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 ) Volcano plot of proteome analysis . Plotted is the negative log10 of the p - value ( y - axis ) versus the log2 LFQ ratio of SPPL2c overexpressing and control cells ( x - axis ) of a given identified protein . A permutation based FDR correction for multiple hypotheses was applied ( p = 0 . 05 ; s0 = 0 . 1 ; dashed line ) . Open grey circles indicate proteins not - significantly altered in SPPL2c expressing cells , red circles significantly altered proteins after FDR correction . Filled red circles indicate type IV membrane proteins and filled blue circles with red line and blue names type II proteins altered in SPPL2c expressing cells , n = 6 biological repl . per cell line . C ) Western Blot of selected type IV SPPL2c candidate substrates . Endogenous protein levels of VAPA , VAPB , VAMP8 , Syntaxin 8 , 18 ( Stx8 & Stx18 ) and Membrin ( encoded by the gene Golgi SNAP Receptor Complex Member 2 , GOSR2 ) were analyzed in HEK293 cells ectopically expressing wild type SPPL2c ( SPPL2c WT ) or a catalytically inactive SPPL2c ( SPPL2c D / A ) . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as controls . VAPA , VAPB , VAMP8 , Stx8 and Membrin protein levels were decreased in SPPL2c expressing cells but not in control or SPPL2c D / A expressing cells . For Stx18 a cleavage product was detected in SPPL2c WT expressing cells . Expression levels of SPPL2c are depicted and Calnexin serves as loading control .

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

Figure 1A) Volcano plot of proteome analysis. Plotted is the negative log10 of the p-value (y-axis) versus the log2 LFQ ratio of SPPL2c overexpressing and control cells (x-axis) of a given identified protein. A permutation based FDR correction for multiple hypotheses was applied (p = 0.05; s0 = 0.1; dashed line). Open grey circles indicate proteins not-significantly altered in SPPL2c expressing cells, red circles significantly altered proteins after FDR correction. Filled red circles indicate type IV membrane proteins and filled blue circles with red line and blue names type II proteins altered in SPPL2c expressing cells, n=6 biological repl. per cell line.C) Western Blot of selected type IV SPPL2c candidate substrates. Endogenous protein levels of VAPA, VAPB, VAMP8, Syntaxin 8, 18 (Stx8 & Stx18) and Membrin (encoded by the gene Golgi SNAP Receptor Complex Member 2, GOSR2) were analyzed in HEK293 cells ectopically expressing wild type SPPL2c (SPPL2c WT) or a catalytically inactive SPPL2c (SPPL2c D/A). Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. VAPA, VAPB, VAMP8, Stx8 and Membrin protein levels were decreased in SPPL2c expressing cells but not in control or SPPL2c D/A expressing cells. For Stx18 a cleavage product was detected in SPPL2c WT expressing cells. Expression levels of SPPL2c are depicted and Calnexin serves as loading control.


Figure 2A ) SPPL2c overexpression causes a decrease on SPPL3 levels . Endogenous levels of SPPL3 were assessed in cells ectopically expressing wild type SPPL2c ( SPPL2c WT ) or catalytically inactive SPPL2c ( SPPL2c D / A ) by Western Blot . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as controls . Note that SPPL3 expression is reduced in cells expressing SPPL2c wt . Data information : Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlB ) SPPL2c impairs secretion and maturation of glycan - modifying enzymes . Protein levels of endogenous EXTL3 , B4GALT1 , OGFOD3 and GnT - V were monitored in lysates of cells treated with control siRNA ( siCtr ) or with siRNA targeting SPPL3 ( siSPPL3 ) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot . In addition , the secreted forms ( sEXTL3 , sB4GALT1 , sOGFOD3 , sGnTV , ) were analyzed in the corresponding conditioned media using Western Blot . Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down , but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3 . Data information : Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlC ) Processing of ectopically expressed GnT - V with an N - terminal Flag - and C - terminal V5 - tag ( Flag - GnTV - V5 ) was analyzed in lysates and conditioned media ( sup . ) of either SPPL2c wt or SPPL2c D / A expressing cells by Western Blot . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as controls . In addition , maturation of the glycoproteins Nicastrin ( NCT ) , Lamp2 and SPPL2a was monitored by detection of the respective mature ( mat ) and immature ( im ) version . Note that secretion of GnT - V is impaired in SPPL2c wt expressing cells and immature glycoproteins accumulate . Data information : Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlD ) SPPL2c induces mislocalization of GnT - V . Ectopically expressed GnT - V was visualized by immunofluorescence staining using the anti - V5 antibody either in control cells ( Ctr ) or in cells co - expressing either SPPL2c wt or SPPL2c D / A . The ER was stained using the anti - BiP antibody and the Golgi with the anti - TGN46 antibody . While in control cells GnT - V mainly localizes to the Golgi , its localization shifts to more ER - like structures in SPPL2c expressing cells . Scale bar 5 µm .

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

Figure 2A) SPPL2c overexpression causes a decrease on SPPL3 levels. Endogenous levels of SPPL3 were assessed in cells ectopically expressing wild type SPPL2c (SPPL2c WT) or catalytically inactive SPPL2c (SPPL2c D/A) by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. Note that SPPL3 expression is reduced in cells expressing SPPL2c wt. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlB) SPPL2c impairs secretion and maturation of glycan-modifying enzymes. Protein levels of endogenous EXTL3, B4GALT1, OGFOD3 and GnT-V were monitored in lysates of cells treated with control siRNA (siCtr) or with siRNA targeting SPPL3 (siSPPL3) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot. In addition, the secreted forms (sEXTL3, sB4GALT1, sOGFOD3, sGnTV,) were analyzed in the corresponding conditioned media using Western Blot. Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down, but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlC) Processing of ectopically expressed GnT-V with an N-terminal Flag- and C-terminal V5-tag (Flag-GnTV-V5) was analyzed in lysates and conditioned media (sup.) of either SPPL2c wt or SPPL2c D/A expressing cells by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. In addition, maturation of the glycoproteins Nicastrin (NCT), Lamp2 and SPPL2a was monitored by detection of the respective mature (mat) and immature (im) version. Note that secretion of GnT-V is impaired in SPPL2c wt expressing cells and immature glycoproteins accumulate. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlD) SPPL2c induces mislocalization of GnT-V. Ectopically expressed GnT-V was visualized by immunofluorescence staining using the anti-V5 antibody either in control cells (Ctr) or in cells co-expressing either SPPL2c wt or SPPL2c D/A. The ER was stained using the anti-BiP antibody and the Golgi with the anti-TGN46 antibody. While in control cells GnT-V mainly localizes to the Golgi, its localization shifts to more ER-like structures in SPPL2c expressing cells. Scale bar 5 µm.


Figure 3Protein levels of the validated SPPL2c substrates VAPA ( A ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control and overexpression of SPPL2c WT was used as a positive control . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as additional controls for antibody specificity . Processing and maturation of ectopically expressed GnT - V and maturation of endogenous SPPL2a were monitored by Western Blotting . None of the substrates ' knock down fully recapitulated the effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing cells . Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D / A was confirmed using a SPPL2c specific antibody . Protein levels of the validated SPPL2c substrates VAPB ( B ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control and overexpression of SPPL2c WT was used as a positive control . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as additional controls for antibody specificity . Processing and maturation of ectopically expressed GnT - V and maturation of endogenous SPPL2a were monitored by Western Blotting . None of the substrates ' knock down fully recapitulated the effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing cells . Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D / A was confirmed using a SPPL2c specific antibody . Protein levels of the validated SPPL2c substrates VAMP8 ( C ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control and overexpression of SPPL2c WT was used as a positive control . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as additional controls for antibody specificity . Processing and maturation of ectopically expressed GnT - V and maturation of endogenous SPPL2a were monitored by Western Blotting . None of the substrates ' knock down fully recapitulated the effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing cells . Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D / A was confirmed using a SPPL2c specific antibody . Protein levels of the validated SPPL2c substrates Syntaxin 8 ( Stx8 ) ( D ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control and overexpression of SPPL2c WT was used as a positive control . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as additional controls for antibody specificity . Processing and maturation of ectopically expressed GnT - V and maturation of endogenous SPPL2a were monitored by Western Blotting . None of the substrates ' knock down fully recapitulated the effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing cells . Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D / A was confirmed using a SPPL2c specific antibody . Protein levels of the validated SPPL2c substrates Syntaxin 18 ( Stx18 ) ( E ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control and overexpression of SPPL2c WT was used as a positive control . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as additional controls for antibody specificity . Processing and maturation of ectopically expressed GnT - V and maturation of endogenous SPPL2a were monitored by Western Blotting . None of the substrates ' knock down fully recapitulated the effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing cells . Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D / A was confirmed using a SPPL2c specific antibody . Protein levels of the validated SPPL2c substrates Membrin ( F ) were reduced using specific siRNAs . Non - targeting control siRNA ( Ctr ) served as negative control and overexpression of SPPL2c WT was used as a positive control . Non - transfected cells ( - ) or stably transfected but non - induced SPPL2c wt cells ( n / i ) served as additional controls for antibody specificity . Processing and maturation of ectopically expressed GnT - V and maturation of endogenous SPPL2a were monitored by Western Blotting . None of the substrates ' knock down fully recapitulated the effect on GnT - V or on endogenous glycoproteins observed in SPPL2c overexpressing cells . Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D / A was confirmed using a SPPL2c specific antibody .

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

Figure 3Protein levels of the validated SPPL2c substrates VAPA (A) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.Protein levels of the validated SPPL2c substrates VAPB (B) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.Protein levels of the validated SPPL2c substrates VAMP8 (C) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.Protein levels of the validated SPPL2c substrates Syntaxin 8 (Stx8) (D) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.Protein levels of the validated SPPL2c substrates Syntaxin 18 (Stx18) (E) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.Protein levels of the validated SPPL2c substrates Membrin (F) were reduced using specific siRNAs. Non-targeting control siRNA (Ctr) served as negative control and overexpression of SPPL2c WT was used as a positive control. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as additional controls for antibody specificity. Processing and maturation of ectopically expressed GnT-V and maturation of endogenous SPPL2a were monitored by Western Blotting. None of the substrates' knock down fully recapitulated the effect on GnT-V or on endogenous glycoproteins observed in SPPL2c overexpressing cells. Calnexin served as loading control and ectopic expression of SPPL2c WT and SPPL2c D/A was confirmed using a SPPL2c specific antibody.


Figure 4B ) Processing of endogenous Syntaxin 5 ( Stx5 ) and Syntaxin 6 ( Stx6 ) by SPPL2c wt by Western Blot . Non - transfected cells ( - ) , stably transfected but non - induced SPPL2c wt cells ( n / i ) and induced cells stably expressing SPPL2c D / A served as controls . Results are derived from one experiment , thus expression ( SPPL2c ) and loading ( Calnexin ) control are the same . Note , that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not . C ) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT - V , as well maturation of the endogenous glycoproteins Nicastrin ( NCT ) and SPPL2a was analyzed in Western Blot . Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody . While GnT - V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression , SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells . D ) Syntaxin 5 localization is impaired by overexpression of SPPL2c . Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression ( Ctr ) and in cells overexpressing SPPL2c wt or SPPL2c D / A using immunofluorescence . BiP was used as a marker protein of the ER and TGN46 as Golgi marker . White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining . Scale bar 5 µm . E ) Syntaxin 5 knock down induces mislocalization of GnT - V similar to SPPL2c overexpression Cells ectopically expressing GnT - V were treated either with non - specific siRNA ( siCtr ) or with siRNA targeting Syntaxin 5 ( siStx5 ) . GnT - V was visualized in immunofluorescence using anti - V5 antibody targeting its C - terminal tag and either BIP was co - stained as ER marker protein or TGN46 as Golgi marker protein . Scale bar 5 µm .

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

Figure 4B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.C) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells.D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm.E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.


Figure 5After seeding cells were cultured for 72h and expression of catalytically active ( wt ) or non - active ( D / A ) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated . Non - transfected cells ( Ctr ) or non - induced SPPL2c cells ( n / i ) served as controls . The ER was stained with the anti - BiP antibody , the cis / medial - Golgi with the anti - Giantin , the trans - Golgi Network with the anti - TGN46 antibody and all Golgi subcompartments with the CAB45 - specific antibody in immunofluorescence . Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis - Golgi change compared to controls . Scale bar 5 µm .

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

Figure 5After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.


Figure 6A ) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c , human testis and mouse testis tissue . For human and mouse tissue , 5 μg protein of each total testis preparation is loaded . The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1 : 50 ( 0 . 1 μg total protein ) . B ) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL / 6J ( Black6 ) mice . Endogenous Nicastrin ( NCT ) and SPPL2a were analysed in membrane preparations from testis , kidney , brain , spleen and liver in littermate male mice . Note that immature proteins ( im ) accumulate in testis compared to other tissues . SPPL2c protein is only detected in testis tissue . C ) Analysis of Syntaxin 8 ( Stx8 ) in SPPL2c - / - mice compared to wildtype ( wt ) littermates . Membrane isolates from whole testis were analyzed for Syntaxin 8 ( Stx8 ) and SPPL2c expression . TMEM192 serves as loading control . D ) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192 . Bars represent mean values of n = 3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice . Statistical significance was calculated applying an unpaired , two - sided Student ' s t - test . * p ≤ 0 . 05 . Note that Syntaxin 8 is accumulating in SPPL2c - / - mice . E ) Real - time qPCR analysis of Syntaxin 8 ( Stx8 ) mRNA levels in testis of SPPL2c - / - and the wildtype mice normalized to that of Tuba1a . The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n = 6 individuals from two independent experiments ± SD . For statistical analysis an unpaired , two - sided Student ' s t - test was performed . ns = not significant . F ) Immunohistochemical stainings of cross - sectioned seminiferous tubules of SPPL2c + / + and SPPL2c - / - mice using anti - Cab45 antibody . Stages VII / VIII are depicted . Higher magnifications are indicated with dashed lines and black arrows indicate pre - acrosomal structures . Scale bar 50 µm . G ) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c - / - mice compared to SPPL2c + / + ( wt ) . Each circle represents one lectin . The lectins above the dashed line ( light blue ) have a significant p - value < 0 . 05 . Lectins on the left side of the blot demonstrate reduced binding while UEA - I on the right side has increased binding . n = 4 animals per genotype , two - sided , unpaired Student ' s t - Test .

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

Figure 6A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein).B) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL/6J (Black6) mice. Endogenous Nicastrin (NCT) and SPPL2a were analysed in membrane preparations from testis, kidney, brain, spleen and liver in littermate male mice. Note that immature proteins (im) accumulate in testis compared to other tissues. SPPL2c protein is only detected in testis tissue.C) Analysis of Syntaxin 8 (Stx8) in SPPL2c-/- mice compared to wildtype (wt) littermates. Membrane isolates from whole testis were analyzed for Syntaxin 8 (Stx8) and SPPL2c expression. TMEM192 serves as loading control.D) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192. Bars represent mean values of n=3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice. Statistical significance was calculated applying an unpaired, two-sided Student's t-test. * p≤0.05. Note that Syntaxin 8 is accumulating in SPPL2c-/- mice.E) Real-time qPCR analysis of Syntaxin 8 (Stx8) mRNA levels in testis of SPPL2c-/- and the wildtype mice normalized to that of Tuba1a. The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n=6 individuals from two independent experiments ± SD. For statistical analysis an unpaired, two-sided Student's t-test was performed. ns = not significant.F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm.G) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c-/- mice compared to SPPL2c+/+ (wt). Each circle represents one lectin. The lectins above the dashed line (light blue) have a significant p-value<0.05. Lectins on the left side of the blot demonstrate reduced binding while UEA-I on the right side has increased binding. n=4 animals per genotype, two-sided, unpaired Student's t-Test.


Figure 1 ( A ) Western blotting for phospho - Thr172 - AMPK ( pAMPK ) , total AMPK , phospho - Ser79 - ACC ( pACC ) , and total ACC 24h after treatment . Quantification represents the pAMPK / AMPK and pACC / ACC ratios relative to the non - treated ( NT ) control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( B ) Phase contrast images of fibers 72h after treatment . Scale bars represent 0 . 25mm ( C ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( D - E ) RT - qPCR analysis of the mRNA levels of MyoD ( D 24h after treatment relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( D - E ) RT - qPCR analysis of the mRNA levels o myogenin ( E ) 24h after treatment relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (A) Western blotting for phospho-Thr172-AMPK (pAMPK), total AMPK, phospho-Ser79-ACC (pACC), and total ACC 24h after treatment. Quantification represents the pAMPK/AMPK and pACC/ACC ratios relative to the non-treated (NT) control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (B) Phase contrast images of fibers 72h after treatment. Scale bars represent 0.25mm  (C) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (D-E) RT-qPCR analysis of the mRNA levels of MyoD (D 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (D-E) RT-qPCR analysis of the mRNA levels o myogenin (E) 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 2 ( A - B ) Rates of glucose consumption ( A measured in the media 24h after treatment relative to the non - treated ( NT ) control from three independent experiments ( n = 3 ) . Error bars represent the SEM Data information : Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls

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

Figure 2 (A-B) Rates of glucose consumption (A measured in the media 24h after treatment relative to the non-treated (NT) control from three independent experiments (n = 3). Error bars represent the SEM Data information: Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls


Figure 3 ( A ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K ( n = 3 ) and pS6 / S6 ( n = 4 ) ratios relative to the non - treated ( NT ) control Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( B ) Radiographic analysis of de novo protein synthesis using 35S - labelling . Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

Figure 3 (A) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K (n = 3) and pS6/S6 (n = 4) ratios relative to the non-treated (NT) control Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls (B) Radiographic analysis of de novo protein synthesis using 35S-labelling. Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 6 ( A ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( B - C ) Rates of glucose consumption ( B measured in the media 24h after treatment relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( B - C ) Rates o lactate production ( C ) measured in the media 24h after treatment relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( D ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( E ) Western blotting of iNOS , tubulin , phospho - Thr172 AMPKα ( pAMPK ) , total AMPKα ( AMPK ) , phospho - Ser79 - ACC ( pACC ) and total ACC . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( F ) RT - qPCR analysis of iNOS mRNA levels relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( G ) Nitric oxide ( NO ) levels in the culture media of cells relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

Figure 6 (A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (B-C) Rates of glucose consumption (B measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (B-C) Rates o lactate production (C) measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (D) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (E) Western blotting of iNOS, tubulin, phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC (pACC) and total ACC. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls (F) RT-qPCR analysis of iNOS mRNA levels relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls (G) Nitric oxide (NO) levels in the culture media of cells relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 7 ( A ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( B ) Western blotting for iNOS and tubulin protein levels 24h after treatment . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( C ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

Figure 7 (A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls  (B) Western blotting for iNOS and tubulin protein levels 24h after treatment. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls  (C) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 8 ( A ) Representative images of overall hind limb musculature . Scale bar represents 1cm ( B ) Western blotting for phospho - Ser79 - ACC ( pACC ) and total ACC ( C ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls ( D - E ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( D mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls ( D - E ) RT - qPCR analysis o MuRF1 ( E ) mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8 (A) Representative images of overall hind limb musculature. Scale bar represents 1cm  (B) Western blotting for phospho-Ser79-ACC (pACC) and total ACC  (C) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls  (D-E) RT-qPCR analysis of Atrogin-1/MAFbx (D mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls  (D-E) RT-qPCR analysis o MuRF1 (E) mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


Figure 9 ( A ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls ( B - C ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( B mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls ( B - C ) RT - qPCR analysis o MuRF1 ( C ) mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9 (A) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls  (B-C) RT-qPCR analysis of Atrogin-1/MAFbx (B mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls  (B-C) RT-qPCR analysis o MuRF1 (C) mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


Figure 3Genes with higher dominant PAS usage ( DPU ) ( A ) or with lower APA Shannon index ( B ) express at higher levels ( liver shown as example ) . The number of genes in each group is indicated on top . Mann - Whitney U test significance levels are indicated for neighboring groups : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . The boxplots show the Q1 to Q3 quartile values ( the box limits ) , the median ( the horizontal green lines ) and values within the 1 . 5 * IQR ( the whiskers ) . Spearman correlations between gene expression level and dominant PAS usage ( DPU ) in each tissue for all genes ( blue ) , genes with DPU equal to or above 90 % and genes with DPU less than 90 % . Genes with DPU less than 90 % exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue . An empty square indicates that the P value of Spearman ' s rho is above 0 . 01 . ( ESC : embryonic stem cells , CER : cerebellum , COR : cerebral cortex , HEA : heart , KID : kidney , LIV : liver , LUN : lung , MUS : muscle , SPL : spleen . )

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

Figure 3Genes with higher dominant PAS usage (DPU) (A) or with lower APA Shannon index (B) express at higher levels (liver shown as example). The number of genes in each group is indicated on top. Mann-Whitney U test significance levels are indicated for neighboring groups: *P < 0.05; **P < 0.01; ***P < 0.001. The boxplots show the Q1 to Q3 quartile values (the box limits), the median (the horizontal green lines) and values within the 1.5 * IQR (the whiskers).Spearman correlations between gene expression level and dominant PAS usage (DPU) in each tissue for all genes (blue), genes with DPU equal to or above 90% and genes with DPU less than 90%. Genes with DPU less than 90% exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue. An empty square indicates that the P value of Spearman's rho is above 0.01. (ESC: embryonic stem cells, CER: cerebellum, COR: cerebral cortex, HEA: heart, KID: kidney, LIV: liver, LUN: lung, MUS: muscle, SPL: spleen.)


Figure 4Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons . Same : isoforms enriched in same cellular location . Diff : isoforms enriched in different cellular locations , i . e . one isoform in neuropil and another one in soma . ( Fisher exact test : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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

Figure 4Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons. Same: isoforms enriched in same cellular location. Diff: isoforms enriched in different cellular locations, i.e. one isoform in neuropil and another one in soma. (Fisher exact test: *P < 0.05; **P < 0.01; ***P < 0.001).


Figure 72D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL / 6J ( BL6 ) or SPRET / EiJ ( SPR ) allelic PAS usages from 5 , 264 genes ( see Methods ) . 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5 , 264 genes . All clustering was performed hierarchically using Pearson correlations between samples .

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

Figure 72D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL/6J (BL6) or SPRET/EiJ (SPR) allelic PAS usages from 5,264 genes (see Methods). 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5,264 genes. All clustering was performed hierarchically using Pearson correlations between samples.


Figure 1Representative Ca2 + signals in sperm from a patient with deafness - infertility syndrome lacking functional CatSper channels ( CATSPER2 - / - ) ( red ) and a healthy donor ( black ) , evoked by progesterone , PGE1 , NH4Cl , or ionomycin . NH4Cl increases the intracellular pH . Bar graph : Amplitudes ( n = 4 ; mean ± SD ) of Ca2 + signals in CATSPER2 - / - spermRepresentative monovalent CatSper currents in CATSPER2 - / - sperm ( blue , green , orange , purple , brown ) and in sperm from a healthy donor ( black ) and corresponding current - voltage relationship ( right ) . The membrane voltage was stepped from - 100 mV to + 100 mV in increments of 10 mV from a holding potential of - 80 mV . Outward and inward current amplitudes ( mean ± SD ) at + 100 mV and - 100 mV , respectively , in CATSPER2 - / - sperm ( color code : panel B ) and sperm from healthy donors ( black ) . Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2 - / - sperm form DIS patients using antibodies directed against CatSper 3 ( D ) or CatSper 4 ( E ) ; DNA was labelled with DAPI ( blue ) . Scale bars represent 10 µm . 3D - STORM image in xy - projection of sperm from a healthy donor labelled with the anti - CatSper 3 antibody ( left ) . Axial projection of the boxed region ( right ) . Scale bars represent 5 µm in xy - projections and 200 nm in axial projections . 3D - STORM images in xy - projection of CATSPER2 - / - sperm ( left ) labelled with the anti - CatSper 3 ( upper panel ) and anti - CatSper 4 ( lower panel ) antibody . Axial projection of the boxed regions ( right ) . Scale bars represent 5 µm in xy - projections and 200 nm in axial projections .

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

Figure 1Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- spermRepresentative monovalent CatSper currents in CATSPER2-/- sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black) and corresponding current-voltage relationship (right). The membrane voltage was stepped from -100 mV to +100 mV in increments of 10 mV from a holding potential of -80 mV. Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2-/- sperm (color code: panel B) and sperm from healthy donors (black).Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm.3D-STORM image in xy-projection of sperm from a healthy donor labelled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.3D-STORM images in xy-projection of CATSPER2-/- sperm (left) labelled with the anti-CatSper 3 (upper panel) and anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.


Figure 2Dark - field microscopy of a single sperm ; shown are single frames obtained at t = 0 , 48 , 96 , and 136 ms . Scale bar = 25 µmDark - field imaging of a sperm population ; left : single frames at t = 185 , 363 , 540 , and 718 ms . Sperm selected for analysis are highlighted ( 1 - 4 ) . Right : Temporal change in the brightness ( blinking ) of sperm heads . The blue lines correspond to the time - points of the single frames . Scale bar = 25 µm . Representative distribution of rotation frequencies of freely swimming non - capacitated ( 0 mM bicarbonate ; black ; n = 218 ) and capacitated sperm ( 25 mM bicarbonate ; red ; n = 232 ) determined by dark - field imaging . Rolling frequency ( mean ± SD ) of non - capacitated ( 0 mM bicarbonate , n = 1 , 455 ) and capacitated sperm ( 25 mM bicarbonate , n = 1 , 097 ) . Rolling frequency ( mean ± SD ) of freely swimming sperm in 0 ( n = 1 , 175 ) , 0 . 2 ( n = 832 ) , and 1 % ( w / v ) ( n = 599 ) methyl cellulose . . Bright - field images of an optically trapped sperm cell , obtained at t = 0 , 55 , 155 , and 185 ms . Scale bar represents 10 µm . Representative time - course of the rotation frequency of trapped sperm ( each sperm cell is represented by a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . . Distribution of rotation frequencies in trapped sperm ( n = 32 ; mean frequency ± SD 6 . 0 ± 2 . 1 Hz ) . Image series of a sperm cell trapped parallel to the optical axis ; images were obtained at t = 0 , 95 , 165 , and 205 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm . Representative distribution of rotation frequencies of freely swimming CatSper - deficient sperm incubated under non - capacitating ( 0 mM bicarbonate ; black ; n = 73 ) and capacitating conditions ( 25 mM bicarbonate ; red ; n = 272 ) . Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm incubated under non - capacitating ( 0 mM bicarbonate , n = 1009 ) and capacitating ( 25 mM bicarbonate , n = 946 ) conditions . Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm in 0 ( n = 457 ) , 0 . 2 ( n = 389 ) , and 1 % ( w / v ) ( n = 187 ) methyl cellulose . Image series of a CATSPER2 - / - sperm cell optically trapped perpendicular to the optical axis ; images were obtained at t = 0 , 75 , 150 , and 225 ms . Scale bar represents 10 µm . Image series of a CATSPER2 - / - sperm cell optically trapped parallel to the optical axis ; images were obtained at t = 0 , 60 , 195 , and 320 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm . Representative time courses of the rotation frequencies of optically trapped CATSPER2 - / - sperm ( each sperm is represented in a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis .

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

Figure 2Dark-field microscopy of a single sperm; shown are single frames obtained at t = 0, 48, 96, and 136 ms. Scale bar = 25 µmDark-field imaging of a sperm population; left: single frames at t = 185, 363, 540, and 718 ms. Sperm selected for analysis are highlighted (1-4). Right: Temporal change in the brightness (blinking) of sperm heads. The blue lines correspond to the time-points of the single frames. Scale bar = 25 µm.Representative distribution of rotation frequencies of freely swimming non-capacitated (0 mM bicarbonate; black; n = 218) and capacitated sperm (25 mM bicarbonate; red; n = 232) determined by dark-field imaging.Rolling frequency (mean ± SD) of non-capacitated (0 mM bicarbonate, n = 1,455) and capacitated sperm (25 mM bicarbonate, n = 1,097).Rolling frequency (mean ± SD) of freely swimming sperm in 0 (n = 1,175), 0.2 (n = 832), and 1 % (w/v) (n = 599) methyl cellulose. .Bright-field images of an optically trapped sperm cell, obtained at t = 0, 55, 155, and 185 ms. Scale bar represents 10 µm.Representative time-course of the rotation frequency of trapped sperm (each sperm cell is represented by a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis..Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz).Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t = 0, 95, 165, and 205 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm.Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272).Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm incubated under non-capacitating (0 mM bicarbonate, n = 1009) and capacitating (25 mM bicarbonate, n = 946) conditions.Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm in 0 (n = 457), 0.2 (n = 389), and 1 % (w/v) (n = 187) methyl cellulose.Image series of a CATSPER2-/- sperm cell optically trapped perpendicular to the optical axis; images were obtained at t = 0, 75, 150, and 225 ms. Scale bar represents 10 µm. Image series of a CATSPER2-/- sperm cell optically trapped parallel to the optical axis; images were obtained at t = 0, 60, 195, and 320 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm. Representative time courses of the rotation frequencies of optically trapped CATSPER2-/- sperm (each sperm is represented in a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.