Figure 2A Cell volume of WT and Y / T DKO clones at steady state measured using DHM . Each dot represents a single cell . Error bars represent mean ± 95 % CI . Kruskal - Wallis test with Dunn ' s post - hoc . Data from three independent experiments . * * * p < 0 . 001 , p = 0 . 0917 ( Y / T DKO # 1 vs # 2 ) . B Comparison of cell volume of WT and LATS1 / 2 DKO clones at steady state obtained using DHM . Each dot represents a single cell . Data from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis test with Dunn ' s post - hoc . * * p = 0 . 0018 ( WT vs LATS1 / 2 DKO # 1 ) , * * * p < 0 . 001 ( WT vs LATS1 / 2 DKO # 2 ) , p = 0 . 0557 ( LATS1 / 2 DKO # 1 vs # 2 ) . C Comparison of cell volume of WT and MST1 / 2 DKO at steady state obtained using DHM . Each dot represents a single cell . Data from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U test . p = 0 . 2731 . D HEK293A NF2 KO cells response to 100mbar cyclic hydrostatic pressure . Each dot represents a single cell and Error bars represent mean ± 95 % CI . Data pooled from three independent experiments . Mann - Whitney U test . p = 0 . 4072 . E Comparison of cell volume of WT and TEAD KO clones at steady state obtained using DHM . Each dot represents a single cell . Data from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis test with Dunn ' s post - hoc . p > 0 . 9999 for all comparisons . F Maxima and minima are identified to calculate average percentage change in cell volume in response to hydrostatic pressure using measurements obtained by DHM . G Representative WT and Y / T DKO single cell volume change in response to hydrostatic pressure recorded by DHM . H Average change in cell volume in response to hydrostatic pressure in WT and Y / T DKO clones . Each dot represents a single cell . Images and data were acquired at the onset of oscillating pressure . Data from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis test with Dunn ' s post hoc . * * * p < 0 . 001 ( for both comparisons ) , p = 0 . 1450 ( Y / T DKO # 1 vs # 2 ) . I Representative images of Flipper - TR labelled HEK293A WT ( top ) , Y / T DKO # 1 ( middle ) and Y / T DKO # 2 ( bottom ) are shown obtained using fluorescence lifetime imaging microscopy ( FLIM ) ( ex . 485 , em . 600 ) J Average fluorescence lifetime of Flipper - TR probe quantified using fluorescence lifetime imaging microscope ( FLIM ) ( ex . 485 , em . 600 ) . HEK293A wildtype and Y / T DKO clones # 1 and # 2 were labelled with 2μM Flipper - TR for 15 minutes prior to imaging . The average lifetime measurements were obtained by fitting the photon histogram with a double exponential and the longest lifetime was extracted to report plasma membrane tension , Each dot represents fluorescence lifetime of a single cell and data shown is pooled from three independent experiments ( mean ± 95 % CI ) . Kruskal - Wallis test with Dunn ' s post hoc . * * p = 0 . 0027 ( WT vs Y / T DKO # 1 ) , * p = 0 . 0136 ( WT vs Y / T DKO # 2 ) , p > 0 . 9999 ( Y / T DKO # 1 vs # 2 ) . K Representative immunofluorescence images showing MYC - tagged YAP expression in WT YAP re - expressing cells and S94A mutant YAP re - expressing cells compared to Y / T DKO vector control . 95 % were positive for expression of YAP or YAP mutant . Scale bar = 20µm . L Western blot showing total YAP , TAZ and CYR61 levels in WT YAP and S94A mutant YAP re - expressing cells compared to WT and Y / T DKO vector control . M Average change in cell volume in response to hydrostatic pressure in WT YAP and S94A mutant YAP re - expressing cells relative to Y / T DKO . Each dot represents a single cell . Error bars represent mean ± 95 % CI . Data pooled from three independent experiments . Kruskal - Wallis test with Dunn ' s post hoc . p < 0 . 001 ( vector vs . WT YAP ) , p = 0 . 4912 ( vector vs . S94A YAP ) , p = 0 . 0327N Average change in cell volume in response to 100mbar cyclic hydrostatic pressure in WT and TEAD KO clones . Each dot represents a single cell . Data pooled from three independent experiments . Error bars represent mean ± 95 % CI . Kruskal - Wallis test with Dunn ' s post hoc . * * * p < 0 . 001 ( for both comparisons ) , p > 0 . 9999 ( TEAD KO # 1 vs # 2 ) . O WT and Y / T DKO cell volume were measured at steady - state and the average change in cell volume was quantified using the DHM as in previous experiments , these are labelled ( - ) . Cells were then subjected to cyclic hydrostatic pressure for 2 hours and their cell volume was quantified to determine whether prior exposure to hydrostatic pressure ( ' pre - conditioning ' ) would change their cell volume response , these are labelled ( + ) . Each dot represents a single cell and error bars represent 95 % CI . Graphs include data obtained from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . ns = p > 0 . 9999 . P The average percentage change in cell volume was quantified under the same experimental conditions as in " O " . The average change in cell volume in response to cyclic hydrostatic pressure of cells with no previous exposure to hydrostatic pressure ( labelled as ( - ) ) was quantified using DHM and compared to those with prior exposure to hydrostatic pressure ( labelled ( + ) ) . Each dot represents a single cell and error bars represent 95 % CI . Graphs include data obtained from four independent experiments . Kruskal - Wallis with Dunn ' s post hoc . * * p = 0 . 006 ( WT ) , p = 0 . 4173 ( Y / T DKO ) , ns = p > 0 . 9999 .

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

Figure 2A Cell volume of WT and Y/T DKO clones at steady state measured using DHM. Each dot represents a single cell. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. Data from three independent experiments. ***p<0.001, p=0.0917 (Y/T DKO #1 vs #2).B Comparison of cell volume of WT and LATS1/2 DKO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. **p=0.0018 (WT vs LATS1/2 DKO #1), ***p<0.001 (WT vs LATS1/2 DKO #2), p=0.0557 (LATS1/2 DKO #1 vs #2).C Comparison of cell volume of WT and MST1/2 DKO at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.2731.D HEK293A NF2 KO cells response to 100mbar cyclic hydrostatic pressure. Each dot represents a single cell and Error bars represent mean ± 95% CI. Data pooled from three independent experiments. Mann-Whitney U test. p=0.4072.E Comparison of cell volume of WT and TEAD KO clones at steady state obtained using DHM. Each dot represents a single cell. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post-hoc. p>0.9999 for all comparisons.F Maxima and minima are identified to calculate average percentage change in cell volume in response to hydrostatic pressure using measurements obtained by DHM.G Representative WT and Y/T DKO single cell volume change in response to hydrostatic pressure recorded by DHM.H Average change in cell volume in response to hydrostatic pressure in WT and Y/T DKO clones. Each dot represents a single cell. Images and data were acquired at the onset of oscillating pressure. Data from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post hoc. ***p < 0.001 (for both comparisons), p=0.1450 (Y/T DKO #1 vs #2).I Representative images of Flipper-TR labelled HEK293A WT (top), Y/T DKO #1 (middle) and Y/T DKO #2 (bottom) are shown obtained using fluorescence lifetime imaging microscopy (FLIM) (ex. 485, em. 600)J Average fluorescence lifetime of Flipper-TR probe quantified using fluorescence lifetime imaging microscope (FLIM) (ex. 485, em. 600). HEK293A wildtype and Y/T DKO clones #1 and #2 were labelled with 2μM Flipper-TR for 15 minutes prior to imaging. The average lifetime measurements were obtained by fitting the photon histogram with a double exponential and the longest lifetime was extracted to report plasma membrane tension, Each dot represents fluorescence lifetime of a single cell and data shown is pooled from three independent experiments (mean±95% CI). Kruskal-Wallis test with Dunn's post hoc. **p=0.0027 (WT vs Y/T DKO#1), *p=0.0136 (WT vs Y/T DKO #2), p>0.9999 (Y/T DKO#1 vs #2).K Representative immunofluorescence images showing MYC-tagged YAP expression in WT YAP re-expressing cells and S94A mutant YAP re-expressing cells compared to Y/T DKO vector control. 95% were positive for expression of YAP or YAP mutant. Scale bar = 20µm.L Western blot showing total YAP, TAZ and CYR61 levels in WT YAP and S94A mutant YAP re-expressing cells compared to WT and Y/T DKO vector control.M Average change in cell volume in response to hydrostatic pressure in WT YAP and S94A mutant YAP re-expressing cells relative to Y/T DKO. Each dot represents a single cell. Error bars represent mean ± 95% CI. Data pooled from three independent experiments. Kruskal-Wallis test with Dunn's post hoc. p<0.001 (vector vs. WT YAP), p=0.4912 (vector vs. S94A YAP), p=0.0327N Average change in cell volume in response to 100mbar cyclic hydrostatic pressure in WT and TEAD KO clones. Each dot represents a single cell. Data pooled from three independent experiments. Error bars represent mean ± 95% CI. Kruskal-Wallis test with Dunn's post hoc. ***p < 0.001 (for both comparisons), p>0.9999 (TEAD KO #1 vs #2).O WT and Y/T DKO cell volume were measured at steady-state and the average change in cell volume was quantified using the DHM as in previous experiments, these are labelled (-). Cells were then subjected to cyclic hydrostatic pressure for 2 hours and their cell volume was quantified to determine whether prior exposure to hydrostatic pressure ('pre-conditioning') would change their cell volume response, these are labelled (+). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal-Wallis with Dunn's post hoc. ns= p>0.9999. P The average percentage change in cell volume was quantified under the same experimental conditions as in "O". The average change in cell volume in response to cyclic hydrostatic pressure of cells with no previous exposure to hydrostatic pressure (labelled as (-)) was quantified using DHM and compared to those with prior exposure to hydrostatic pressure (labelled (+)). Each dot represents a single cell and error bars represent 95% CI. Graphs include data obtained from four independent experiments. Kruskal-Wallis with Dunn's post hoc. **p=0.006 (WT), p=0.4173 (Y/T DKO), ns= p>0.9999.


Figure 3C 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 ) .

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

Figure 3C 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).


Figure 5A 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 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 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 ) .

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

Figure 5A 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 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 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).


Figure 1A , 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 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 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 ) , 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 ) , 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 .

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

Figure 1A, 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 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 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), 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), 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.


Figure 2B 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 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 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 .

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

Figure 2B 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 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 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.


Figure 3B - 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 .

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

Figure 3B-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.


Figure 4A 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 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 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 .

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

Figure 4A 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 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 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.


Figure 5A , 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 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 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 ) .

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

Figure 5A, 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 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 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).


Figure 6A , 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 cells . 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 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 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 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 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 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 .

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

Figure 6A, 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 cells.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 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 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 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 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 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.


Figure 7A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A 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.


Figure 8A 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A 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.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.


Figure 9A 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 . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A 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.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.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.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).


Figure 10A 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10A 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.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.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).


Figure 1 ( 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 ) and are representative for two independent experiments .

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

Figure 1(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) and are representative for two independent experiments.


Figure 2 ( 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 ) 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 .

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Figure 2(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) 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.


Figure 3 ( 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 .

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

Figure 3(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.


Figure 4Phosphoflow 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 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 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 .

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

Figure 4Phosphoflow 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 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 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.


Figure 5 ( 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 . 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 ) 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 .

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

Figure 5(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.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) 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.


Figure 6 ( 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 ) .

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Figure 6(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).


Figure 1Coomassie stained SDS - PAGE of McjD - C547 reconstituted in liposomes . The amount reconstituted was evaluated against detergent purified proteinChromatogram 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 squareThe 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

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

Figure 1Coomassie stained SDS-PAGE of McjD-C547 reconstituted in liposomes. The amount reconstituted was evaluated against detergent purified proteinChromatogram 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 squareThe 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


Figure 2Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicatedApparent 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

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

Figure 2Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicatedApparent 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


Figure 3Representative 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

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Figure 3Representative 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


Figure 4Representative 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

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

Figure 4Representative 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


Figure 5Representative 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 )

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

Figure 5Representative 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)


Figure 1B 18F - FDG PET - MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX . C H & E staining of FNB and the resection specimen . Scale bars represent 50 µm . 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 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 . 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 - based copy number profiles for ID188 ( p7 ) and ID211 ( p6 ) organoids . 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 ) .

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

Figure 1B 18F-FDG PET-MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX.C H&E staining of FNB and the resection specimen. Scale bars represent 50 µm.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 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.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-based copy number profiles for ID188 (p7) and ID211 (p6) organoids.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).