Figure 7 ( A ) Left panel : In tor1 - 1 avo3 ∆ 1274 - 1430 cells rapamycin specifically inhibits TORC2 but not TORC1 ( Gaubitz et al . , 2015 ) . tor1 - 1 avo3 ∆ 1274 - 1430 cells were treated with cycloheximide ( CHX ) and rapamycin or vehicle ( methanol ) for the indicated times . Right panel : WT or Orm2 mutant cells were treated with CHX for the indicated times . The protein levels of FLAG - Orm2 or of the indicated FLAG - Orm2 mutants from total cell lysates were analyzed by SDS - PAGE and Western blotting with the indicated antibodies . ( B ) Graphs display the FLAG - Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0 , 90 and 180 min after the addition of CHX . Each experiment was repeated at least three times . FLAG - Orm2 levels were normalized to Pgk1 loading controls , and each timepoint was related to t = 0 min ( set to 1 ) . Data are presented as mean ± standard deviation . ( C ) Denaturing immunoprecipitation ( IP ) of FLAG - Orm2 , FLAG - Orm2 - 3A and FLAG - Orm2 - 3D . The control strain expresses untagged Orm2 . Input and elutions ( with FLAG peptides ) were analyzed by SDS - PAGE and Western blot with the indicated antibodies . ( D ) Epifluorescence and phase contrast microscopy of living tul1 ∆ mutants expressing GFP - Orm2 , GFP - Orm2 - 3A or GFP - Orm2 - 3D ( green ) with dsRed - HDEL ( red ) . Arrowheads point to GFP - Orm2 accumulations outside the endoplasmic reticulum ( ER ) . Scale bars = 5 µm . ( E ) SDS - PAGE and Western blot analysis with the indicated antibodies of input and elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from cells expressing FLAG - Orm2 - K25 , 33R , FLAG - Orm2 - K25 , 33R - 3A or FLAG - Orm2 - K25 , 33R - 3D . The control strain expresses untagged Orm2 . ( F ) The levels of the long chain bases ( LCB ) C18 - dihydrosphingosine ( C18 - DHS ) and C18 - phytosphingosine ( C18 - PHS ) and of two major ceramide ( Cer ) species in lipid extracts from WT , tul1 ∆ , Orm2 - K25 , 33R , Orm2 - 3A and Orm2 - 3D mutants were measured using LC - MS and quantified using spiked non - yeast LCBs / ceramides as internal standard . Data are normalized to WT levels of the most abundant LCB / ceramide species ( set to 1 ) and presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - test . p values are given above the respective bars .

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

Figure 7(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.(B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.(C) Denaturing immunoprecipitation (IP) of FLAG-Orm2, FLAG-Orm2-3A and FLAG-Orm2-3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS-PAGE and Western blot with the indicated antibodies.(D) Epifluorescence and phase contrast microscopy of living tul1∆ mutants expressing GFP-Orm2, GFP-Orm2-3A or GFP-Orm2-3D (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 µm.(E) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-K25,33R, FLAG-Orm2-K25,33R-3A or FLAG-Orm2-K25,33R-3D. The control strain expresses untagged Orm2.(F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


Figure 1 ( B ) When transposon insertion libraries subjected to ampicillin resistance selection , a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor ( pBR322 bla : : Im7 L53A I54A ) relative to a strain containing just pBR322 . Transposon insertion frequency increased 21 - , 24 - , and 8 - fold in the pstS , pstA , and pstC genes , respectively , compared to the insertion frequency found with no ampicillin selection .

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

Figure 1 (B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.


Figure 2 ( A ) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla : : Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect , pBR322 and pBR322 bla : : Im7 L53A I54A were transformed into strains containing in - frame deletion mutants of each of the pst genes ( pstS , pstC , and pstA , respectively ) . The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla : : Im7 L53A I54A , but did not shower higher ampicillin resistance in the presence of pBR322 . ( B ) Levels of β - lactamase ( Bla ) and β - lactamase - Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β - lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes ( CL248 ( pstS : : Tn5 ) , CL249 ( pstC : : Tn5 ) , CL250 ( pstA : : Tn5 ) ) . Maltose binding protein ( MBP ) was used as a control . ( C ) β - lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay . Data are represented as mean ± SEM from three technical replicates ( unpaired t - test , * * P < 0 . 01 , * * * P < 0 . 001 comparison to MG1655 ; ns , not significant ) . ( D ) To test if levels of the Im7 L53A I54A protein itself , in the absence of the fusion , were also higher in the pstA mutant , expression of the Im7 L53A I54A protein from the IPTG - inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0 - 5 mM . Periplasmic fractions were isolated and examined by SDS - PAGE and subsequent Coomassie staining to determine overall protein expression patterns . Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7 . MBP was used as a loading control . Im7 L53A I54A was increased in the pstA strain relative to the WT control strain . Coomassie staining revealed that a number of periplasmic proteins , including UgpB , were also induced in the pstA strain .

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

Figure 2 (A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322.  (B) Levels of β-lactamase (Bla) and β-lactamase-Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β-lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes (CL248 (pstS::Tn5), CL249 (pstC::Tn5), CL250 (pstA::Tn5)). Maltose binding protein (MBP) was used as a control.  (C) β-lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay. Data are represented as mean ± SEM from three technical replicates (unpaired t-test,** P<0.01, *** P<0.001 comparison to MG1655; ns, not significant).  (D) To test if levels of the Im7 L53A I54A protein itself, in the absence of the fusion, were also higher in the pstA mutant, expression of the Im7 L53A I54A protein from the IPTG-inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0-5 mM. Periplasmic fractions were isolated and examined by SDS-PAGE and subsequent Coomassie staining to determine overall protein expression patterns. Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7. MBP was used as a loading control. Im7 L53A I54A was increased in the pstA strain relative to the WT control strain. Coomassie staining revealed that a number of periplasmic proteins, including UgpB, were also induced in the pstA strain.


Figure 3 ( A ) MG1655 WT , single mutants of pstA and ugpB , and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322 - based folding biosensor constructs . The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations . Strains containing pBR322 bla : : Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation ( compare uppermost left and right panels ) . This resistance is completely lost upon transducing in a ugpB mutation ( compare next panels down ) , showing that ugpB is responsible for the resistance gained by the pstA mutation . ( B ) Ampicillin sensitivity conferred by a ugpB deletion mutant ( CL543 ) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB ( pCL1 ) . ( C ) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein . Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain ( CL543 ) from pCDFTrc and pTrc99a vectors , respectively , both of which have an IPTG - inducible promoter . Periplasmic fractions were isolated and examined by Western blotting . " - " indicates strains containing empty vector and " + " indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB . MBP was used as a loading control . Data information : These experiments were repeated twice with similar results and one representative is shown .

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

Figure 3 (A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation.  (B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1).  (C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown.


Figure 4 ( A ) UgpB overexpression confers bile salt resistance , and a ugpB deletion mutant is slightly CHO sensitive . ( B ) CHO - induced MDH protein aggregation analyzed by monitoring light scattering . ( C ) CHO - induced MDH protein aggregation analyzed by a solubility assay using centrifugation ( cfg ) at 16 , 000 g for 20 min . The supernatant ( Sup ) represents the soluble fraction and the pellet the insoluble fraction ; 12 µM MDH was used as a model aggregation - prone substrate ( see Methods ) . Addition of increasing quantities of UgpB increasingly solubilizes MDH . The molar ratios of MDH : UgpB that were used ( left to right ) are as follows : 1 : 0 , 1 : 0 . 1 , 1 : 0 . 2 , 1 : 0 . 5 , and 1 : 1 . 5 - fold more of the pellet fraction was loaded relative to the supernatant fraction . ( D ) The supernatant / pellet assay was performed using a 1 : 1 MDH : chaperone ratio for Spy , SurA , and OsmY . The pellet fraction was concentrated 5 - fold . ( E ) 12 µM of MDH alone and a 1 : 1 ratio of either MDH : UgpB or MDH : MBP were incubated at 37 ° C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above ( C ) . The pellet fraction was concentrated 5 - fold . In some samples ( + G3P ) , 120 µM of G3P was added prior to the addition of any other protein . Addition of G3P inactivates UgpB ' s ability to inhibit protein aggregation . ( F ) 800 μg of E . coli total lysate and 60 µM of UgpB / MBP were used for the supernatant / pellet assay . 600 µM of G3P was added before adding proteins . The pellet fraction was concentrated 10 - fold . As before , addition of UgpB inhibited aggregation , but only in the absence of G3P . ( G ) The anti - aggregation activity of tryptophan single mutant ( W169S ) and double mutant ( W169S / W172S ) was examined by CHO - induced MDH aggregation assay . Light scattering values were monitored . ( I ) The anti - aggregation activity of the UgpB variants ( E66A and R374V ) was examined by CHO - induced MDH aggregation assay . CHO - induced MDH protein aggregation analyzed by monitoring light scattering . ( J ) Solubility of MDH is also monitored by supernatant / pellet assay . 12 µM of MDH alone and a 1 : 1 ratio of MDH : UgpB proteins , 120 µM of G3P were used . 15 mM CHO were used for all the aggregation and supernatant / pellet assays .

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

Figure 4(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive. (B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering.  (C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction.  (D) The supernatant/pellet assay was performed using a 1:1 MDH:chaperone ratio for Spy, SurA, and OsmY. The pellet fraction was concentrated 5-fold.  (E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation.  (F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P.  (G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored.  (I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering.  (J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used. 15 mM CHO were used for all the aggregation and supernatant/pellet assays.


Figure 6 ( A ) Acid denatures UgpB . The secondary structure of 2 µM UgpB was monitored by circular dichroism ( CD ) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7 . ( B ) G3P binds to acid - stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum , either at pH7 or after transition from pH 2 to 7 ( see Methods ) . Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift , indicating G3P binding . ( C ) The anti - aggregation activity of UgpB is monitored by CHO - induced MDH aggregation assay . Light scattering values were monitored . The anti - aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P ( 120 µM ) can again inactivate its chaperone activity . ( D ) Addition of G3P to UgpB inhibits its anti - aggregation chaperone activity . 12 µM of MDH and a 1 : 1 ratio of UgpB : MDH were used in a CHO - induced aggregation assay . At 30 min , the indicated concentrations of G3P were added to solutions containing MDH , and MDH aggregation was monitored by light scattering . Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan

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

Figure 6(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7. (B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding.  (C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity.  (D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity . 12 µM of MDH and a 1:1 ratio of UgpB:MDH were used in a CHO-induced aggregation assay. At 30 min, the indicated concentrations of G3P were added to solutions containing MDH, and MDH aggregation was monitored by light scattering. Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan fluorescence


Figure 1A , B Intracellular NAD levels and NAD / NADH ratio in DC and age - matched healthy control fibroblasts . All values are presented as mean ± SD of four and eight replicates in A and B , respectively . Student ' s t test was performed on DC cells vs controls . Immunoblots of the expression of NAD synthesis and consuming proteins , and their activities in DC and control cells . Protein levels are normalized to GAPDH . E Immunoblots of the expression of NAD synthesis and consuming proteins Quantification values are presented as mean ± SD of four controls versus five DC samples as shown in D . Student ' s t test was performed on DC cells vs controls . Con : control fibroblasts . F Effects of NR and NAM supplementation ( 24 hours ) on the NAD levels in DC fibroblasts . All values are presented as mean ± SD of four replicates . One - Way ANOVA was performed on DC cells in indicated conditions .

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

Figure 1A, B Intracellular NAD levels and NAD/NADH ratio in DC and age-matched healthy control fibroblasts. All values are presented as mean ± SD of four and eight replicates in A and B, respectively. Student's t test was performed on DC cells vs controls.Immunoblots of the expression of NAD synthesis and consuming proteins, and their activities in DC and control cells. Protein levels are normalized to GAPDH.E Immunoblots of the expression of NAD synthesis and consuming proteins Quantification values are presented as mean ± SD of four controls versus five DC samples as shown in D. Student's t test was performed on DC cells vs controls. Con: control fibroblasts.F Effects of NR and NAM supplementation (24 hours) on the NAD levels in DC fibroblasts. All values are presented as mean ± SD of four replicates. One-Way ANOVA was performed on DC cells in indicated conditions.


Figure 2Quantitative RT - PCR and immunoblots of the expression of CD38 and SARM1 in DC and age - matched health control fibroblasts . All values are presented as mean ± SD of three replicates in quantitative RT - PCR . Protein levels are normalized to GAPDH , and mean ( ± SD ) quantification values of 4 controls vs 5 DC samples are shown . Student ' s t test was performed on DC cells vs controls . Irrelevant intervening lanes have been removed for clarity ( full blots are available online as Source Data ) . Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor , KU - 55933 for 48 hours . Mean ( ± SD ) quantification values of three DC lines with and without ATMi treatment are shown . P values on the basis of Student ' s t test . The efficacy of CD38 shRNA ( sh - 1 and sh - 2 ) was verified by quantitative RT - PCR in DC fibroblasts and by RT - PCR and immunoblots in A549 cells . The relative CD38 mRNA values in the CD38 knockdown cells were normalized to the scrambled shRNA . All values are presented as mean ± SD of three replicates . Student ' s t test was performed on DC and A549 cells in indicated conditions . Intracellular NAD levels in the scramble and CD38 knockdown DC fibroblasts . The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib ( 400nM ) and the SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . All values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC cells in indicated conditions . immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts . The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib ( 400nM ) and the SIRT1 inhibitor EX 527 ( 1 μM ) for 24 hours . Data are representative four replicates . All values are presented as mean ± SD of four replicates . One - way ANOVA was performed on DC cells in indicated conditions .

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

Figure 2Quantitative RT-PCR and immunoblots of the expression of CD38 and SARM1 in DC and age-matched health control fibroblasts. All values are presented as mean ± SD of three replicates in quantitative RT-PCR. Protein levels are normalized to GAPDH, and mean (± SD) quantification values of 4 controls vs 5 DC samples are shown. Student's t test was performed on DC cells vs controls. Irrelevant intervening lanes have been removed for clarity (full blots are available online as Source Data).Immunoblots and quantification of indicated proteins in DC fibroblasts treated with DMSO and with 2 μM ATM inhibitor, KU-55933 for 48 hours. Mean (± SD) quantification values of three DC lines with and without ATMi treatment are shown. P values on the basis of Student's t test.The efficacy of CD38 shRNA (sh-1 and sh-2) was verified by quantitative RT-PCR in DC fibroblasts and by RT-PCR and immunoblots in A549 cells. The relative CD38 mRNA values in the CD38 knockdown cells were normalized to the scrambled shRNA. All values are presented as mean ± SD of three replicates. Student's t test was performed on DC and A549 cells in indicated conditions.Intracellular NAD levels in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.immunoblots of the expression of indicated proteins in the scramble and CD38 knockdown DC fibroblasts. The CD38 knockdown DC fibroblasts were treated with the PARP1 inhibitor Olaparib (400nM) and the SIRT1 inhibitor EX 527 (1 μM) for 24 hours. Data are representative four replicates. All values are presented as mean ± SD of four replicates. One-way ANOVA was performed on DC cells in indicated conditions.


Figure 3Intracellular NAD levels in brain tissues from 6 - month - old G1 and G3 Tert - / - female mice . immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6 - month - old G1 and G3 Tert - / - female mice . The CD38 antibody was validated using the CD38 - / - mouse brain tissues . C Quantification of immunoblots from the indicated proteins in B . Data information : All values are presented as mean ± SD of G1 Tert - / - mouse brain tissues versus G3 Tert - / - mouse brain tissues . n = 4 in each group . P values on the basis of Student ' s t test . D NADase activity in G1 and G3 Tert - / - mouse brain lysate was determined at various time points during the reaction . NADase activity was not detectable in the extracts treated with the CD38 inhibitor , 78c , at a concentration of 50nM .

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Figure 3Intracellular NAD levels in brain tissues from 6-month- old G1 and G3 Tert-/- female mice.immunoblots of the expression of NAD consuming related proteins and their activities in brain tissues from 6-month- old G1 and G3 Tert-/- female mice. The CD38 antibody was validated using the CD38-/- mouse brain tissues.C Quantification of immunoblots from the indicated proteins in B. Data information: All values are presented as mean ± SD of G1 Tert-/- mouse brain tissues versus G3 Tert-/- mouse brain tissues. n=4 in each group. P values on the basis of Student's t test.D NADase activity in G1 and G3 Tert-/- mouse brain lysate was determined at various time points during the reaction. NADase activity was not detectable in the extracts treated with the CD38 inhibitor, 78c, at a concentration of 50nM.


Figure 4A Representative immunoblots of the expression of PGC1 - 1α in DC cells treated with vehicle or 3 mM NR . Quantification of the indicated proteins is from three immunoblots . B - D Cellular and mitochondrial ROS in DC and age - matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates . Cellular and mitochondrial ROS in DC and age - matched healthy control cells in the scramble and CD38 knockdown DC cells ( D ) were measured by flow cytometry from three replicates . E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR . White arrows : mitochondria . Yellow arrow : autophagosome - like structures with engulfed mitochondria . Enlarged image within white frame is shown . F - H Quantifications of percentage of damaged mitochondria per cell ( F ) , mitochondrial length ( G ) , and mitochondrial diameter ( H ) . A minimum of 200 mitochondria counted per group .

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Figure 4A Representative immunoblots of the expression of PGC1-1α in DC cells treated with vehicle or 3 mM NR. Quantification of the indicated proteins is from three immunoblots.B-D Cellular and mitochondrial ROS in DC and age-matched healthy control cells supplemented with vehicle and NR were measured by flow cytometry from three replicates.Cellular and mitochondrial ROS in DC and age-matched healthy control cells in the scramble and CD38 knockdown DC cells (D) were measured by flow cytometry from three replicates.E Representative images captured by transmission electron microscopy of DC1 cells treated with vehicle or NR. White arrows: mitochondria. Yellow arrow: autophagosome-like structures with engulfed mitochondria. Enlarged image within white frame is shown. F-H Quantifications of percentage of damaged mitochondria per cell (F), mitochondrial length (G), and mitochondrial diameter (H). A minimum of 200 mitochondria counted per group.


Figure 5Representative images of mitophagy showing co - localization between the mitophagy dye ( red ) and the lysosome dye ( green ) in DC and control fibroblasts treated with vehicle or 3 mM NR . White arrows : mitophagy . B The mean value of fluorescence intensity / cells in each image was scored . At least 15 images ( ~ 200 cells ) were counted per group . The relative values in each group was normalized to Con1 . C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR . Quantification of immunoblots from the indicated proteins is from three replicates .

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Figure 5Representative images of mitophagy showing co-localization between the mitophagy dye (red) and the lysosome dye (green) in DC and control fibroblasts treated with vehicle or 3 mM NR. White arrows: mitophagy. B The mean value of fluorescence intensity/cells in each image was scored. At least 15 images (~ 200 cells) were counted per group. The relative values in each group was normalized to Con1. C Representative immunoblots of the expression of PINK1 and PARKIN in DC cells treated with vehicle or 3 mM NR. Quantification of immunoblots from the indicated proteins is from three replicates.


Figure 6A PCR amplification efficiency at the telomere in the mock - and FPG - treated DC and age - matched healthy control fibroblasts supplemented with vehicle or 3 mM NR . All values are presented as mean ± SD of three replicates . The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene . B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR . Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d , with or without 3 mM NR treatment . At left , DNA molecular mass markers , in kilobase pairs ( Kb ) . C , D Representative images of γH2AX ( green ) , telomere immuno - FISH ( red ) in DC cells treated with vehicle or 3 mM NR . White arrows : co - localization of γH2AX foci and telomeric DNA ( or TIF ) . Enlarged views of co - localizing foci are shown at the right panel and in Figure EV1 . Percentage of DC and control cells with indicated TIFs per nuclei . ~ 100 cells per condition were scored .

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Figure 6A PCR amplification efficiency at the telomere in the mock- and FPG-treated DC and age-matched healthy control fibroblasts supplemented with vehicle or 3 mM NR. All values are presented as mean ± SD of three replicates. The relative telomere PCR amplification in each sample was normalized to the 36B4 reference gene.B Telomere restriction fragment analysis in DC and control fibroblasts treated with vehicle or 3 mM NR. Genomic DNA was isolated from the indicated cell lines at 0 d or 14 d, with or without 3 mM NR treatment. At left, DNA molecular mass markers, in kilobase pairs (Kb).C, D Representative images of γH2AX (green), telomere immuno-FISH (red) in DC cells treated with vehicle or 3 mM NR. White arrows: co-localization of γH2AX foci and telomeric DNA (or TIF). Enlarged views of co-localizing foci are shown at the right panel and in Figure EV1. Percentage of DC and control cells with indicated TIFs per nuclei. ~ 100 cells per condition were scored.


Figure 7A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR . Each data point is represented as mean ± SD of three replicates . B , C Representative images of BrdU ( red ) and DAPI ( blue ) staining of DC and age - matched healthy control fibroblasts treated with vehicle or NR . Percentage of BrdU - positive cells per condition . Each dot represents the percentage of cells with BrdU staining per image . ~ 400 cells were counted per condition . All values are presented as mean ± SD . D , E Representative images of SPiDER - β - gal ( green ) and DAPI ( blue ) staining of DC and age - matched healthy control cells treated with vehicle or 3 mM NR . Percentage of SA - β - gal - positive cells per condition . Each dot represents the percentage of cells with SA - β - gal staining per image . ~ 400 cells were counted per condition . All values are presented as mean ± SD . F The levels of IL - 6 , IL - 8 , and MCP - 1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR . All values are represented as mean ± SD of three replicates . G , H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR . Quantification of the indicated proteins are presented as mean ± SD from three immunoblots .

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Figure 7A Cumulative population doubling analysis of the proliferation of representative scramble and CD38 knockdown DC fibroblasts or DC fibroblasts treated with vehicle or 3 mM NR. Each data point is represented as mean ± SD of three replicates.B, C Representative images of BrdU (red) and DAPI (blue) staining of DC and age-matched healthy control fibroblasts treated with vehicle or NR. Percentage of BrdU -positive cells per condition. Each dot represents the percentage of cells with BrdU staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.D, E Representative images of SPiDER-β-gal (green) and DAPI (blue) staining of DC and age-matched healthy control cells treated with vehicle or 3 mM NR. Percentage of SA-β-gal-positive cells per condition. Each dot represents the percentage of cells with SA-β-gal staining per image. ~400 cells were counted per condition. All values are presented as mean ± SD.F The levels of IL-6, IL-8, and MCP-1 in both supernatant and cell lysate of indicated DC fibroblasts treated with vehicle or 3 mM NR. All values are represented as mean ± SD of three replicates.G, H Representative immunoblots of p21 and p16 in DC fibroblasts treated with vehicle or 3 mM NR. Quantification of the indicated proteins are presented as mean ± SD from three immunoblots.


Figure 1A - G Double immunolabelling of PCNT - PCNT - ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes on ( A ) zygotene , ( B ) pachytene , ( C ) diplotene , ( D ) diakinesis , ( E ) prometaphase I , ( F ) metaphase I and ( G ) telophase I . Chromatin has been stained with DAPI ( blue ) . Scale bar in G represents 10 μm .

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Figure 1A-G Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes on (A) zygotene, (B) pachytene, (C) diplotene, (D) diakinesis, (E) prometaphase I, (F) metaphase I and (G) telophase I. Chromatin has been stained with DAPI (blue). Scale bar in G represents 10 μm.


Figure 2A - F Double immunolabelling of PCNT - PCNT - ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) interkinesis , ( B ) early prophase II , ( C ) late prophase II , ( D ) prometaphase II , ( E ) metaphase II and ( F ) telophase II . Chromatin has been stained with DAPI ( blue ) . Scale bar in F represents 10 μm .

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Figure 2A-F Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) interkinesis, (B) early prophase II, (C) late prophase II, (D) prometaphase II, (E) metaphase II and (F) telophase II. Chromatin has been stained with DAPI (blue). Scale bar in F represents 10 μm.


Figure 3A - L Double immunolabelling of CETN3 ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) leptotene , ( B ) zygotene , ( C ) pachytene , ( D ) metaphase I , ( E ) telophase I , ( F ) early interkinesis ( F ) , late interkinesis ( G ) , prophase II ( H ) , metaphase II ( I ) , telophase II ( J ) , round spermatid ( K ) , and elongated spermatid ( L ) . Amplifications at 300 % magnification for the centrosomic CETN3 signals are presented for each image . Chromatin has been stained with DAPI ( blue ) . Scale bar in L represents 10 μm .

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Figure 3A-L Double immunolabelling of CETN3 (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) leptotene, (B) zygotene, (C) pachytene, (D) metaphase I, (E) telophase I, (F) early interkinesis (F), late interkinesis (G), prophase II (H), metaphase II (I), telophase II (J), round spermatid (K), and elongated spermatid (L). Amplifications at 300% magnification for the centrosomic CETN3 signals are presented for each image. Chromatin has been stained with DAPI (blue). Scale bar in L represents 10 μm.


Figure 4A - J Double immunolabelling of PLK1S137P ( green ) and SYCP3 ( red ) on squashed WT mouse spermatocytes at ( A ) pachytene , ( C ) diplotene , ( E ) diakinesis , ( G ) metaphase I and ( I ) metaphase II . Chromatin has been stained with DAPI ( blue ) . Fourth column shows the double Immunolabelling of PLK1 - S137P ( green ) and γ - Tubulin ( magenta ) on squashed WT mouse spermatocytes at ( B ) pachytene , ( D ) diplotene , ( F ) diakinesis , ( H ) metaphase I and ( J ) metaphase II . Insets correspond to centrosomes at 200 % magnification . White arrows indicate the location of centrosomes . Scale bar in J represents 10µm .

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Figure 4A-J Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) pachytene, (C) diplotene, (E) diakinesis, (G) metaphase I and (I) metaphase II. Chromatin has been stained with DAPI (blue). Fourth column shows the double Immunolabelling of PLK1-S137P (green) and γ-Tubulin (magenta) on squashed WT mouse spermatocytes at (B) pachytene, (D) diplotene, (F) diakinesis, (H) metaphase I and (J) metaphase II. Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in J represents 10µm.


Figure 5A - L First column shows the triple immunolabelling of CETN3 ( green ) , PCNT ( red ) and SYCP3 ( grey ) on squashed WT mouse spermatocytes . Second column shows the correspondent labellings for Plk1 ( ∆ / ∆ ) . Third column shows signals for BI2536 - treated spermatocytes . Images are shown for ( A , E , I ) zygotene , ( B , F , J ) pachytene , ( C , G , K ) diplotene and ( D , H , L ) diakinesis . Amplifications at 300 % magnification for all labellings are displayed for each image . M - O Quantification of the percentage of cells without PCNT at centrosomes ( PCNT - ) in ( M ) zygotene , ( N ) pachytene and ( O ) diplotene in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules . Data are presented as mean ± SD for three biological replicates for each condition .

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Figure 5A-L First column shows the triple immunolabelling of CETN3 (green), PCNT (red) and SYCP3 (grey) on squashed WT mouse spermatocytes. Second column shows the correspondent labellings for Plk1(∆/∆). Third column shows signals for BI2536-treated spermatocytes. Images are shown for (A, E, I) zygotene, (B, F, J) pachytene, (C, G, K) diplotene and (D, H, L) diakinesis. Amplifications at 300% magnification for all labellings are displayed for each image. M-O Quantification of the percentage of cells without PCNT at centrosomes (PCNT-) in (M) zygotene, (N) pachytene and (O) diplotene in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules. Data are presented as mean ± SD for three biological replicates for each condition.


Figure 6A - C Immunolabelling of γ - Tubulin and SYCP3 during prophase I in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Images are show for pachytene , diplotene and diakinesis in Control ( A ) , Plk1 ( ∆ / ∆ ) ( B ) and BI2536 - treated spermatocytes ( C ) . First columns show the double immunolabelling of PCM - PCNT - ( green ) and SYCP3 ( red ) , and second columns shows γ - Tubulin ( γ - Tub , magenta ) and SYCP3 ( red ) . Chromatin has been stained with DAPI ( blue ) . Amplifications at 300 % magnification for all labellings are displayed for each image . Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules ( D ) Immunolabelling of CETN3 and γ - Tubulin in control and Plk1 ( ∆ / ∆ ) . Double immunolabelling of CETN3 ( red ) and γ - Tubulin ( green ) in pachytene ( a - b ) and diakinesis ( c - d ) . Chromatin has been stained with DAPI ( blue ) . ( E ) Quantification of the number of cells without γ - Tubulin at centrosomes ( γ - Tub - ) in pachytene and diplotene spermatocytes in control , Plk1 ( ∆ / ∆ ) and BI2536 - treated spermatocytes . Data represent average of two biological replicates per condition .

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Figure 6A-C Immunolabelling of γ-Tubulin and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Images are show for pachytene, diplotene and diakinesis in Control (A), Plk1(∆/∆) (B) and BI2536-treated spermatocytes (C). First columns show the double immunolabelling of PCM -PCNT- (green) and SYCP3 (red), and second columns shows γ-Tubulin (γ-Tub, magenta) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Amplifications at 300% magnification for all labellings are displayed for each image. Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules(D) Immunolabelling of CETN3 and γ-Tubulin in control and Plk1(∆/∆). Double immunolabelling of CETN3 (red) and γ-Tubulin (green) in pachytene (a-b) and diakinesis (c-d). Chromatin has been stained with DAPI (blue).(E) Quantification of the number of cells without γ-Tubulin at centrosomes (γ-Tub -) in pachytene and diplotene spermatocytes in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data represent average of two biological replicates per condition.


Figure 7A - D Control : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I ( A ) and metaphase II ( B ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) on control squashed spermatocytes at metaphase I ( C ) and metaphase II ( D ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . E - H Plk1 ( ∆ / ∆ ) mouse model : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) in monopolar spindle I ( E ) and monopolar spindle II ( F ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) labelling in monopolar spindle I ( G ) and monopolar spindle II ( H ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . I - L BI2536 - treated spermatocytes : Double immunolabelling of SYCP3 ( cyan ) and α - Tubulin ( red ) at monopolar spindle I ( I ) and monopolar spindle II ( J ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) labelling in monopolar spindle I ( K ) and monopolar spindle II ( L ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules . M - P MLN8237 - treated spermatocytes : Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) in monopolar spindle I ( M ) and monopolar spindle II ( N ) . Double immunolabelling of PCNT ( green ) and α - Tubulin ( red ) in monopolar spindle I ( O ) and monopolar spindle II ( P ) . Chromatin has been stained with DAPI ( pseudo coloured in grey ) . Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules . Q - R Quantitative analysis of the incidence of monopolar spindles during the first ( Q ) and second ( R ) meiotic divisions for each experimental condition . Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Experiments were conducted in three different biological replicates .

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

Figure 7A-D Control: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I (A) and metaphase II (B). Double immunolabelling of PCNT (green) and α-Tubulin (red) on control squashed spermatocytes at metaphase I (C) and metaphase II (D). Chromatin has been stained with DAPI (pseudo coloured in grey). E-H Plk1 (∆/∆) mouse model: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) in monopolar spindle I (E) and monopolar spindle II (F). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (G) and monopolar spindle II (H). Chromatin has been stained with DAPI (pseudo coloured in grey). I-L BI2536-treated spermatocytes: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) at monopolar spindle I (I) and monopolar spindle II (J). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (K) and monopolar spindle II (L). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules. M-P MLN8237-treated spermatocytes: Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (M) and monopolar spindle II (N). Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (O) and monopolar spindle II (P). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules. Q-R Quantitative analysis of the incidence of monopolar spindles during the first (Q) and second (R) meiotic divisions for each experimental condition. Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Experiments were conducted in three different biological replicates.


Figure 8A - B AURKP distribution in control , Plk1 ( Δ / Δ ) , 100µM 8h BI2536 - treated and 10µM 8h MLN8237 - treated spermatocytes . ( A ) Double immunolabelling of AURKP ( grey / green ) and α - Tubulin ( red ) in control , Plk1 ( Δ / Δ ) , BI2536 - treated and MLN8237 - treated spermatocytes . Images are shown for bipolar spindles I and II for each condition . ( B ) Monopolar spindles I and II are shown for Plk1 ( Δ / Δ ) , BI2536 - treated and MLN8237 - treated spermatocytes . Chromatin has been stained with DAPI ( blue ) . . ( C ) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signals for each experimental condition . Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent the average of two biological replicates per condition . Data are mean ± SD . ( D ) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting ( blue bars ) or lacking ( green bars ) centrosomic AURKP signal for each experimental condition . Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules . Data represent the average of two biological replicates per condition . Data are mean ± SD . ( E ) Quantitative analysis of AURKP signal at cell poles . Experiments were conducted for two biological replicates . Number of cells analysed : control ( n = 32 ) , Plk1 ( Δ / Δ ) ( n = 27 ) , BI2536 - treated spermatocytes ( n = 27 ) . Data are mean ± SD ; ( * * * * ) p < 0 . 0001 , Student ' s t - test .

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

Figure 8A-B AURKP distribution in control, Plk1(Δ/Δ), 100µM 8h BI2536-treated and 10µM 8h MLN8237-treated spermatocytes. (A) Double immunolabelling of AURKP (grey/green) and α-Tubulin (red) in control, Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Images are shown for bipolar spindles I and II for each condition. (B) Monopolar spindles I and II are shown for Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Chromatin has been stained with DAPI (blue)..(C) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting (blue bars) or lacking (green bars) centrosomic AURKP signals for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. (D) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting (blue bars) or lacking (green bars) centrosomic AURKP signal for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD. (E) Quantitative analysis of AURKP signal at cell poles. Experiments were conducted for two biological replicates. Number of cells analysed: control (n=32), Plk1(Δ/Δ) (n=27), BI2536-treated spermatocytes (n= 27). Data are mean ± SD; (****) p<0.0001, Student's t-test.


Figure 9 ( A ) Triple immunolabelling of CEP250 ( green ) , CETN3 ( magenta ) and SYCP3 ( grey ) in ( a - b ) late diplotene and ( c - d ) metaphase I in control and Plk1 ( Δ / Δ ) spermatocytes . ( B ) Double immunolabelling of KIF11 ( grey / green ) , and SYCP3 ( red ) in ( a ) control spindle , ( b ) Plk1 ( Δ / Δ ) monopolar spindle , ( c ) BI2536 monopolar spindle and ( d ) MLN8237 monopolar spindle . Chromatin has been stained with DAPI ( bue ) . Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules .

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

Figure 9(A) Triple immunolabelling of CEP250 (green), CETN3 (magenta) and SYCP3 (grey) in (a-b) late diplotene and (c-d) metaphase I in control and Plk1(Δ/Δ) spermatocytes.(B) Double immunolabelling of KIF11 (grey/green), and SYCP3 (red) in (a) control spindle, (b) Plk1(Δ/Δ) monopolar spindle, (c) BI2536 monopolar spindle and (d) MLN8237 monopolar spindle. Chromatin has been stained with DAPI (bue). Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules.


Figure 1H . Endogenous levels of Stim1 in testes from C57BL / 6 wt and splice deficient 10A mice , using an antibody detecting the N - term of STIM1 , detecting STIM1A ( A , red arrow ) , STIM1 and its glycosylated ( wt ) and unglycosylated ( wt gl . ) form .

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

Figure 1H. Endogenous levels of Stim1 in testes from C57BL/6 wt and splice deficient 10A mice, using an antibody detecting the N-term of STIM1, detecting STIM1A (A, red arrow), STIM1 and its glycosylated (wt) and unglycosylated (wt gl.) form.


Figure 2A . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar in MEF Stim1 / Stim2 - / - cells transfected with Stim1 - ( black trace , n = 111 ) , Stim1A ( red trace , n = 116 ) , the combination Stim1 with Stim1A ( blue trace , n = 86 ) or with vector only ( grey trace , n = 79 ) . B . Quantification of changes in ratio of resting , influx rate ( Δratio / time ) , Δpeak and Δplateau measured in A . * * * p < 0 . 001 , Kruskal - Wallis Anova . C . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura 2 ratio ) in HEK293 cells co - transfected with Orai1 and either Stim1 - ( black trace , n = 119 ) or Stim1A IRES - mCherry ( red trace , n = 118 ) . D . Quantification of changes in ratio of resting , influx rate ( Δratio / time ) , Δpeak and Δplateau measured in C . * * p < 0 . 01 * * * p < 0 . 001 Mann - Whitney test . E . Data information : Data were obtained from three biological replicates ( e . g . transfections ) with each three measured dishes ( technical replicates ) with multiple cells each ( yielding a total number n ) and is shown as mean ± s . e . m . I . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura 2 ratio ) in primary astrocytes from C57BL / 6 wt ( CTRL , black trace , n = 49 ) and splice deficient C57BL / 6 10ACon mice ( 10ACon , red trace , n = 82 ) . J . Quantification of parameters determined in [ I ] . * * * p < 0 . 001 Mann - Whitney test . Data information : D were obtained from three biological replicates ( e . g . transfections ) with each three measured dishes ( technical replicates ) with multiple cells each ( yielding a total number n ) and is shown as mean ± s . e . m .

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

Figure 2A. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar in MEF Stim1/Stim2-/- cells transfected with Stim1- (black trace, n=111), Stim1A (red trace, n=116), the combination Stim1 with Stim1A (blue trace, n=86) or with vector only (grey trace, n=79). B. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in A. *** p<0.001, Kruskal-Wallis Anova. C. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in HEK293 cells co-transfected with Orai1 and either Stim1- (black trace, n=119) or Stim1A IRES-mCherry (red trace, n=118). D. Quantification of changes in ratio of resting, influx rate (Δratio/time), Δpeak and Δplateau measured in C. ** p<0.01 *** p<0.001 Mann-Whitney test. E. Data information: Data were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.I. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura 2 ratio) in primary astrocytes from C57BL/6 wt (CTRL,black trace, n=49) and splice deficient C57BL/6 10ACon mice (10ACon, red trace, n=82). J. Quantification of parameters determined in [I]. *** p<0.001 Mann-Whitney test. Data information: D were obtained from three biological replicates (e.g. transfections) with each three measured dishes (technical replicates) with multiple cells each (yielding a total number n) and is shown as mean±s.e.m.


Figure 3C . Images showing representative HEK293 cells expressing Orai1 - GFP ( green = donor molecule ) or either STIM1 - mCherry ( upper panel = acceptor molecule ) or STIM1A - mCherry ( lower panel = acceptor molecule ) after stimulation with TG . Also shown is the overlay of the two channels , as well as the FRET signal .

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

Figure 3C. Images showing representative HEK293 cells expressing Orai1-GFP (green = donor molecule) or either STIM1-mCherry (upper panel = acceptor molecule) or STIM1A-mCherry (lower panel = acceptor molecule) after stimulation with TG. Also shown is the overlay of the two channels, as well as the FRET signal.


Figure 4B . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar with constructs as indicated expressed in HEKO1 . STIM1 ( black , n = 145 ) , STIM1A ( red , n = 157 ) and STIM1A _ D503A ( n = 185 ) . C . Relative fluorescence intensities of mCherry - tagged constructs measured in ( B ) . D . Quantification of changes in resting ratio , TG peak ( ∆ ratio ) and SOCE parameters measured in B . E . Data information : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( each multiple cells ) and shown as mean ± s . e . m for traces and as scatter plots with the underlying boxes showing the means for individual parameters . E . Average traces showing whole - cell current density ( CD ) over time extracted at - 80 mV in HEKO1 cells transfected with STIM1 ( black ) , STIM1A ( red ) or STIM1A _ D503A ( blue ) . F . Average maximum CDs recorded from cells measured in E ( n within bars ) . G Data information : * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( each multiple cells ) and shown as mean ± s . e . m for traces and as scatter plots with the underlying boxes showing the means for individual parameters . G . Average current - voltage ( I - V ) relationship of all cells recorded in E .

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

Figure 4B. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKO1. STIM1 (black, n=145), STIM1A (red, n=157) and STIM1A_D503A (n=185). C. Relative fluorescence intensities of mCherry-tagged constructs measured in (B). D. Quantification of changes in resting ratio, TG peak (∆ ratio) and SOCE parameters measured in B. E. Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.E. Average traces showing whole-cell current density (CD) over time extracted at -80 mV in HEKO1 cells transfected with STIM1 (black), STIM1A (red) or STIM1A_D503A (blue). F. Average maximum CDs recorded from cells measured in E (n within bars). G Data information: *P<0.05, **P<0.01 ***P<0.001 Kruskal-Wallis Anova with Dunn's multiple comparisons test. Data points (total n) were obtained from three biological replicates with each three technical replicates (each multiple cells) and shown as mean±s.e.m for traces and as scatter plots with the underlying boxes showing the means for individual parameters.G. Average current-voltage (I- V) relationship of all cells recorded in E.


Figure 5F . Traces showing average changes ( mean ± s . e . m . ) in intracellular Ca2 + ( Fura2 ratio ) over time in response to perfusion of different [ Ca2 + ] o as indicated in the upper bar with constructs as indicated expressed in HEKS1 / S2 - / - . G . Quantification of the relative reduction of ratio / time or ratio for cells ( 135 H Data information : , * * * P < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparisons test Data points ( total n ) were obtained from three biological replicates with each three technical replicates ( with multiple cells ) and shown as mean ± s . e . m for traces ( F ) and as scatter plots with the underlying boxes showing the means for individual parameters ( G ) . H , I . Average traces ( mean ± s . e . m . ) showing whole - cell current density ( CD ) over time extracted at - 80 mV in in HEKS1 / S2 - / - cells co - transfected either with STIM1 ( black ) or STIM1A ( red ) and ORAI1 ( H ) or with ORAI1 R77E ( I ) and recorded using extracellular solution containing 2 mM Ca2 + . J . Average maximum CDs recorded from cells measured in [ H , I ] ( n within bars ) . Data information : I , * P < 0 . 05 , * * P < 0 . 01 , unpaired T test with Welch ' s correction . For patch - clamp experiments , number in bars indicate measured cells , which were from three independent transfections .

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

Figure 5F. Traces showing average changes (mean±s.e.m.) in intracellular Ca2+ (Fura2 ratio) over time in response to perfusion of different [Ca2+]o as indicated in the upper bar with constructs as indicated expressed in HEKS1/S2-/-. G. Quantification of the relative reduction of ratio/time or ratio for cells (135 H Data information: , ***P<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test Data points (total n) were obtained from three biological replicates with each three technical replicates (with multiple cells) and shown as mean±s.e.m for traces (F) and as scatter plots with the underlying boxes showing the means for individual parameters (G).H,I. Average traces (mean±s.e.m.) showing whole- cell current density (CD) over time extracted at -80 mV in in HEKS1/S2-/- cells co-transfected either with STIM1 (black) or STIM1A (red) and ORAI1 (H) or with ORAI1 R77E (I) and recorded using extracellular solution containing 2 mM Ca2+. J. Average maximum CDs recorded from cells measured in [H,I] (n within bars). Data information: I , *P<0.05, **P<0.01, unpaired T test with Welch's correction. For patch-clamp experiments, number in bars indicate measured cells, which were from three independent transfections.


Figure 6B . Hits from ( A ) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation ( BiFC ) via flow cytometry . HEKS1 / S2 - / - were transfected with STIM1 - YFPC ( black ) or STIM1A - YFPC ( red ) in combination with POI - YFPN and screened for YFP + cells via FACS , results ( mean ± SD ) from three transfections with each 10 . 000 sorted cells . D . Ratio nuclear NFAT - GFP vs . cytosolic GFP intensity normalized to t = 0 ( mean ± s . e . m . ) after transfection of SH - SY5Y S1 - / - cells with STIM ( n = 42 ) , STIM1A ( n = 82 ) , STIM1A _ D503A ( n = 49 ) or vector only ( ØSTIM , n = 15 ) IRES mCherry and induction of SOCE after stimulation with 1 µM TG . Data ( total n as indicated ) from at least three independent transfections .

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Figure 6B. Hits from (A) tested for interaction with commercially available constructs quantified with bimolecular fluorescence complementation (BiFC) via flow cytometry. HEKS1/S2-/- were transfected with STIM1-YFPC (black) or STIM1A-YFPC (red) in combination with POI-YFPN and screened for YFP+ cells via FACS, results (mean ± SD) from three transfections with each 10.000 sorted cells.D. Ratio nuclear NFAT-GFP vs. cytosolic GFP intensity normalized to t=0 (mean±s.e.m.) after transfection of SH-SY5Y S1-/- cells with STIM (n=42), STIM1A (n=82), STIM1A_D503A (n=49) or vector only (ØSTIM, n=15) IRES mCherry and induction of SOCE after stimulation with 1 µM TG. Data (total n as indicated) from at least three independent transfections.


Figure 7A . Time course of translocation ( Nuclear NFAT - GFP signal / cytosolic signal normalized to TG addition at t = 0 , mean ± s . e . m , for each time point ) after transfection of indicated constructs and preincubation with 1 µM PF - 04957325 ( analyzed cells : STIM1 + PF n = 75 , STIM1A + PF n = 50 , ØSTIM + PF n = 9 ) Data ( total n as indicated ) from at least three independent transfections . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparisons test . B . PF - 04957325 induced difference of the nuclear NFAT - GFP signal vs . cytosolic signal from ( A ) to mean of control without the blocker ( see Fig . 6D ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Mann - Whitney test of differences . . C Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , 2way Anova Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each . C . Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment , shown as mean ± s . e . m , ØSTIM + 6Bnz - cAMP / 8 - pCPT - cAMP n = 15 / 14 cells ; STIM1 + 6Bnz - cAMP / 8 - pCPT - cAMP : n = 87 / 66 , STIM1A + 6Bnz - cAMP : n = 78 / 67 ; from three independent transfections . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , , Kruskal - Wallis Anova with Dunn ' s multiple comparison test Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each . G . Normalized fluorescence signal change ( mean ± s . e . m ) of R - FlincA , SH - SY5Y S1 - / - cells expressing STIM1 ( n = 20 ) or STIM1A ( n = 22 ) IRES GFP variants and the indicator were stimulated by TG ( 1 μM ) . To avoid photobleaching , values were only recorded after 5 and 30 min , respectively The mutant R - Flinc ( n = 15 ) was measured in SH - SY5Y wt cells after TG stimulation . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Kruskal - Wallis Anova with Dunn ' s multiple comparison test Data ( total number of cells , n ) were obtained from at least three independent transfections with several technical replicates each .

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

Figure 7A. Time course of translocation (Nuclear NFAT-GFP signal/cytosolic signal normalized to TG addition at t=0, mean±s.e.m, for each time point) after transfection of indicated constructs and preincubation with 1 µM PF-04957325 (analyzed cells: STIM1 + PF n = 75, STIM1A + PF n = 50, ØSTIM + PF n = 9) Data (total n as indicated) from at least three independent transfections. * p< 0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparisons test. B. PF-04957325 induced difference of the nuclear NFAT-GFP signal vs. cytosolic signal from (A) to mean of control without the blocker (see Fig. 6D). * p< 0.05, **p<0.01, ***p<0.001,Mann-Whitney test of differences.. C Data information: *p<0.05, **p<0.01, ***p<0.001, 2way Anova Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.C. Endpoint value with addition of PF or indicated cAMP analoga loaded for 30 min as AM esters before the experiment, shown as mean±s.e.m , ØSTIM + 6Bnz-cAMP/8-pCPT-cAMP n = 15/14 cells; STIM1 + 6Bnz-cAMP/8-pCPT-cAMP: n = 87/66, STIM1A + 6Bnz-cAMP : n = 78/67; from three independent transfections. Data information: *p<0.05, **p<0.01, ***p<0.001, , Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.G. Normalized fluorescence signal change (mean±s.e.m) of R-FlincA, SH-SY5Y S1-/- cells expressing STIM1 (n=20) or STIM1A (n=22) IRES GFP variants and the indicator were stimulated by TG (1 μM). To avoid photobleaching, values were only recorded after 5 and 30 min, respectively The mutant R-Flinc (n=15) was measured in SH-SY5Y wt cells after TG stimulation. Data information: *p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis Anova with Dunn's multiple comparison test Data (total number of cells, n) were obtained from at least three independent transfections with several technical replicates each.


Figure 3MST analysis of the binding interactions between Sir4 H ‑ BRCT and Cy5 - labeled phospho ( filled circles ) and non - phospho peptides ( empty circles ) from Esc1 , Ty5 and Ubp10 . KD = equilibrium dissociation constant . ΔFnorm values were divided by the amplitude of the saturation level , resulting in the fraction bound ( from 0 to 1 ) for each data point . Data are represented as the mean ± SEM from ≥ 3 independent measurements .

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

Figure 3MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1, Ty5 and Ubp10. KD = equilibrium dissociation constant. ΔFnorm values were divided by the amplitude of the saturation level, resulting in the fraction bound (from 0 to 1) for each data point. Data are represented as the mean ± SEM from ≥3 independent measurements.


Figure 4Example images of Nup49 - RFP Sir4 - GFP in wild - type ( wt ) cells ( GA - 10372 ; row 1 ) ; sir4 RKR cells ( GA - 10373 ; row 2 ) ; wt cells transformed with either empty plasmid ( row 3 ) , plasmid overexpressing wt Sir4 H ‑ BRCT ( row 4 ) , or plasmid overexpressing RKR mutant Sir4 H ‑ BRCT ( row 5 ) . The bar indicates 2 μm . Quantification of mean fluorescence intensity of Sir4 - GFP using > 100 cells . Error bars show the SD from 3 independent experiments . Quantification of cells containing Sir4 clusters at the nuclear envelope ( NE ) . Data are represented as mean ± SD from 3 independent experiments where > 100 cells were counted . Top : Schematic representation of the TEL7L : : URA3 reporter used to assay silencing at telomeres . Bottom : wt cells ( GA - 503 ) transformed with either an empty plasmid ( row 1 ) , plasmid overexpressing wt Sir4 H ‑ BRCT ( row 2 ) , or plasmid overexpressing RKR mutant Sir4 H ‑ BRCT ( row 3 ) . Cells lacking endogenous sir4 ( GA - 5822 ) ( row 4 ) . Cells were grown in the absence of uracil and plated as 1 : 5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days . Top : Schematic representation of the HMR - EΔB : : TRP1 reporter gene locus used for assaying silencing at the HMR locus . Bottom : wt cells ( GA - 485 ) transformed with plasmids as in panel D . Cells lacking endogenous sir4 ( GA - 6888 ) ( row 4 ) . Cells were grown in the absence of tryptophan and plated as 1 : 5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days .

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Figure 4Example images of Nup49-RFP Sir4-GFP in wild-type (wt) cells (GA-10372; row 1); sir4 RKR cells (GA-10373; row 2); wt cells transformed with either empty plasmid (row 3), plasmid overexpressing wt Sir4 H‑BRCT (row 4), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 5). The bar indicates 2 μm.Quantification of mean fluorescence intensity of Sir4-GFP using >100 cells. Error bars show the SD from 3 independent experiments. Quantification of cells containing Sir4 clusters at the nuclear envelope (NE). Data are represented as mean ± SD from 3 independent experiments where >100 cells were counted.Top: Schematic representation of the TEL7L::URA3 reporter used to assay silencing at telomeres. Bottom: wt cells (GA-503) transformed with either an empty plasmid (row 1), plasmid overexpressing wt Sir4 H‑BRCT (row 2), or plasmid overexpressing RKR mutant Sir4 H‑BRCT (row 3). Cells lacking endogenous sir4 (GA-5822) (row 4). Cells were grown in the absence of uracil and plated as 1:5 serial dilutions onto control YPAD or YPAD lacking uracil plates and imaged after 2 days. Top: Schematic representation of the HMR-EΔB::TRP1 reporter gene locus used for assaying silencing at the HMR locus. Bottom: wt cells (GA-485) transformed with plasmids as in panel D. Cells lacking endogenous sir4 (GA-6888) (row 4). Cells were grown in the absence of tryptophan and plated as 1:5 serial dilutions onto YPAD or YPAD lacking tryptophan plates and imaged after 2 days.


Figure 5Volcano plot showing proteins that interact with wt Sir4 H ‑ BRCT but not with the RKR mutant ( upper right quadrant ) . MST analysis of the binding interactions between Sir4 H ‑ BRCT and Cy5 - labeled phospho ( filled circles ) and non - phospho peptides ( empty circles ) from Tom1 and Cbf1 . KD represents the equilibrium dissociation constant . The KD for interactions with the non - phospho peptides could not be determined ( n . d . ) . ΔFnorm [ 0 / 00 ] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence . Data are represented as the mean ± SEM from at least 3 independent measurements . Extracts from asynchronous cultures of strains Sir4 - Myc Tom1 - HA ( GA - 10645 ) , Sir4 - RKR - Myc Tom1 - HA ( GA - 10646 ) , Tom1 - HA ( GA - 10647 ) and Sir4 - Myc ( GA - 10199 ) were subjected to anti - Myc IP and western blotting with indicated antibodies in the presence and absence of lambda phosphatase .

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

Figure 5Volcano plot showing proteins that interact with wt Sir4 H‑BRCT but not with the RKR mutant (upper right quadrant).MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Tom1 and Cbf1. KD represents the equilibrium dissociation constant. The KD for interactions with the non-phospho peptides could not be determined (n.d.). ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are represented as the mean ± SEM from at least 3 independent measurements.Extracts from asynchronous cultures of strains Sir4-Myc Tom1-HA (GA-10645), Sir4-RKR-Myc Tom1-HA (GA-10646), Tom1-HA (GA-10647) and Sir4-Myc (GA-10199) were subjected to anti-Myc IP and western blotting with indicated antibodies in the presence and absence of lambda phosphatase.


Figure 6MST analysis of the binding interactions between Dbf4 H ‑ BRCT and Cy5 - labeled phospho ( filled circles ) and non - phospho peptides ( empty circles ) from Esc1 and Ubp10 . KD = equilibrium dissociation constant , which could not be determined ( n . d . ) for non - phospho peptides . ΔFnorm [ 0 / 00 ] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence . Data are the mean ± SEM from ≥ 3 independent measurements .

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Figure 6MST analysis of the binding interactions between Dbf4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Esc1 and Ubp10. KD = equilibrium dissociation constant, which could not be determined (n.d.) for non-phospho peptides. ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are the mean ± SEM from ≥3 independent measurements.


Figure 1B GFP + EBs derived from CT and RA cultures at day 10 ; scale bar : 100 μm . C Representative FC plots depicting percentage of GFP + cells obtained at day 15 , from CT and RA cultures in a typical experiment . D Heat map demonstrating enrichment of cardiac genes in GFP + fractions ( CT + , RA + ) compared to GFP − fractions ( CT − , RA − ) at day 31 . E , F Heat map of a select list of genes ( E ) upregulated and ( F ) downregulated in RA + compared to CT + at day 31 . Fold change > 2 .

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Figure 1B GFP+ EBs derived from CT and RA cultures at day 10; scale bar: 100 μm.C Representative FC plots depicting percentage of GFP+ cells obtained at day 15, from CT and RA cultures in a typical experiment.D Heat map demonstrating enrichment of cardiac genes in GFP+ fractions (CT+, RA+) compared to GFP− fractions (CT−, RA−) at day 31.E, F Heat map of a select list of genes (E) upregulated and (F) downregulated in RA+ compared to CT+ at day 31. Fold change > 2.


Figure 2A - C qPCR of selected transcripts at day 31 to validate ( A ) enrichment of cardiac markers in GFP + fractions against GFP − fractions , ( B ) upregulation of atrial and ( C ) downregulation of ventricular genes in RA + compared to CT + ( n = 3 ) . Data information : Data are presented as mean ± SEM . In ( A - C ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired t - test . In ( A ) , for TNNT2 , P = 0 . 0001 for CT − against CT + and P = 0 . 0002 for RA − against RA + ; for NKX2 . 5 , P = 0 . 00005 for CT − against CT + and P = 0 . 00007 for RA − against RA + . In ( B ) , P = 0 . 0006 for NPPA and P = 0 . 0002 for PITX2 . In ( C ) , P = 0 . 02 for HEY2 and P = 0 . 007 for IRX4 .

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Figure 2A-C qPCR of selected transcripts at day 31 to validate (A) enrichment of cardiac markers in GFP+ fractions against GFP− fractions, (B) upregulation of atrial and (C) downregulation of ventricular genes in RA+ compared to CT+ (n = 3).Data information: Data are presented as mean ± SEM. In (A-C), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), for TNNT2, P = 0.0001 for CT− against CT+ and P = 0.0002 for RA− against RA+; for NKX2.5, P = 0.00005 for CT− against CT+ and P = 0.00007 for RA− against RA+. In (B), P = 0.0006 for NPPA and P = 0.0002 for PITX2. In (C), P = 0.02 for HEY2 and P = 0.007 for IRX4.


Figure 3B Representative APs of day 31 CMs from control ( CT ) and RA - treated ( RA ) groups at 1 Hz . C - E RMP , APAmax and APAplat ( C ) , dV / dtmax ( D ) and APD20 , APD50 and APD90 of CT and RA CMs ( E ) . F Plot showing all measured APAplat values of CT and RA CMs . G Representative APs of CT and RA CMs at 0 . 5 - 4 Hz . H Average APAplat at 0 . 5 - 4 Hz . Please note that the AP differences in morphology are present at all measured frequencies .

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Figure 3B Representative APs of day 31 CMs from control (CT) and RA-treated (RA) groups at 1 Hz.C-E RMP, APAmax and APAplat (C), dV/dtmax (D) and APD20, APD50 and APD90 of CT and RA CMs (E).F Plot showing all measured APAplat values of CT and RA CMs.G Representative APs of CT and RA CMs at 0.5-4 Hz.H Average APAplat at 0.5-4 Hz. Please note that the AP differences in morphology are present at all measured frequencies.


Figure 4A , B Line plot illustrating relative mRNA levels of ( A ) COUP - TFI and ( B ) COUP - TFII in VM and AM differentiations from day 5 through day 9 ( left ) and in GFP + CMs at day 31 ( right ) ; n = 3 . Data information : Data are presented as mean ± SEM . In ( A , B ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired t - test . In ( A ) , left panel , P = 0 . 03 , 0 . 02 , 0 . 03 , 0 . 005 and 0 . 0004 for comparison of COUP - TFI expression between AM and VM at days 5 , 6 , 7 , 8 and 9 of differentiation . In ( A ) , right panel , P = 0 . 0002 for comparison of COUP - TFI expression at day 31 between AM and VM . In ( B ) , left panel , P = 0 . 02 , 0 . 03 , 0 . 006 , 0 . 004 and 0 . 003 for comparison of COUP - TFII expression between AM and VM at days 5 , 6 , 7 , 8 and 9 of differentiation . In ( B ) , right panel , P = 0 . 0001 for comparison of COUP - TFII expression at day 31 between AM and VM . CT = control differentiation ; hESC - atrial ( AM ) ; hESC - ventricular ( VM ) . C , D COUP - TFI ( C ) and COUP - TFII ( D ) immunofluorescence at day 31 in AM ( top ) and VM ( bottom ) . Scale bars : 40 μm .

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

Figure 4A, B Line plot illustrating relative mRNA levels of (A) COUP-TFI and (B) COUP-TFII in VM and AM differentiations from day 5 through day 9 (left) and in GFP+CMs at day 31 (right); n = 3.Data information: Data are presented as mean ± SEM. In (A, B), *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. In (A), left panel, P = 0.03, 0.02, 0.03, 0.005 and 0.0004 for comparison of COUP-TFI expression between AM and VM at days 5, 6, 7, 8 and 9 of differentiation. In (A), right panel, P = 0.0002 for comparison of COUP-TFI expression at day 31 between AM and VM. In (B), left panel, P = 0.02, 0.03, 0.006, 0.004 and 0.003 for comparison of COUP-TFII expression between AM and VM at days 5, 6, 7, 8 and 9 of differentiation. In (B), right panel, P = 0.0001 for comparison of COUP-TFII expression at day 31 between AM and VM. CT = control differentiation; hESC-atrial (AM); hESC-ventricular (VM).C, D COUP-TFI (C) and COUP-TFII (D) immunofluorescence at day 31 in AM (top) and VM (bottom). Scale bars: 40 μm.


Figure 5A , B mRNA expression of COUP - TFI and COUP - TFII following shRNA - mediated knockdown of ( A ) COUP - TFI or ( B ) COUP - TFII in hESC - atrial cardiomyocytes ( AM ) at day 30 . C , D mRNA expression of ion channel genes KCNA5 , KCNJ3 and KCNJ5 after knockdown of ( C ) COUP - TFI or ( D ) COUP - TFII in AM at day 30 . E , F Schematic of NR2F binding sites in ( E ) KCNA5 and ( F ) KCNJ3 promoters . G , H ChIP - qPCR analysis at day 30 shows enriched binding of COUP - TFI and COUP - TFII to the promoter region of ( G ) KCNA5 and ( H ) KCNJ3 , compared to IgG in AM .

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Figure 5A, B mRNA expression of COUP-TFI and COUP-TFII following shRNA-mediated knockdown of (A) COUP-TFI or (B) COUP-TFII in hESC-atrial cardiomyocytes (AM) at day 30.C, D mRNA expression of ion channel genes KCNA5, KCNJ3 and KCNJ5 after knockdown of (C) COUP-TFI or (D) COUP-TFII in AM at day 30.E, F Schematic of NR2F binding sites in (E) KCNA5 and (F) KCNJ3 promoters.G, H ChIP-qPCR analysis at day 30 shows enriched binding of COUP-TFI and COUP-TFII to the promoter region of (G) KCNA5 and (H) KCNJ3, compared to IgG in AM.