS1 / 2 DKO LatB ) . G Fold difference in average change in volume in response to cyclic 0 . 1Hz 100mbar hydrostatic pressure from ( F ) normalised against control . Each dot represents a single error bars represent mean ± 95 % CI . Mann - Whitney U . p = 0 . 1472 ( WT vs . Y / T DKO ) , * * p = 0 . 0015 ( WT vs LATS1 / 2 DKO ) .

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

C Representative immunofluorescence images showing Phalloidin-labelled actin (red) and Hoechst (blue) in Latrunculin B (LatB) (0.5µM) or Cytochalasin D (CytD) (2µM)-treated HEK293A cells compared to control across Hippo pathway genome-edited cells as shown. Scale bar = 20μm. D Average change in cell volume obtained by DHM in response to hydrostatic pressure with 2µM Cytochalasin D (CytD) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. **p=0.0044 (WT con vs CytD), p*=0.0409 (Y/T DKO con vs CytD), p>0.9999 (LATS1/2 DKO con vs CytD), ***p=0.0003 (WT con vs Y/T DKO con), ***p<0.001 (WT con vs Y/T DKO CytD), ***p<0.001 (Y/T DKO CytD vs LATS1/2 DKO CytD). E Fold difference in average change in cell volume in response to dynamic hydrostatic pressure from (H) normalised against untreated cells. Each dot represents a single cell and error bars are 95% CI. Mann-Whitney U test. **p=0.0069 (WT vs. Y/T DKO), **p=0.0031 (WT vs LATS1/2 DKO). F Average change in cell volume in response to hydrostatic pressure with 0.5µM Latrunculin B (Lat B) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. ***p<0.001 (all comparisons), p=0.0972 (LATS/12 DKO con vs LatB), *p=0.0216 (Y/T DKO LatB vs LATS1/2 DKO LatB). G Fold difference in average change in cell volume in response to cyclic 0.1Hz 100mbar hydrostatic pressure from (F) normalised against control. Each dot represents a single cell and error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.1472 (WT vs. Y/T DKO), **p=0.0015 (WT vs LATS1/2 DKO).


A Confocal IF labelled with Hoechst ( Blue ) internalised Transferrin ( Trf ) . were allowed to internalize for ten minutes , fixed acid stripped to remove surface bound Trf . Hydrostatic pressure ( H . P . ) promotes transferrin uptake in WT LATS1 / 2 DKO . Scale bar = 20µm . B of transferrin uptake in WT , Y / T DKO LATS1 / 2 DKO treated with hydrostatic pressure compared to steady state . Each dot represents a single error bars represent mean ± 95 % CI . Data from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . Steady - state statistical is shown in blue , transferrin uptake induced by pressure is shown in black . * * * p < 0 . 001 ( WT con vs + H . P . ) , p = 0 . 2375 ( Y / T DKO con vs + H . P . ) , * * * p < 0 . 001 ( LATS1 / 2 DKO con vs + H . P . ) , p = 0 . 2764 ( WT con vs Y / T DKO con ) , p = 0 . 0808 ( WT con vs LATS1 / 2 DKO con ) , * * * p = 0 . 0004 ( Y / T DKO con vs LATS1 / 2 DKO con ) .

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A Confocal IF images labelled with Hoechst (Blue) and internalised Transferrin (Trf). Cells were allowed to internalize for ten minutes, fixed and acid stripped to remove surface bound Trf. Hydrostatic pressure (H.P.) promotes transferrin uptake in WT and LATS1/2 DKO cells. Scale bar = 20µm. B Quantification of transferrin uptake in WT, Y/T DKO and LATS1/2 DKO cells treated with hydrostatic pressure compared to steady state. Each dot represents a single cell and error bars represent mean ± 95% CI. Data from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. Steady-state statistical analysis is shown in blue, transferrin uptake induced by pressure is shown in black. ***p<0.001 (WT con vs +H.P.), p=0.2375 (Y/T DKO con vs +H.P.), ***p<0.001 (LATS1/2 DKO con vs +H.P.), p=0.2764 (WT con vs Y/T DKO con), p=0.0808 (WT con vs LATS1/2 DKO con), ***p=0.0004 (Y/T DKO con vs LATS1/2 DKO con).


C Confocal IF myc tagged AP180C ( green ) showing internalised Trf ( red ) stained with Hoechst ( blue ) . AP180C inhibits transferrin uptake in WT Y / T DKO . Scale bar = 20µm . D of transferrin uptake in AP180C positive compared to vector control from as in C . Each dot represents a single from four independent experiments error bars represent mean ± 95 % CI . Mann - Whitney U . * * * p < 0 . 001 ( for both comparisons ) .

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C Confocal IF images myc tagged AP180C (green) cells showing internalised Trf (red) and stained with Hoechst (blue). AP180C inhibits transferrin uptake in WT and Y/T DKO cells. Scale bar = 20µm. D Quantification of transferrin uptake in AP180C positive cells compared to vector control from images as in C. Each dot represents a single cell from four independent experiments and error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001 (for both comparisons).


E Confocal IF of HEK293A WT showing subcellular localisation of YAP ( red ) in AP180C ( green ) negative positive WT LATS1 / 2 DKO . also stained with Hoechst ( blue ) . F of - to - of YAP in AP180C positive WT LATS1 / 2 DKO compared to vector control from as in E . Each dot represents a single pooled from four independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U . * * * p = 0 . 0008 ( WT ) , p = 0 . 9318 ( LATS1 / 2 DKO ) .

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E Confocal IF images of HEK293A WT cells showing subcellular localisation of YAP (red) in AP180C (green) negative and positive WT and LATS1/2 DKO cells. Cells also stained with Hoechst (blue). F Quantification of cytoplasmic-to-nuclear ratio of YAP in AP180C positive WT and LATS1/2 DKO cells compared to vector control from images as in E. Each dot represents a single cell pooled from four independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p=0.0008 (WT), p=0.9318 (LATS1/2 DKO).


A , B X - gal showed NB - 3 / LacZ expression in sham - operated ( A ) injured ( B ) spinal cords from adult NB - 3 + / − mice .

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A, B X-gal staining showed NB-3/LacZ expression in the sham-operated (A) and injured (B) spinal cords from adult NB-3+/−mice.


C , D Western blot ( C ) ( D ) of NB - 3 expression in lesion post - injury . NB - 3 expression were normalized by b - tubulin . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - . Data were analyzed from 10 independent experiments each including 2 mice per group .

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C, D Western blot analysis (C) and quantification (D) of NB-3 expression in the lesion area post-injury. NB-3 expression levels were normalized by b-tubulin. Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 10 independent experiments each including 2 mice per group.


E , F Single - labeling immunostaining showed NB - 3 expression in sham - operated ( E ) injured ( F ) spinal cords from adult NB - 3 + / + mice .

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E, F Single-labeling immunostaining showed NB-3 expression in the sham-operated (E) and injured (F) spinal cords from adult NB-3+/+mice.


G - K Co - immunostaining of NB - 3 ( green ) , PDGFR - β ( red ) , GFAP ( blue ) on serial sagittal sections from same NB - 3 + / + spinal cord 14 days post - injury . ( G1 - K1 ) High - magnification of boxed areas in ( G - K ) , respectively . L Co - immunostaining of NB - 3 ( green ) , PDGFR - β ( red ) , GFAP ( blue ) on sagittal sections from NB - 3 − / − spinal cord 14 days post - injury . ( L1 ) High - magnification image of boxed in ( L ) . arrows indicate lesion sites .

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G-K Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (G1-K1) High-magnification images of boxed areas in (G-K), respectively.L Co-immunostaining of NB-3 (green), PDGFR-β (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (L1) High-magnification image of the boxed area in (L). The arrows indicate the lesion sites.


M - Q Co - immunostaining of NB - 3 ( green ) , Fibronectin ( red ) , GFAP ( blue ) on serial sagittal sections from same NB - 3 + / + spinal cord 14 days post - injury . ( M1 - Q1 ) High - magnification of boxed areas in ( M - Q ) , respectively . R Co - immunostaining of NB - 3 ( green ) , Fibronectin ( red ) , GFAP ( blue ) on sagittal sections from NB - 3 − / − spinal cord 14 days post - injury . ( R1 ) High - magnification image of boxed in ( R ) . arrows in ( G - R ) indicate lesion sites .

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M-Q Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on serial sagittal sections from the same NB-3+/+spinal cord 14 days post-injury. (M1-Q1) High-magnification images of boxed areas in (M-Q), respectively.R Co-immunostaining of NB-3 (green), Fibronectin (red), and GFAP (blue) on sagittal sections from the NB-3−/− spinal cord 14 days post-injury. (R1) High-magnification image of the boxed area in (R). The arrows in (G-R) indicate the lesion sites.


B NB - 3 expression in corticospinal neurons 14 28 days post - injury ( dpi ) . FG ( blue ) was applied to retrogradely label corticospinal neurons . C of of NB - 3 in ( B ) . * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - . intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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B NB-3 expression in corticospinal neurons 14 and 28 days post-injury (dpi). FG (blue) was applied to retrogradely label corticospinal neurons.C Quantification of the fluorescence intensity of NB-3 in (B). **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


D , E Western blot ( D ) ( E ) of NB - 3 expression in sensorimotor cortex after SCI . NB - 3 expression were normalized by b - tubulin . * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA followed by Bonferroni ' s post - . Data were analyzed from 3 independent experiments each including 3 mice per group .

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D, E Western blot analysis (D) and quantification (E) of NB-3 expression in the sensorimotor cortex after SCI. NB-3 expression levels were normalized by b-tubulin. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni's post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.


F - K Induced NB - 3 expression at distal terminations of post - traumatic corticospinal axons in reactive astrocytes at lesion border 14 ( H I ) 28 days post - injury ( dpi ) ( J K ) . BDA ( red ) was applied to anterogradely label corticospinal axons . GFAP ( blue ) was used to label reactive astrocytes . ( G ) High - magnification image of boxed in ( F ) . ( I K ) High - magnification x - y - z reconstruction of boxed areas in ( H J ) , respectively , show co - localization of NB - 3 GFAP in astrocytes at lesion border co - localization of NB - 3 in BDA - labeled axons GFAP - positive astrocytes at contact surface of corticospinal axons astrocytes ( arrowheads ) . arrows in ( H J ) indicate lesion sites . asterisks in ( H J ) indicate terminations of BDA - labeled projecting axons .

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F-K Induced NB-3 expression at the distal terminations of post-traumatic corticospinal axons and in reactive astrocytes at the lesion border 14 (H and I) and 28 days post-injury (dpi) (J and K). BDA (red) was applied to anterogradely label corticospinal axons. GFAP (blue) was used to label reactive astrocytes. (G) High-magnification image of the boxed area in (F). (I and K) High-magnification and x-y-z reconstruction images of boxed areas in (H and J), respectively, show co-localization of NB-3 and GFAP in astrocytes at the lesion border and co-localization of NB-3 in BDA-labeled axons and GFAP-positive astrocytes at the contact surface of corticospinal axons and astrocytes (arrowheads). The arrows in (H and J) indicate the lesion sites. The asterisks in (H and J) indicate the terminations of the BDA-labeled projecting axons.


B - K Representative of serial sagittal sections showing main BDA - labeled corticospinal axons in NB - 3 + / + ( B - D ) NB - 3 − / − ( E - G ) mice 14 after complete spinal transection . ( H - J ) High - magnification of areas in ( E - G ) ( single asterisks ) , respectively . ( K ) High - magnification image of in ( E ) ( double asterisk ) . arrows indicate lesion sites ; dashed lines indicate regenerative corticospinal axons extending into distal spinal cord ; single asterisks in ( E - G ) indicate lesion epicenters ; double asterisk in ( E ) indicates axons extending from caudal lesion border . Scale bars , 400 µm ( B - G ) 100 µm ( H - K ) . L of index of BDA - labeled axons at certain distances from lesion border in ( B - G ) . Data are represented as mean ± . * p < 0 . 05 , * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 25 mice per group .

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B-K Representative images of serial sagittal sections showing the main BDA-labeled corticospinal axons in NB-3+/+ (B-D) and NB-3−/− (E-G) mice 14 weeks after complete spinal transection. (H-J) High-magnification images of the areas in (E-G) (single asterisks), respectively. (K) High-magnification image of the area in (E) (double asterisk). The arrows indicate the lesion sites; the white dashed lines indicate the regenerative corticospinal axons extending into the distal spinal cord; the single asterisks in (E-G) indicate the lesion epicenters; the double asterisk in (E) indicates the axons extending from the caudal lesion border. Scale bars, 400 µm (B-G) and 100 µm (H-K).L Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (B-G). Data are represented as mean ± SEM. *p < 0.05, and **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 25 mice per group.


A Time course of change in BMS scores in NB - 3 + / + NB - 3 − / − mice as measured with BMS rating scale after spinal cord complete transection . * p < 0 . 05 ; two - way ANOVA followed by Bonferroni ' s post - . n = 25 mice per group .

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A Time course of the change in BMS scores in NB-3+/+ and NB-3−/−mice as measured with the BMS rating scale after spinal cord complete transection. *p < 0.05; two-way ANOVA followed by Bonferroni's post-test. n= 25 mice per group.


C Electrophysiology transmission across spinal lesion site . In each group of NB - 3 + / + NB - 3 − / − mice , electrophysiological responses in caudal spinal cord were recorded in condition of sham - operated or 12 post - injury . latencies amplitudes of responses were analyzed quantified in each group . n . s . , not significant , * p < 0 . 05 , * * p < 0 . 01 ; two - way ANOVA followed by Bonferroni ' s post - . n = 10 mice per group .

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C Electrophysiology transmission across the spinal lesion site. In each group of NB-3+/+ and NB-3−/−mice, the electrophysiological responses in the caudal spinal cord were recorded in the condition of sham-operated or 12 weeks post-injury. The latencies and amplitudes of responses were analyzed and quantified in each group. n.s., not significant, *p < 0.05, and **p < 0.01; two-way ANOVA followed by Bonferroni's post-test. n= 10 mice per group.


E Representative of coronal sections showing synapse formation between glutamatergic corticospinal terminals spinal neurons in NB - 3 + / + NB - 3 − / − mice . Corticospinal axons , neurons , glutamatergic terminals of corticospinal axons were labeled with BDA ( red ) , Tuj1 ( blue ) vGlut1 / 2 ( green ) , respectively . Each group showed data from rostral caudal spinal cord in condition of sham - operated or 12 post - injury . Scale bar , 20 μm . F of number of glutamatergic corticospinal synapses per neuron in each group . n . s . , not significant , * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 15 mice per group .

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E Representative images of coronal sections showing synapse formation between the glutamatergic corticospinal terminals and spinal neurons in NB-3+/+ and NB-3−/−mice. Corticospinal axons, neurons, and glutamatergic terminals of corticospinal axons were labeled with BDA (red), Tuj1 (blue) and vGlut1/2 (green), respectively. Each group showed the data from the rostral and caudal spinal cord in the condition of sham-operated or 12 weeks post-injury. Scale bar, 20 μm.F Quantification of the number of glutamatergic corticospinal synapses per neuron in each group. n.s., not significant, **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 15 mice per group.


A , B Western blot ( A ) ( B ) of NB - 3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control ( LV - nc - GFP ) that encoding NB - 3 shRNA ( LV - NB - 3 shRNA - GFP ) . NB - 3 expression were normalized by b - tubulin . * * * p < 0 . 001 ; one sample t - . Data were analyzed from 5 independent experiments .

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A, B Western blot analysis (A) and quantification (B) of NB-3 expression in cultured pyramidal neurons infected with lentivirus encoding negative control (LV-nc-GFP) and that encoding NB-3 shRNA (LV-NB-3 shRNA-GFP). NB-3 expression levels were normalized by b-tubulin. ***p < 0.001; one sample t-test. Data were analyzed from 5 independent experiments.


C - E Sagittal sections showing BDA - labeled corticospinal axons in injured spinal cords following cortical injection of LV - nc - GFP ( C ) LV - NB - 3 shRNA - GFP ( D ) . High - magnification image ( E ) of in ( D ) ( asterisk ) . F , J of index of BDA - labeled axons at certain distances from lesion border in ( C , D , G , H ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 19 mice per group in ( F ) , n = 17 mice per group in ( J ) .

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C-E Sagittal sections showing the BDA-labeled corticospinal axons in injured spinal cords following cortical injection of LV-nc-GFP (C) and LV-NB-3 shRNA-GFP (D). High-magnification image (E) of the area in (D) (asterisk).F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 19 mice per group in (F), and n= 17 mice per group in (J).


G - I Sagittal sections showing corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( G ) LV - NB - 3 shRNA - GFP ( H ) into lesion sites . High - magnification image ( I ) of in ( H ) ( asterisk ) . arrows in ( C - E G - H ) indicate lesion sites ; asterisks in ( D H ) indicate lesion epicenters ; dashed lines in ( D H ) indicate regenerative corticospinal axons extending into distal spinal cord . F , J of index of BDA - labeled axons at certain distances from lesion border in ( C , D , G , H ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 19 mice per group in ( F ) , n = 17 mice per group in ( J ) .

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G-I Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (G) and LV-NB-3 shRNA-GFP (H) into the lesion sites. High-magnification image (I) of the area in (H) (asterisk). The arrows in (C-E and G-H) indicate the lesion sites; the asterisks in (D and H) indicate the lesion epicenters; the white dashed lines in (D and H) indicate the regenerative corticospinal axons extending into the distal spinal cord.F, J Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (C, D, G, and H). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 19 mice per group in (F), and n= 17 mice per group in (J).


A , B NB - 3 - Fc - coated bead aggregation ( A ) . BSA - coated beads as a negative control ( B ) .

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A, B NB-3-Fc-coated bead aggregation assay (A). BSA-coated beads as a negative control (B).


C Co - immunoprecipitation of NB - 3 - Myc with NB - 3 - HA from co - transfected COS1 .

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C Co-immunoprecipitation of NB-3-Myc with NB-3-HA from co-transfected COS1 cells.


D , E Cis interactions of NB - 3 - Myc with NB - 3 - HA in COS1 . ( E ) High - magnification image of boxed in ( D ) . were co - stained for Myc ( red ) , HA ( green ) , DAPI ( blue ) . arrowheads indicate surface co - localization of NB - 3 - Myc NB - 3 - HA .

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D, E Cis interactions of NB-3-Myc with NB-3-HA in COS1 cells. (E) High-magnification image of the boxed area in (D). Cells were co-stained for Myc (red), HA (green), and DAPI (blue). The arrowheads indicate cell surface co-localization of NB-3-Myc and NB-3-HA.


F , G Trans interactions between NB - 3 expressed in COS1 treated NB - 3 - Fc . were co - stained for Myc ( red ) , Fc or NB - 3 - Fc ( green ) , DAPI ( blue ) . arrows indicate co - localization of NB - 3 - Myc NB - 3 - Fc at ( G ) .

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F, G Trans interactions between NB-3 expressed in COS1 cells and treated NB-3-Fc. Cells were co-stained for Myc (red), Fc or NB-3-Fc (green), and DAPI (blue). The arrows indicate co-localization of NB-3-Myc and NB-3-Fc at the cell membrane (G).


H co - localization of NB - 3 - Myc NB - 3 - HA separately expressed in 293T COS1 . were co - stained for Myc ( red ) , HA ( green ) , DAPI ( blue ) .

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H Cell membrane co-localization of NB-3-Myc and NB-3-HA separately expressed in 293T and COS1 cells. Cells were co-stained for Myc (red), HA (green), and DAPI (blue).


I , J NB - 3 expression in cultured pyramidal neurons astrocytes . Pyramidal neurons were co - stained for NB - 3 , Ctip2 Tuj1 ( I ) . Astrocytes were co - stained for NB - 3 , GFAP , DAPI ( J ) .

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I, J NB-3 expression in cultured pyramidal neurons and astrocytes. Pyramidal neurons were co-stained for NB-3, Ctip2 and Tuj1 (I). Astrocytes were co-stained for NB-3, GFAP, and DAPI (J).


K Western blot of NB - 3 in cultured pyramidal neurons astrocytes . NB - 3 expression were normalized by b - tubulin expressed relative to that of neurons . * * * p < 0 . 001 ; one sample t - . Data were analyzed from 3 independent experiments .

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K Western blot analysis and quantification of NB-3 in cultured pyramidal neurons and astrocytes. NB-3 expression levels were normalized by b-tubulin and expressed relative to that of neurons. ***p < 0.001; one sample t-test. Data were analyzed from 3 independent experiments.


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

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

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

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

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

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

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

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

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

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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 CHL1 from spinal cord lesion areas of NB - 3 + / + NB - 3 − / − mice .

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

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

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

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

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

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

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

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

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

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

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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).


( B ) Flow cytometry of WT to Il21r - / - splenic SWHEL B over time . ( C ) WT to Il21r - / - SWHEL B within CTV division peaks . Data information : Data in ( B ) is representative of 8 - 9 biological replicates ( n = 8 - 9 ) pooled from two independent experiments with statistical by one - way ANOVA with Tukey ' s post - . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 . Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) are representative for two independent experiments .

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(B) Flow cytometry analysis of WT to Il21r-/- splenic SWHEL B cells over time. (C) WT to Il21r-/- SWHEL B cell ratio within CTV division peaks. Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( E ) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks . Data information : Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) are representative for two independent experiments .

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(E) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks. Data information: Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( B ) Flow cytometry showing WT to Il21r - / - SWHEL B of that have been in S phase thus incorporated BrdU . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical by one - sample t . * p ≤ 0 . 05 .

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(B) Flow cytometry analysis showing WT to Il21r-/- SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( D ) of proportion of BrdU positive WT Il21r - / - SWHEL B with 2N content 4 , 10 12 hours post BrdU on day 3 . 5 post immunization . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical by one - sample t . * p ≤ 0 . 05 .

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(D) Ratio of the proportion of BrdU positive WT and Il21r-/- SWHEL B cells with 2N DNA content 4, 10 and 12 hours post BrdU pulse on day 3.5 post immunization. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( C ) Rate of de novo cycle entry 10 hours post in vivo IL - 21 ( 2 µg ) or saline . of WT to Il21r - / - not in S / G2 at time of ( BrdU - ) containing > 2N content 10 hours after is shown . Data information : Data are representative of 10 - 12 biological replicates ( n = 10 - 12 ) from two independent experiments . Statistical by one - sample t . * p ≤ 0 . 05 .

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(C) Rate of de novo cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA content 10 hours after pulse is shown. Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.


Phosphoflow of naïve WT or Il21r - / - B following in vitro culture for 3 hours with or without IL - 21 ( 20 ng / mL ) / or in presence of BCR cross - linking ( biotinylated anti - Igκ + anti - Igλ avidin - mediated cross - linking ) or agonistic anti - CD40 . ( A ) Exemplary p - AKT ( S473 ) ( B ) of p - AKT median ( MedFI ) . ( C ) Exemplary p - S6 ( Ser235 / 236 ) ( D ) of frequency of p - S6 positive Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical by one - way ANOVA with Tukey ' s post - ( D ) , t ( B , E ) * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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Phosphoflow analysis of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR cross-linking (biotinylated anti-Igκ + anti-Igλ and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT (S473) staining and (B) quantification of p-AKT median fluorescence intensity (MedFI). (C) Exemplary p-S6 (Ser235/236) staining and (D) quantification of frequency of p-S6 positive cells Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( F , In vitro culture of CTV labeled SWHEL B with or without IL - 21 ( 20 ng / mL ) anti - CD40 for three days by flow cytometry . ( F ) Exemplary CTV division peaks .

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(F, In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (F) Exemplary CTV division peaks.


G ) In vitro culture of CTV labeled SWHEL B with or without IL - 21 ( 20 ng / mL ) anti - CD40 for three days by flow cytometry . ( G ) of proportion SWHEL B that had divided thus diluted CTV . Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical by two - way ANOVA with correction for multiple comparison using Šidák method ( G ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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G) In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (G) Quantification of the proportion SWHEL B that had divided and thus diluted CTV. Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( B , C ) of splenic OTII Tfh by flow cytometry . ( B ) Tfh ( C ) Proportion of Il21Gfp / + OTII Tfh transcribing Il21 locus thus expressing GFP . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . Statistical by one - way ANOVA with Tukey ' s post - ( B , C ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(B, C) Analysis of splenic OTII Tfh cells by flow cytometry. (B) Tfh cell count and (C) Proportion of Il21Gfp/+ OTII Tfh cells transcribing the Il21 locus and thus expressing GFP. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. Statistical analysis by one-way ANOVA with Tukey's post-test (B, C). * p ≤ 0.05; ** p ≤ 0.01.


of splenic WT Il21r - / - SWHEL B by flow cytometry . ( D ) CTV profile of ( E ) absolute in pooled CTV division peaks ( 0 - 4 , 5 - 8 , 9 - 11 + ) . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . D , show concatenated data representative of 3 - 5 biological replicates . Data in E were analyzed after log transformation using Multiple paired t p values corrected for multiple comparisons using Holm - Šídák method . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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Analysis of splenic WT and Il21r-/- SWHEL B cells by flow cytometry. (D) CTV profile of and (E) absolute cell count in pooled CTV division peaks (0-4, 5-8, 9-11+). Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, show concatenated data representative of 3-5 biological replicates. Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.


( F ) surface expression ( median , MedFI ) of CD38 , IgD FAS across CTV division peaks . ( G ) Representative flow cytometry plot of plasma differentiation indicated by CD138 expression . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . F G show concatenated data representative of 3 - 5 biological replicates .

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(F) Cell surface expression (median fluorescence intensity, MedFI) of CD38, IgD and FAS across individual CTV division peaks. (G) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. F and G show concatenated data representative of 3-5 biological replicates.


( A - E ) of splenic SWHEL B PC by flow cytometry . ( A ) Proportion of SWHEL B with a FAS + GL7 + GC phenotype , ( B ) SWHEL GC B ( C ) representation of IL - 21R deficient among total SWHEL GC B . ( D ) CD98 + CD138 + _ SWHEL PC ( E ) representation of IL - 21R deficient among total SWHEL PC . Data information : Data are representative of 6 - 10 biological replicates ( n = 6 - 10 ) from two independent experiments . Data in B , D were analyzed after log transformation with with statistical by Multiple paired t ( A , B , D ) p - values corrected for multiple comparisons using Holm - Šídák method or One - way ANOVA with Tukey ' s post - ( C ) or t ( E ) .

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

(A-E) Analysis of splenic SWHEL B cells and PC by flow cytometry. (A) Proportion of SWHEL B cells with a FAS+GL7+ GC phenotype, (B) SWHEL GC B cell count and (C) representation of IL-21R deficient cells among total SWHEL GC B cells. (D) CD98+ CD138+_SWHEL PC count and (E) representation of IL-21R deficient cells among total SWHEL PC. Data information: Data are representative of 6-10 biological replicates (n = 6-10) from two independent experiments. Data in B, D were analyzed after log transformation with with statistical analysis by Multiple paired t tests (A, B, D) and p-values corrected for multiple comparisons using Holm-Šídák method or One-way ANOVA with Tukey's post-test (C) or t test (E).


Coomassie stained SDS - PAGE of McjD - C547 reconstituted in liposomes . amount reconstituted was evaluated against detergent purified

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Coomassie stained SDS-PAGE of McjD-C547 reconstituted in liposomes. The amount reconstituted was evaluated against detergent purified protein


Chromatogram of McjD C547 ( black ) labelled with Alexa 555 ( blue ) Alexa 647 ( red ) maleimide fluorophore using a Superdex 200 - exclusion column . fraction containing highest degree of labeling is marked by a

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Chromatogram of McjD C547 (black) labelled with Alexa 555 (blue) and Alexa 647 (red) maleimide fluorophore using a Superdex 200 size-exclusion column. The fraction containing the highest degree of labeling is marked by a square


ATPase activity of labelled variants C547 , Y64C , L67C was determined in liposomes compared to wild - type , wt . labelled McjD variants show basal - ligand - induced ATPase activities in liposomes comparable to that of wild type , indicating that labelling does not interfere with their activity . Error bars were calculated from two replicates from two independent reconstitutions ( mean ± standard deviation , n = 2 ) . ruler character of FRET using fluorophore pair Alexa555 / Cy5 was supported by data of dsDNA in Figure EV1B , where different base - pair distances between both dyes correlate directly with apparent FRET

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The ATPase activity of all labelled variants C547, Y64C, L67C was determined in liposomes and compared to wild-type, wt. All labelled McjD variants show basal- and ligand-induced ATPase activities in liposomes comparable to that of wild type protein, indicating that labelling does not interfere with their activity. Error bars were calculated from two replicates from two independent reconstitutions (mean ± standard deviation, n=2). The ruler character of FRET using the fluorophore pair Alexa555/Cy5 was supported by data of dsDNA in Figure EV1B, where different base-pair distances between both dyes correlate directly with the apparent FRET efficiency


Confocal single molecule with ALEX of labeled McjD in detergent under different conditions as

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Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicated


Apparent ligand binding affinity values of McjD for AMPPNP ( top ) , ATP ( middle ) , ADP - vanadate ( bottom ) . Data points were obtained from areas of fits from E * histograms considering ligand - free / ( ligand - free + ligand - bound ) at indicated substrate concentrations . Extraction of Kd values from a fit to data is described in Material Methods section . Data for detergent - solubilized McjD for additional biochemical conditions is shown in Figure

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Apparent ligand binding affinity values of McjD for AMPPNP (top), ATP (middle), and ADP-vanadate (bottom). Data points were obtained from the areas of fits from the E* histograms considering the ratio ligand-free/(ligand-free+ligand-bound) at the indicated substrate concentrations. Extraction of Kd values from a fit to the data is described in the Material and Methods section. Data for detergent-solubilized McjD for additional biochemical conditions is shown in Figure EV2


Representative time traces ( blue , donor signal ; red , acceptor signal ; black , FRET signal ; yellow , fit ) of McjD labeled with Alexa555 Alexa647 - maleimide with indicated concentrations of corresponding substrate : ( B ) McjD L67C with 10 mM ATP 200 µM MccJ25 , McjD Y64C with 10 mM ATP 200 µM MccJ25 , McjD C547 with 10 mM ATP . respective right panels are projections of accumulated time traces in absence presence of substrate for each variant . We note that differences between setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted . Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts distinct TEV cleavage for liposomes compared to

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Representative fluorescence time traces (blue, donor signal; red, acceptor signal; black, FRET signal; yellow, fit) of McjD labeled with Alexa555 and Alexa647-maleimide with indicated concentrations of corresponding substrate: (B) McjD L67C with 10 mM ATP and 200 µM MccJ25, McjD Y64C with 10 mM ATP and 200 µM MccJ25, and McjD C547 with 10 mM ATP. The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant. We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted. Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to detergent


Representative FRET time traces ( black , FRET signal ; yellow , fit ) of McjD NBD C547 labeled with Alexa 555 Alexa 647 - maleimide under apo conditions at 10 ms time resolution . Approximately ~ 85 % of apo - McjD traces show no significant fluctuations beyond shot - noise . Apo - McjD is predominantly in low FRET thus open state with E * values < 0 . 3 Within complete data set of apo - McjD ( N = 80 traces ) comprising donor acceptor , ~ 15 % , show infrequent fluctuations to a higher FRET efficiency state with a lifetime of ( C ) 82 ± 25 ms where E * values > 0 . 5 are observed during short dwells . data set represents a total recording time of 4 . 6 mins with a temporal resolution of 10 ms . To exclude unwanted influence on McjD due to surface - immobilization , experimental verification is provided that ATP - induced NBD switching occurs similarly with detergent solubilized McjD on surface in Figure

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

Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide under apo conditions at 10 ms time resolution. Approximately ~85% of all apo-McjD traces show no significant fluctuations beyond shot-noise. Apo-McjD is predominantly in the low FRET and thus open state with E* values <0.3 Within the complete data set of apo-McjD (N = 80 traces) comprising donor and acceptor, ~15%, show infrequent fluctuations to a higher FRET efficiency state with a lifetime of (C) 82 ± 25 ms where E* values > 0.5 are observed during short dwells. The data set represents a total recording time of 4.6 mins with a temporal resolution of 10 ms. To exclude unwanted influence on McjD due to surface-immobilization, experimental verification is provided that ATP-induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure EV4


Representative FRET time traces ( black , FRET signal ; yellow , fit ) of McjD NBD C547 labeled with Alexa 555 Alexa 647 - maleimide in apo conditions at 10 ms time resolution . This panel shows stable ATP - loaded ( left ) ATP - free ( right ) McjD Representative FRET time traces that show switching between ATP free McjD ( E * ~ 0 . 3 ) ATP - loaded ( high FRET > 0 . 5 ) at ~ 4s s ( left ) ~ 1 . 4 s ( right ) . traces show photobleaching events of acceptor after ~ 10 s ( left ) ~ 3 . 3 s ( right ) Representative FRET time traces that show switching between ATP - loaded ( high FRET > 0 . 5 ) ATP free McjD ( E * ~ 0 . 3 ) at 2 s ( left ) 5 s ( right ) . traces show photobleaching events after ca . 40 s ( left , donor - bleaching ) 70 s ( right , acceptor - bleaching )

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Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide in apo conditions at 10 ms time resolution. This panel shows stable ATP-loaded (left) and ATP-free (right) McjD Representative FRET time traces that show switching between ATP free McjD (E* ~ 0.3) and ATP-loaded (high FRET >0.5) at ~4s s (left) and ~1.4 s (right). The traces show photobleaching events of the acceptor after ~10 s (left) and ~3.3 s (right) Representative FRET time traces that show switching between ATP-loaded (high FRET >0.5) and ATP free McjD (E* ~ 0.3) at 2 s (left) and 5 s (right). The traces show photobleaching events after ca. 40 s (left, donor-bleaching) and 70 s (right, acceptor-bleaching)


B 18F - FDG PET - MRI at time of diagnosis in interim staging after one cycle of FOLFIRINOX .

B 18F-FDG PET-MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX.


C H & E of FNB resection specimen . Scale bars represent 50 µm .

C H&E staining of FNB and the resection specimen. Scale bars represent 50 µm.


D Phase - contrast of organoids ( upper panel ) respective 2D cultures ( isolated from biopsy before FOLFIRINOX ( ID188 ) or after neoadjuvant therapy from resected cancer ( ID211 ) . Scale bar represents 200µm .

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D Phase-contrast images of organoids (upper panel) and respective 2D cultures (isolated from the biopsy before FOLFIRINOX (ID188) or after the neoadjuvant therapy from the resected cancer (ID211). Scale bar represents 200µm.


E IF of actin filaments ( red ) nuclei ( blue ) of organoids in left panel H & E of embedded sectioned organoids in right panel ( ID188 p30 , ID211 p30 ) .

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E IF staining of the actin filaments (red) and nuclei (blue) of organoids in the left panel and H&E staining of embedded and sectioned organoids in the right panel (ID188 p30, ID211 p30).


F Normalized expression ( log2 scale ) for gene pairs evaluated during Purity independent subtyping of tumors ( PurIST ) . Normalized expression ( log2 scale ) , gene 1 = basal - like , gene 2 = classical .

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F Normalized expression (log2 scale) for gene pairs evaluated during Purity independent subtyping of tumors (PurIST). Normalized expression (log2 scale), gene 1 = basal-like, gene 2 = classical.


G Gene Set Enrichment ( GSEA ) of pre - ( ID188 , p7 ) post - treatment ( ID211 , p6 ) PDO lines . Normalized enrichment score ( NES ) adjusted p - value is depicted .

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G Gene Set Enrichment Analysis (GSEA) of pre-(ID188, p7) and post-treatment (ID211, p6) PDO lines. Normalized enrichment score (NES) and the adjusted p-value is depicted.


H Whole exome sequencing - copy number profiles for ID188 ( p7 ) ID211 ( p6 ) organoids .

H Whole exome sequencing-based copy number profiles for ID188 (p7) and ID211 (p6) organoids.


I Allele frequencies of miss - nonsense SNVs Indels which are shared between both organoid lines ( purple ) , private to ID188 ( red ) or private to ID211 ( blue ) .

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I Allele frequencies of miss- and nonsense SNVs and Indels which are shared between both organoid lines (purple), private to ID188 (red) or private to ID211 (blue).


A Dose response curves of ID188 ( p27 - 29 ) ID211 ( p26 - 28 ) organoids treated with FOLFIRINOX over three days . ATP was measured with CellTiter - Glo assays . Shown is mean ± SD of three independent experiments .

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A Dose response curves of ID188 (p27-29) and ID211 (p26-28) organoids treated with FOLFIRINOX over three days. ATP was measured with CellTiter-Glo assays. Shown is the mean ± SD of three independent experiments.


C Plot of delta AUC in ID188 ( p5 + 27 ) versus ID211 ( p9 + 11 ) over 415 drugs . Dashed lines demark threshold of delta AUC > 0 . 3 or < - 0 . 3 . D of screening hits as defined in C . AUC is color coded .

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C Plot of the delta AUC in ID188 (p5+27) versus ID211 (p9+11) cells over 415 drugs. Dashed lines demark the threshold of delta AUC >0.3 or <-0.3. D Heatmap of the screening hits as defined in C. AUC is color coded.


E - J Dose response curves of ID188 ID211 organoids ( ID188 p27 - 29 , ID211 p26 - 28 ) or 2D lines ( ID188 p5 + 29 , ID211 p9 + 13 ) treated with ( E F ) : Cobimetinib or ( H I ) : Biminetinib . ( G ) Cobimetinib ( J ) Biminetinib : GI50 values . Shown is mean ± SD of three independent experiments .

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E - J Dose response curves of ID188 and ID211 organoids (ID188 p27-29, ID211 p26-28) or 2D lines (ID188 p5+29, ID211 p9+13) treated with (E and F): Cobimetinib or (H and I): Biminetinib. (G) Cobimetinib and (J) Biminetinib: GI50 values. Shown is the mean ± SD of three independent experiments.


K Illustration of a cohort of ( n = 15 ) with available 2D PDAC lines . This cohort includes ID188 ( pre - FFX ( FOLFIRINOX ) , red ) ID211 ( post - FFX , blue ) lines . These lines were screened for sensitivity towards three MEKi . L 15 human PDAC 2D lines were screened for MEKi ( as indicated ) sensitivity . determined AUC was variance scaled z - scores are depicted . ID188 ID211 identity is color coded .

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K Illustration of a cohort of patients (n=15) with available primary 2D PDAC cell lines. This cohort includes the ID188 (pre-FFX (FOLFIRINOX), red) and ID211 (post-FFX, blue) lines. These lines were screened for sensitivity towards three MEKi. L 15 primary human PDAC 2D cell lines were screened for MEKi (as indicated) sensitivity. The determined AUC was variance scaled and the z-scores are depicted. The ID188 and ID211 identity is color coded.


Treatment of HeLa with DFP ( 1mM for 24 h ) reduces conjugation of SUMO - 2 / 3 ( n = 5 , biological replicates ; * * , p < 0 . 01 ; paired t - ) . Treatment of HeLa with DFP ( 1mM for 24 h ) does not change global SUMO - 1 - ylation ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - ) .

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Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).


Treatment of HeLa with DFP ( 1mM for 24 h ) leads to increased of SENP3 ( n = 8 , biological replicates ; * * p < 0 . 01 ; paired t - ) . Treatment of HeLa with DFP ( 1mM for 24 h ) does not change of SENP5 ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - ) .

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Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).


DFP treatment increases SENP3 stability . DFP ( 1mM for 24 h ) - treated HeLa were treated with cycloheximide ( CHX ; 50μg / ml ) for indicated durations in a chase experiment . Lysate samples were separated on different lanes in same gel immunoblotted as indicated .

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DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.


Treatment of HeLa with DFP ( 1mM for 24 h ) decreases of CHIP ( n = 8 , biological replicates ; * * * p < 0 . 001 ; paired t - ) . Treatment of HeLa with DFP ( 1mM for 24 h ) leads to decreased of CHIP mRNA ( n = 3 , biological replicates ; * p < 0 . 05 ; unpaired t - ) .

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Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).


Treatment of DFP ( 1mM for 24 h ) - treated HeLa with 50μM MG132 for final 6 h does not recover reduced of CHIP induced by DFP . Histogram shows relative of CHIP upon four different conditions ( n = 3 , biological replicates ; * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; repeated measures of variance followed by Tukey ' s multiple comparisons ) .

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Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).


CHIP knockdown increases of SENP3 but not SENP5 in HeLa . Nsi or CHIPi ( Nsi , non - specific siRNA ; CHIPi , CHIP siRNA ; , 20 nM ) was transfected into HeLa for 48 h . Lysate samples were blotted as indicated . CHIP knockdown prevents DFP - induced increase in SENP3 . Nsi , SENP3i or CHIPi ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; CHIPi , CHIP siRNA ; siRNA , 20nM ) was transfected into HeLa for 48 h . Two days post - transfection were treated with DFP for a further 24 h .

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

CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.   CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.


SENP3 knockdown abolishes DFP - induced increase in LC3 - II in HeLa . Nsi or SENP3i ( I ) was transfected into HeLa . Two days post - transfection were treated with DFP for a further 24 h . Whole lysate samples were blotted as indicated ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; siRNA , 20nM ; DFP , 1mM for 24 h ; values normalized to control value ; n = 3 , biological replicates ; * * * , p < 0 . 001 ; N . S . , not significant , p > 0 . 05 ; paired t - ) .

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SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).


Nsi or SENP3i ( II ) was transfected into HeLa with stable expression of mito - Keima grown on 6 - well plates . Two days post - transfection were treated with DFP ( 1mM ; 24 h ) . transfected with Nsi treated with CQ ( 50μM ; 24 h ) in absence or presence of DFP ( 1mM ; 24 h ) were included as a negative control for mitophagy induction . show overlaid transmitted of unfixed , live obtained using an EVOS - fl inverted LED microscope ( Red : mitophagic ; Scale bar , 100 μm ) . Histogram shows percentages of mitophagic under 6 different conditions ( n = 3 , biological replicates ; * * * * , p < 0 . 0001 ; Ordinary one - way ANOVA followed by Tukey ' s multiple comparisons ) .

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

Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).


SENP3 knockdown abolishes DFP - induced mitophagic autolysosomes . Nsi or SENP3i ( I ) together with mito - pHfluorin were transfected into HeLa in absence or presence of DFP ( 24 h ) , were analyzed using mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or indicating occurrences of mitophagy marked by arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Magenta : SENP3 ; Scale bar , 10 μm ; n = 28 per condition from two experiments ; * * * * p < 0 . 0001 ; unpaired t - ) .

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

SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).


Fis1 knockdown abolishes DFP - induced LC3 - II . Fis1 siRNA was introduced into HeLa for 48 h , were treated with DFP for further 24 h ( Nsi , non - specific siRNA ; Fis1i , Fis1 siRNA ; siRNA , 20nM ; DFP , 1mM ) . Whole lysate samples were blotted as indicated . Values are presented as mean ± are normalized to control value ( n = 3 , biological replicates ; * * , p < 0 . 01 ; paired t - ) .

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

Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).


Fis1 knockout abolishes DFP - induced LC3 - II . Fis1 + / + or Fis1 - / - were treated with DFP for 24 h . Whole lysate samples were blotted as indicated . Values are presented as mean ± are normalized to control value ( n = 6 , biological replicates ; * , p < 0 . 05 ; paired t - ) .

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

Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).