C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively . D Frequency of meiosis I metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - C were used to perform a metaphase spread as shown in Fig 1H and 1I . n = 3 mice for each genotype . The total numbers of counted MI cells in the four types of spermatocytes were 89 , 73 , 132 and 104 , respectively . E Frequency of meiosis II metaphase cells with chromosomal abnormalities . The four types of spermatocytes in A - D were used to perform a metaphase spread as shown in Fig 1K and L . n = 3 mice for each genotype . The total numbers of counted MII cells in the four types of spermatocytes were 74 , 87 , 85 and 94 , respectively .

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

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively. D Frequency of meiosis I metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-C were used to perform a metaphase spread as shown in Fig 1H and 1I. n=3 mice for each genotype. The total numbers of counted MI cells in the four types of spermatocytes were 89, 73, 132 and 104, respectively. E Frequency of meiosis II metaphase cells with chromosomal abnormalities. The four types of spermatocytes in A-D were used to perform a metaphase spread as shown in Fig 1K and L. n=3 mice for each genotype. The total numbers of counted MII cells in the four types of spermatocytes were 74, 87, 85 and 94, respectively.


C Frequency of abnormal arrangement in the meiotic metaphase I plate . The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C . n = 3 mice for each genotype . The total numbers of counted metaphase I plates in the four types of spermatocytes were 221 , 181 , 421 and 240 , respectively .

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

C Frequency of abnormal arrangement in the meiotic metaphase I plate. The four types of spermatocytes in A and B were used to perform immunofluorescence as shown in Fig 1B and C. n=3 mice for each genotype. The total numbers of counted metaphase I plates in the four types of spermatocytes were 221, 181, 421 and 240, respectively.


( a ) Effect of steroids on MERS - CoV replication . Vero cells were infected with MERS - CoV at MOI = 0 . 01 in the presence of steroids for 24 h . The viral titer in the cell supernatant was quantified by standard plaque assay using Vero / TMPRSS2 cells . Cell viability in the presence of steroids was quantified by WST assay at 24 hours post - infection ( hpi ) .

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(a) Effect of steroids on MERS-CoV replication. Vero cells were infected with MERS-CoV at MOI = 0.01 in the presence of steroids for 24 h. The viral titer in the cell supernatant was quantified by standard plaque assay using Vero/TMPRSS2 cells. Cell viability in the presence of steroids was quantified by WST assay at 24 hours post-infection (hpi).


( b ) Ciclesonide escape mutant . Vero cells treated with ciclesonide were infected with parental MERS - CoV or ciclesonide escape mutant . Viral titer was measured as described in panel a .

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(b) Ciclesonide escape mutant. Vero cells treated with ciclesonide were infected with parental MERS-CoV or ciclesonide escape mutant. Viral titer was measured as described in panel a.


( c ) Vero cells were infected with the parental MERS - CoV / EMC strain ( Re - EMC / MERS ) or the recombinant mutant strain ( Re - Nsp15 - A25V ) with an amino acid substitution at A25V in NSP15 . Viral titer was measured as described in panel a .

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(c) Vero cells were infected with the parental MERS-CoV/EMC strain (Re-EMC/MERS) or the recombinant mutant strain (Re-Nsp15-A25V) with an amino acid substitution at A25V in NSP15. Viral titer was measured as described in panel a.


( a ) Intracellular SARS - CoV - 2 RNA ( 6 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 1 in the presence of 10 □ μM compounds for 6 h . Cellular viral RNA was quantified by real - time PCR using the E gene primer / probe set .

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(a) Intracellular SARS-CoV-2 RNA (6 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 1 in the presence of 10□μM compounds for 6 h. Cellular viral RNA was quantified by real-time PCR using the E gene primer/probe set.


( b ) Culture Medium SARS - CoV - 2 RNA ( 27 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 0 . 01 in the presence of ciclesonide for 27 h . Viral RNA in culture medium was quantified by real - time PCR using the E gene primer / probe set . Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay .

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(b) Culture Medium SARS-CoV-2 RNA (27 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 0.01 in the presence of ciclesonide for 27 h. Viral RNA in culture medium was quantified by real-time PCR using the E gene primer/probe set. Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay.


Cell height was measured using AFM for HeLa - FUCCI in the indicated phase of mitosis . Below : representative images of synchronised HeLa -

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Cell height was measured using AFM for HeLa-FUCCI in the indicated phase of mitosis. Below: representative images of synchronised HeLa-FUCCI


Representative images of synchronised HeLa stained with an anti - geminin to confirm the indicated phase of mitosis ( scale bar 15 µm ) .

Representative images of synchronised HeLa stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 15 µm).


Nucleus height was measured by immunofluorescence using Hoechst staining


Representative images of synchronised MEFs stained with an anti - geminin to confirm the indicated phase of

Representative images of synchronised MEFs stained with an anti-geminin to confirm the indicated phase of mitosis


Nucleus height of synchronised MEFs ( black dot ) and synchronised MRC - 5 ( blue dot ) were measured by immunofluorescence using Hoechst staining . Data are presented as mean ± s . e . m .

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Nucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.


Nucleus height was measured by immunofluorescence using lamin A / C staining .

Nucleus height was measured by immunofluorescence using lamin A/C staining.


Representative HeLa - FUCCI cells in S - G2 - M ( green ) and G1 phase ( red ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 31 µm .

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Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.


Corresponding percentage of HeLa - FUCCI in mitosis .

Corresponding percentage of HeLa-FUCCI in mitosis.


Representative flow cytometry histograms for EdU positive cells in Ctrl , Blebb and Blebb + AP conditions . Corresponding percentage of EdU positive cells .

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Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions. Corresponding percentage of EdU positive cells.


Analysis of transcription factor activity ( Affymetrix combo protein - DNA array ) for Ctrl , Blebb and Blebb + AP conditions . Each square corresponds to a specific transcription factor ( TF ) activity and is color coded to show the relative activity . ① To identify TFs regulated by nuclear shape , we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation - treated cells ( Blebb ) .

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Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).


TF ' s target genes and GAPDH mRNA levels were analyzed by real - time qPCR . Results are expressed as relative mRNA expression levels .

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TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels.


TEAD activity was analyzed in HeLa cells co - transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD - responsive promoter , and with a renilla plasmid as a gene reporter ; HeLa cells were cultured on 1 and 50 kPa ( n = 3 , t - test * * * p < 0 . 01 ) or treated with blebbistatin and blebbistatin + AP ( n = 6 ; t - test * p < 0 . 5 ) .

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TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5).


AP1 activity was assessed in the indicated conditions using AP1 reporter assay .

AP1 activity was assessed in the indicated conditions using AP1 reporter assay.


Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1 .

Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.


Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . ( b ) Corresponding quantifications of p - Jun Ser63 nuclear intensity .

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Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity.


Representative cells stained for YAP ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( ratio nucleus / cytosol ) .

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Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol).


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Blebb , Blebb + AP condition and in Ctrl , Y27632 and Y27632 + AP .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP.


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Cytochalasin D ( CytoD ) , CytoD + AP condition .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition.


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2 .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2.


Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p - Jun Ser63 ( magenta ) , YAP ( cyan ) and for DNA ( green ) . Additionally , cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity .

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

Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.


Quantifications of YAP activity in Ctrl , Blebb and Blebb + AP conditions , treated or not with importazol ( nucleo - cytoplasmic ratio ) .

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Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio).


Immunoblots of p - YAP ( ser127 ) , YAP , p - LATS ( Thr1079 ) , LATS , p - MST ( Thr1083 ) , MST , Kibra , PTPN14 and GADPH for Ctrl , Blebb and Blebb + AP conditions .

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Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.


Corresponding quantification of p - YAP ( Ser125 ) relative to Ctrl and normalized to GAPDH . Corresponding quantification of p - LATS ( thr1079 ) relative to Ctrl and normalized to GAPDH .

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Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH.


Quantifications of p - Jun Ser63 nuclear intensity in Ctrl , Blebb , and Blebb + AP conditions for cells treated with DMSO , SB203580 , a MAP Kinase inhibitor and SP600125 , a JNK inhibitor .

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Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor.


Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions for cells depleted or not for lamin - A / C . Scale bar = 10 µm . Corresponding quantification of p - Jun Ser63 nuclear intensity .

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Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm. Corresponding quantification of p-Jun Ser63 nuclear intensity.


Results from multiplex ACE2 competition assay are shown for the three spike - derived antigens : RBD , S1 - domain ( S1 ) and homotrimeric spike ( Spike ) . Color - coded beads coated with the respective antigens were co - incubated with biotinylated ACE2 and dilution series of NM1267 ( 8 pM to 126 nM ) followed by measuring residual binding of ACE2 . MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2 - only control . IC50 values were calculated from a four - parametric sigmoidal model . Data are presented as mean ± s . d . of three technical replicates .

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Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.


E Neutralization potency of NM1267 was analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 - mNG infectious clones . Infection rate normalized to virus - only infection control is illustrated as percent of infection ( % Infection ) . IC50 value was calculated from a four - parametric sigmoidal model and data are presented as mean ± s . e . m . of three biological replicates ( n = 3 ) .

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E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).


B For the NeutrobodyPlex , antigen - coated beads comprising RBD , S1 or spike were co - incubated with serum samples from 5 patients and a dilution series of NM1267 ( 1 µM to 6 pM ) ( n = 1 ) . Mean fluorescent intensities ( MFI ) derived from antigen - bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum - only samples , illustrated as MFI ( % control ) , are shown .

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B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.


A , B Total IgGs derived from spike - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . C , D Total IgGs derived from N - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . F

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A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F


A PhosTag - based Western blot probed for YAP reveals increased levels of unphosphorylated ( downshift ) YAP in HEK293A cells in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure ( + ) compared to control ( - ) . B PhosTag - based Western blot probed for TAZ reveals increased levels of unphosphorylated ( downshift ) TAZ in HEK293A cells in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure ( + ) compared to control ( - ) .

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A PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-). B PhosTag-based Western blot probed for TAZ reveals increased levels of unphosphorylated (downshift) TAZ in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-).


C Western blots from cell lysates as in ( A ) and ( B ) show total protein levels in control ( - ) and in cell lysates obtained from cells exposed to hydrostatic pressure ( + ) stimulation in HEK293A cells . Note the decrease in the inhibitory p127 YAP signal as well as increase of YAP / TAZ targets CTGF and CYR61 levels upon hydrostatic pressure .

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C Western blots from cell lysates as in (A) and (B) show total protein levels in control (-) and in cell lysates obtained from cells exposed to hydrostatic pressure (+) stimulation in HEK293A cells. Note the decrease in the inhibitory p127 YAP signal as well as increase of YAP/TAZ targets CTGF and CYR61 levels upon hydrostatic pressure.


D PhosTag - based Western blot probed for YAP reveals increased levels of unphosphorylated ( downshift ) YAP in 143B cells in response to hydrostatic pressure ( + ) compared to control ( - ) .

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D PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in 143B cells in response to hydrostatic pressure (+) compared to control (-).


E Western blot shows total protein levels in control ( - ) and with hydrostatic pressure ( + ) stimulation in 143B cells from cell lysates as in D .

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E Western blot shows total protein levels in control (-) and with hydrostatic pressure (+) stimulation in 143B cells from cell lysates as in D.


F HEK293A cells at steady state ( upper ) or upon cyclic 0 . 1Hz 200mbar hydrostatic pressure ( lower ) were analysed by immunofluorescence . Cells are labelled for Hoechst ( blue ) and YAP ( green ) . Scale bar = 20µm . Graph on the right depict quantification of nuclear - to - cytoplasmic ( Nucl / Cyt ) ratio of YAP in cells as shown on the left . Each dot represents a single cell and data are pooled from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U test . * * * p < 0 . 001 . G 143B cells at steady state ( upper ) or upon cyclic 0 . 1Hz 200mbar hydrostatic pressure ( lower ) . Graph on the right show quantification of nuclear - to - cytoplasmic ( Nucl / Cyt ) ratio of YAP in 143B from images as those shown on the left . Each dot represents a single cell and data are pooled from three independent experiments . Error bars represent mean ± 95 % CI . Mann - Whitney U test . * * * p < 0 . 001 .

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F HEK293A cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower) were analysed by immunofluorescence. Cells are labelled for Hoechst (blue) and YAP (green). Scale bar = 20µm. Graph on the right depict quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in cells as shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001. G 143B cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower). Graph on the right show quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in 143B from images as those shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001.


H Western blot confirming no YAP and TAZ expression in two independent ( # 1 and # 2 ) YAP / TAZ double knockout ( Y / T DKO ) clones .

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

H Western blot confirming no YAP and TAZ expression in two independent (#1 and #2) YAP/TAZ double knockout (Y/T DKO) clones.


I Relative expression levels of YAP / TAZ target genes CYR61 and CTGF in HEK293A WT and Y / T DKO clones in response to cyclic 0 . 1Hz 200mbar hydrostatic pressure . Data from six independent experiments Error bars represent mean ± SD . Kruskal - Wallis test with Dunn ' s post hoc . * * p = 0 . 0661 ( WT CYR61 ) , * * * p = 0 . 0001 ( WT CTGF ) , p = 0 . 3924 ( Y / T DKO # 1 CYR61 ) , p = 0 . 4616 ( Y / T DKO # 1 CTGF ) , p > 0 . 999 ( Y / T DKO # 2 CYR61 ) , p = 0 . 0782 ( Y / T DKO # 2 CTGF ) .

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

I Relative expression levels of YAP/TAZ target genes CYR61 and CTGF in HEK293A WT and Y/T DKO clones in response to cyclic 0.1Hz 200mbar hydrostatic pressure. Data from six independent experiments Error bars represent mean ± SD. Kruskal-Wallis test with Dunn's post hoc. **p=0.0661 (WT CYR61), ***p= 0.0001 (WT CTGF), p=0.3924 (Y/T DKO#1 CYR61), p=0.4616 (Y/T DKO#1 CTGF), p>0.999 (Y/T DKO#2 CYR61), p=0.0782 (Y/T DKO#2 CTGF).


J CYR61 and CTGF gene expression levels induced by hydrostatic pressure in ( I ) of Y / T DKO # 1 and # 2 normalised to WT . Kruskal - Wallis test with Dunn ' s post hoc . * p = 0 . 0259 ( Y / T DKO # 1 CYR61 ) , * * p = 0 . 0007 ( Y / T DKO # 1 CTGF ) , * p = 0 . 0155 ( Y / T DKO # 2 CYR61 ) , * * * p < 0 . 001 ( Y / T DKO # 2 CTGF ) .

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

J CYR61 and CTGF gene expression levels induced by hydrostatic pressure in (I) of Y/T DKO #1 and #2 normalised to WT. Kruskal-Wallis test with Dunn's post hoc. *p=0.0259 (Y/T DKO#1 CYR61), **p=0.0007 (Y/T DKO#1 CTGF), *p=0.0155 (Y/T DKO#2 CYR61), ***p<0.001 (Y/T DKO#2 CTGF).


A 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 ) .

A 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

C 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 .

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

D 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 .

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

E 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 .

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

F 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 .

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

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 ) .

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

H 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 )

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 ) .

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

J 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 .

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

K 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 .

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

L 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 .

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

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=0.0327


N 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

N 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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

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

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


A Confocal IF 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 ) .

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

A 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 ) .

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

C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A ) 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 .

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

A) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Western Blot 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.


A ) 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

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

A) 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 control


B ) 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 control


C ) 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

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

C) 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 control


D ) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) 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.


Protein 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 .

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

Protein 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 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 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 .

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

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 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 .

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.


B ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

C) 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 .

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

D) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) 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.


After 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

After 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.


A ) 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 ) .

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

A) 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 .

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 .

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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 .

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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 .

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 .

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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 .

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.


( 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

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(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

(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

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(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

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(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

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(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


( 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

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(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


( 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

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(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

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(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


( 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

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(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

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(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

(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

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(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

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(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

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(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