( A ) RCC1 . 24 ‐ NeoR incubated with IFN ‐ γ were either left untreated ( w / o ) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing . Subsequently cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 - 4 / A4 in a GM ‐ CSF release assay . EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( B ) or pINCO ‐ Tyrosinase ( C ) were tested likewise . Both substances abrogated recognition of neoR ‐ transfected cells , while HLA ‐ A2 ‐ restricted presentation of the tyrosinase peptide was not

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(A) RCC1.24‐NeoR incubated with IFN‐γ were either left untreated (w/o) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing. Subsequently cells were fixed with 0.5% paraformaldehyde and tested with 20-4/A4 in a GM‐CSF release assay. EBV‐B1.11 cells transfected with pINCO‐NeoR (B) or pINCO‐Tyrosinase (C) were tested likewise. Both substances abrogated recognition of neoR‐transfected cells, while HLA‐A2‐restricted presentation of the tyrosinase peptide was not affected


( D ) ) EBV ‐ B1 . 11 cells were incubated with 200 ng / ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in ( A ) . Presentation of exogenous NeoR on HLA ‐ DP3 is blocked by both substances as well , indicating that NeoR presentation is dependent on lysosomal processing .

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(D) ) EBV‐B1.11 cells were incubated with 200 ng/ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in (A). Presentation of exogenous NeoR on HLA‐DP3 is blocked by both substances as well, indicating that NeoR presentation is dependent on lysosomal processing.


( A ) 293T cells transfected with pINCO ‐ NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot . In addition , supernatant of NeoR ‐ pINCO transfected 293T cells ( 96 h post transfection ) was included to test for release of NeoR into the culture supernatant . At 6 h post transfection , NeoR protein becomes detectable in the cytoplasm , while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal / lysosomal fraction . For further characterization of the various fractions , blots were stripped and re ‐ probed with antibodies specific for the cytoplasmic protein LDH , the nuclear protein c ‐ myc , early endosomes ( Rab ‐ 5 ) , late endosomes and lysosomes ( Lamp ‐ 1 ) and mitochondria ( cytochrome c ) , which co ‐ purify with endosomes / lysosomes .

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(A) 293T cells transfected with pINCO‐NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot. In addition, supernatant of NeoR‐pINCO transfected 293T cells (96 h post transfection) was included to test for release of NeoR into the culture supernatant. At 6 h post transfection, NeoR protein becomes detectable in the cytoplasm, while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal/lysosomal fraction. For further characterization of the various fractions, blots were stripped and re‐probed with antibodies specific for the cytoplasmic protein LDH, the nuclear protein c‐myc, early endosomes (Rab‐5), late endosomes and lysosomes (Lamp‐1) and mitochondria (cytochrome c), which co‐purify with endosomes/lysosomes.


( B ) Endosomal / lysosomal fractions were prepared from pINCO ‐ NeoR ‐ transfected cells 96 h post transfection as in ( A ) . To prevent lysosomal degradation of NeoR , half of the cells were treated with leupeptin for the last 48 h . These fractions were separated on a 27 % Percoll gradient and eight different fractions collected . Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp ‐ 1 , Rab ‐ 5 and cytochrome c . Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal / lysosomal fractions 7 and 8 .

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(B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8.


( C ) To determine where NeoR gains access to the vacuolar system , 293T cells were transfected with pINCO ‐ NeoR , treated with leupeptin to prevent NeoR degradation , and the endosomal / lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation . Starting at 16 h post transfection , NeoR becomes detectable in the Rab ‐ 5 ‐ positive fraction 5 , and then spreads into late endosomal / lysosomal fractions . At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA .

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(C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.


( A ) RCC were transfected with the NeoR ‐ GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence . As compared to untreated cells , cells treated with 3 ‐ MA show a much brighter UV fluorescence .

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(A) RCC were transfected with the NeoR‐GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence. As compared to untreated cells, cells treated with 3‐MA show a much brighter UV fluorescence.


( B ) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR ‐ GFP fusion construct . Compared to untreated cells , cells treated for 24 h with 7 . 5 mM 3 ‐ MA show an about threefold increase in mean fluorescence intensity .

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(B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity.


( C ) Western blot analysis of neoR ‐ transfected cells . Treatment of cells with 7 . 5 mM 3 ‐ MA causes a dramatic increase in NeoR protein levels in only 12 h . The ER ‐ resident protein BiP was used as gel loading control .

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(C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control.


( D ) This increase in total NeoR protein in 3 ‐ MA ‐ treated cells is associated with an increase in cytoplasmic , and a decrease in endosomal / lysosomal NeoR protein levels .

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(D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels.


( E ) Western blot analysis of whole cell lysates prepared from neoR ‐ transfected cells . Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels . Simultaneous treatment of cells with 3 ‐ MA prevents NeoR degradation . BiP was again used as a gel loading control .

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(E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.


( A ) RCC1 . 24 ‐ NeoR ( endogenous ) , and RCC1 . 24 cells to which NeoR protein had been added ( exogenous ) , were treated with different concentrations of 3 ‐ MA and cell surface presentation of the NeoR ‐ derived peptide on HLA ‐ DP3 monitored with the T cell clone 20 - 4 / A4 ‐ specific T cell clone 20 - 4 / A4 . Inhibition of autophagy by 3 ‐ MA causes an almost complete inhibition of endogenous antigen presentation , while presentation of exogenous NeoR is not affected .

O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O

(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected.


( B ) EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( endogenous ) , or incubated with recombinant NeoR protein ( exogenous ) , were treated with the inhibitors of autophagy , wortmannin and 3 ‐ MA , and HLA ‐ DP3 ‐ restricted presentation of the NeoR ‐ derived peptide determined . Inhibition of antigen presentation by leupeptin served as control .

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(B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control.


( C ) Presentation of the HLA ‐ A2 ‐ restricted tyrosinase peptide in EBV ‐ B1 . 11 cells transfected with pINCO ‐ Tyrosinase is not affected by these inhibitors of autophagy .

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(C) Presentation of the HLA‐A2‐restricted tyrosinase peptide in EBV‐B1.11 cells transfected with pINCO‐Tyrosinase is not affected by these inhibitors of autophagy.


FACS analysis of HLA ‐ class I and HLA ‐ DP expression on RCC1 . 24 ( D ) and EBV ‐ B1 . 11 cells ( E ) As compared to untreated cells ( thick ‐ lined histogram ) , treatment of cells with 7 . 5 mM 3 ‐ MA ( shaded histogram ) does not cause down ‐ regulation of HLA ‐ class I or HLA ‐ DP . Isotype control is shown as thin ‐ lined histogram .

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FACS analysis of HLA‐class I and HLA‐DP expression on RCC1.24 (D) and EBV‐B1.11 cells (E) As compared to untreated cells (thick‐lined histogram), treatment of cells with 7.5 mM 3‐MA (shaded histogram) does not cause down‐regulation of HLA‐class I or HLA‐DP. Isotype control is shown as thin‐lined histogram.


Viability of diploid ( SC5314 ) and tetraploid ( RBY18 ) cells grown on PRE - SPO medium at 37 ° C ( n = 5 , error bars represent S . E . M . ) .

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Viability of diploid (SC5314) and tetraploid (RBY18) cells grown on PRE-SPO medium at 37°C (n=5, error bars represent S.E.M.).


A tetraploid β - tubulin ( TUB2 ) - GFP , histone H2B ( HTB ) - RFP strain was cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h . Cell images indicate maximum intensity Z projections for GFP and RFP channels , and merged images of the GFP / RFP and GFP / RFP / DIC channels .

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A tetraploid β-tubulin (TUB2)-GFP, histone H2B (HTB)-RFP strain was cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate maximum intensity Z projections for GFP and RFP channels, and merged images of the GFP/RFP and GFP/RFP/DIC channels.


Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 7 days and plated onto 2 - DOG medium to monitor for GAL1 loss .

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Diploid strain RBY1 and tetraploid strain RBY18 were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 7 days and plated onto 2-DOG medium to monitor for GAL1 loss.


2 - DOGR progeny arising from RBY18 on PRE - SPO medium were DNA stained and their ploidy determined via flow cytometry .

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2-DOGR progeny arising from RBY18 on PRE-SPO medium were DNA stained and their ploidy determined via flow cytometry.


Heatmap of RNA - seq data showing global gene expression changes in diploid ( SC5314 , 2n ) and tetraploid ( RBY18 , 4n ) cells on YPD and PRE - SPO medium over 24 h .

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Heatmap of RNA-seq data showing global gene expression changes in diploid (SC5314, 2n) and tetraploid (RBY18, 4n) cells on YPD and PRE-SPO medium over 24 h.


Heatmap of RNA - seq data for glycolysis and oxidative stress genes in diploid ( 2n ) and tetraploid ( 4n ) cells grown on YPD or PRE - SPO medium over 24 h .

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Heatmap of RNA-seq data for glycolysis and oxidative stress genes in diploid (2n) and tetraploid (4n) cells grown on YPD or PRE-SPO medium over 24 h.


TTC assay results in diploid and tetraploid cells grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively . Cells were grown for 24 h at which point a TTC overlay solution was applied and color allowed to develop for 15 min .

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TTC assay results in diploid and tetraploid cells grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively. Cells were grown for 24 h at which point a TTC overlay solution was applied and color allowed to develop for 15 min.


Diploid and tetraploid strains were spot inoculated onto YPD or PRE - SPO medium supplemented with the pH indicator bromocresol purple and cultured for 24 h at 30 ° C or 37 ° C , respectively .

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Diploid and tetraploid strains were spot inoculated onto YPD or PRE-SPO medium supplemented with the pH indicator bromocresol purple and cultured for 24 h at 30°C or 37°C, respectively.


Analysis of metabolic flux in diploid and tetraploid cells following growth for 24 h on YPD or PRE - SPO medium ( 30 ° C or 37 ° C , respectively ) , using a Seahorse instrument . OCR values represent rates of aerobic respiration while ECAR values represent rates of fermentation .

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Analysis of metabolic flux in diploid and tetraploid cells following growth for 24 h on YPD or PRE-SPO medium (30°C or 37°C, respectively), using a Seahorse instrument. OCR values represent rates of aerobic respiration while ECAR values represent rates of fermentation.


Cell size and mean MitoTracker Green signal intensity in diploid and tetraploid cells grown on YPD medium for 24 h at 30 ° C ( n = 100 and n = 5 for cell volume and MitoTracker Green measurements , respectively , error bars represent S . E . M . ) .

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Cell size and mean MitoTracker Green signal intensity in diploid and tetraploid cells grown on YPD medium for 24 h at 30°C (n=100 and n=5 for cell volume and MitoTracker Green measurements, respectively, error bars represent S.E.M.).


Diploid and tetraploid cells were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h and stained with CellROX Green . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm . Diploid and tetraploid cells were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h , stained with CellROX Green , and signal analyzed via flow cytometry .

O O O O 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-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O

Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Diploid and tetraploid cells were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h, stained with CellROX Green, and signal analyzed via flow cytometry.


Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 ( Cap1 - mNeonGreen ) were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm . Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE - SPO medium . " n . d . " indicates that no cells with activated Cap1 were detected .

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Diploid and tetraploid cells containing a fluorescently labeled version of the transcription factor Cap1 (Cap1-mNeonGreen) were grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm. Quantitation of the percentage of diploid and tetraploid cells with activated Cap1 on YPD or PRE-SPO medium. "n.d." indicates that no cells with activated Cap1 were detected.


Analysis of cell viability in tetraploid cells after culture on PRE - SPO medium ( 37 ° C for 24 h ) when supplemented with ascorbic acid , DTT , or glutathione at the indicated concentrations .

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Analysis of cell viability in tetraploid cells after culture on PRE-SPO medium (37°C for 24 h) when supplemented with ascorbic acid, DTT, or glutathione at the indicated concentrations.


Analysis of GAL1 loss ( 2 - DOG resistant colonies ) in tetraploid strain RBY18 after growth on PRE - SPO medium with or without antioxidants for 7 days at 37 ° C .

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Analysis of GAL1 loss (2-DOG resistant colonies) in tetraploid strain RBY18 after growth on PRE-SPO medium with or without antioxidants for 7 days at 37°C.


Tetraploid cells expressing Cap1 - mNeonGreen were grown on PRE - SPO medium supplemented with 20 mM ascorbic acid , 1 mM DTT , or 1 mM glutathione for 24 h at 37 ° C . Cell images were analyzed to determine the percentage of viable cells with activated Cap1 .

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Tetraploid cells expressing Cap1-mNeonGreen were grown on PRE-SPO medium supplemented with 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione for 24 h at 37°C. Cell images were analyzed to determine the percentage of viable cells with activated Cap1.


Assay to monitor for loss of GAL1 from tetraploid RBY18 overexpressing the indicated genes . Cells were grown for 7 d at 37 ° C on PRE - SPO medium in the presence ( gene OFF ) or absence ( gene ON ) of doxycycline ( DOX ) .

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Assay to monitor for loss of GAL1 from tetraploid RBY18 overexpressing the indicated genes. Cells were grown for 7 d at 37°C on PRE-SPO medium in the presence (gene OFF) or absence (gene ON) of doxycycline (DOX).


Diploid ( 2n ) and tetraploid ( 4n ) strains expressing a Gam - GFP reporter were cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , in the presence ( Gam - GFP ON ) or absence ( Gam - GFP OFF ) of doxycycline ( DOX ) for 24 h . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm .

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Diploid (2n) and tetraploid (4n) strains expressing a Gam-GFP reporter were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence (Gam-GFP ON) or absence (Gam-GFP OFF) of doxycycline (DOX) for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.


Flow cytometric analysis of the tetraploid Gam - GFP reporter strain grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , in the presence or absence of doxycycline for 24 h .

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Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence or absence of doxycycline for 24 h.


Flow cytometric analysis of the tetraploid Gam - GFP reporter strain grown on PRE - SPO medium with or without 20 mM ascorbic acid , 1 mM DTT , or 1 mM glutathione at 37 ° C for 24 h .

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Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on PRE-SPO medium with or without 20 mM ascorbic acid, 1 mM DTT, or 1 mM glutathione at 37°C for 24 h.


10 - fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA - damaging agents hydroxyurea ( HU , 20 mM ) or methyl methanesulfonate ( MMS , 0 . 01 % ) . Cells were grown for 48 h at 30 ° C or 37 ° C and the plates imaged .

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10-fold dilutions of diploid and tetraploid cells were spot inoculated onto SCD medium with or without the DNA-damaging agents hydroxyurea (HU, 20 mM) or methyl methanesulfonate (MMS, 0.01%). Cells were grown for 48 h at 30°C or 37°C and the plates imaged.


Assay to monitor for loss of GAL1 ( 2 - DOG resistant cells ) arising from tetraploid strain RBY18 after culture at 37 ° C on SCD medium alone , SCD supplemented with HU ( 20 mM ) or MMS ( 0 . 01 % ) , or SCD medium with exposure to UV light for five seconds a day for 7 days .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

Assay to monitor for loss of GAL1 (2-DOG resistant cells) arising from tetraploid strain RBY18 after culture at 37°C on SCD medium alone, SCD supplemented with HU (20 mM) or MMS (0.01%), or SCD medium with exposure to UV light for five seconds a day for 7 days.


Tetraploid cells were grown on SCD medium with or without the oxidative - stress inducing agents paraquat ( PQT ; 600 μg / mL ) , hydrogen peroxide ( H2O2 ; 2 mM ) , or piperlongumine ( PL ; 100 μg / mL ) for 24 h at 37 ° C and stained with CellROX Green . Cell images indicate calcofluor white staining ( cell wall ; blue ) , GFP , and a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm .

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Tetraploid cells were grown on SCD medium with or without the oxidative-stress inducing agents paraquat (PQT; 600 μg/mL), hydrogen peroxide (H2O2; 2 mM), or piperlongumine (PL; 100 μg/mL) for 24 h at 37°C and stained with CellROX Green. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.


Tetraploid cells expressing Cap1 - mNeonGreen were grown on SCD medium with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) for 24 h at 37 ° C . Cell images as in ( A ) . Scale bar = 10 μm .

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Tetraploid cells expressing Cap1-mNeonGreen were grown on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C. Cell images as in (A). Scale bar = 10 μm.


Tetraploid cells expressing a Gam - GFP reporter were cultured on SCD medium with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) for 24 h at 37 ° C and analyzed via fluorescence microscopy . Cell images as in ( A ) . Scale bar = 10 μm .

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Tetraploid cells expressing a Gam-GFP reporter were cultured on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C and analyzed via fluorescence microscopy. Cell images as in (A). Scale bar = 10 μm.


Flow cytometric analysis of the tetraploid Gam - GFP reporter strain grown on SCD with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) for 24 h at 37 ° C .

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Flow cytometric analysis of the tetraploid Gam-GFP reporter strain grown on SCD with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) for 24 h at 37°C.


2 - DOG assay to monitor for loss of GAL1 function from tetraploid strain RBY18 after culture on SCD medium with or without PQT ( 600 μg / mL ) , H2O2 ( 2 mM ) , or PL ( 100 μg / mL ) at 37 ° C for 7 d .

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

2-DOG assay to monitor for loss of GAL1 function from tetraploid strain RBY18 after culture on SCD medium with or without PQT (600 μg/mL), H2O2 (2 mM), or PL (100 μg/mL) at 37°C for 7 d.


( A ) Representative images of COS7 cells expressing GFP - COP1 ( green ) co - transfected with empty vector or the indicated TRIB1 - FLAG construct ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . See also Appendix Fig S1B for western blots showing the expression levels of the constructs used .

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(A) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector or the indicated TRIB1-FLAG construct (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm. See also Appendix Fig S1B for western blots showing the expression levels of the constructs used.


( B ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( * * * * p < 0 . 00001 ) .

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(B) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (****p < 0.00001).


( C ) Representative images of COS7 cells showing GFP - COP1 ( green ) and TRIB1 - FLAG ( red ) colocalizing in punctate nuclear structures . Insets show enlarged images of these nuclear puncta . Cells were stained with anti - FLAG and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(C) Representative images of COS7 cells showing GFP-COP1 (green) and TRIB1-FLAG (red) colocalizing in punctate nuclear structures. Insets show enlarged images of these nuclear puncta. Cells were stained with anti-FLAG and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( D ) Representative images of COS7 cells showing that GFP - COP1 ( green ) and TRIB1 - HA ( red ) are diffuse in the nucleoplasm when coexpressed with C / EBPα ( cyan ) . Cells were stained with anti - HA ( Santa Cruz ) , anti - C / EBPα ( Cell Signaling ) , anti - mouse Alexa Fluor 405 , and anti - rabbit Alexa Fluor 680 . Scale bar , 10 µm . See also Fig EV2C for images of cells expressing each construct alone or in pairs .

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(D) Representative images of COS7 cells showing that GFP-COP1 (green) and TRIB1-HA (red) are diffuse in the nucleoplasm when coexpressed with C/EBPα (cyan). Cells were stained with anti-HA (Santa Cruz), anti-C/EBPα (Cell Signaling), anti-mouse Alexa Fluor 405, and anti-rabbit Alexa Fluor 680. Scale bar, 10 µm. See also Fig EV2C for images of cells expressing each construct alone or in pairs.


( A ) Representative images of COS7 cells expressing COP1 - 3xFLAG ( green ) co - transfected with empty vector , GFP , or the indicated GFP - TRIB1 constructs ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(A) Representative images of COS7 cells expressing COP1-3xFLAG (green) co-transfected with empty vector, GFP, or the indicated GFP-TRIB1 constructs (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( B ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of COP1 - 3xFLAG in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( * p < 0 . 01 ; * * p < 0 . 001 ; * * * p < 0 . 0001 ) .

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(B) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of COP1-3xFLAG in (A). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (*p < 0.01; **p < 0.001; ***p < 0.0001).


( C ) Representative images of COS7 cells expressing GFP - COP1 ( green ) co - transfected with empty vector , TRIB1 - FLAG , TRIB2 - FLAG , or TRIB3 - FLAG ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(C) Representative images of COS7 cells expressing GFP-COP1 (green) co-transfected with empty vector, TRIB1-FLAG, TRIB2-FLAG, or TRIB3-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( D ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using the Student t test ( ns , not significant ; * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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(D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 with empty vector was calculated using the Student t test (ns, not significant; ***p < 0.0001; ****p < 0.00001).


( B ) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP - COP1 constructs and untagged TRIB1 .

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(B) Coimmunoprecipitation of COP1 WD40 mutants with TRIB1 from COS7 cells transiently transfected with the indicated GFP-COP1 constructs and untagged TRIB1.


( C ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(C) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( D ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using the Student t test ( * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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(D) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (C). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (***p < 0.0001; ****p < 0.00001).


( B ) Representative images of COS7 cells transiently transfected with GFP - COP1 WT or GFP - COP1 4A ( green ) . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(B) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 4A (green). Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( C ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 in ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using the Student t test ( * * * * p < 0 . 00001 ) .

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(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 in (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance relative to GFP-COP1 WT was calculated using the Student t test (****p < 0.00001).


( D ) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL , COP1 PSL 4A , and TRIB1 tail peptides to compete with FITC - TRIB1 tail for binding to the COP1 WD40 domain ( 376 - 731 ) . The COP1 WD40 domain was preincubated with FITC - TRIB1 tail before addition of the indicated concentrations of unlabeled peptide . The change in polarization is plotted as a function of unlabeled peptide concentration . Curves were fit to a sigmoidal dose - response curve . Data are presented as mean values ± S . E . M . for three independent experiments where each measurement was made in duplicate . .

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(D) Fluorescence polarization competition assay measuring the ability of unlabeled COP1 PSL, COP1 PSL 4A, and TRIB1 tail peptides to compete with FITC-TRIB1 tail for binding to the COP1 WD40 domain (376-731). The COP1 WD40 domain was preincubated with FITC-TRIB1 tail before addition of the indicated concentrations of unlabeled peptide. The change in polarization is plotted as a function of unlabeled peptide concentration. Curves were fit to a sigmoidal dose-response curve. Data are presented as mean values ± S.E.M. for three independent experiments where each measurement was made in duplicate..


( F ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) and empty vector or TRIB1 - FLAG ( red ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

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(F) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and empty vector or TRIB1-FLAG (red). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( G ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( F ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant , * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (F). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant, ***p < 0.0001; ****p < 0.00001).


( A ) Representative images of COS7 cells transiently transfected with GFP - COP1 WT or GFP - COP1 NESmut ( V238A / L242A ) ( green ) and empty vector or 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Cell Signaling ) and anti - rabbit Alexa Fluor 680 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

(A) Representative images of COS7 cells transiently transfected with GFP-COP1 WT or GFP-COP1 NESmut (V238A/L242A) (green) and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling) and anti-rabbit Alexa Fluor 680. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( B ) Representative images of COS7 cells transfected with GFP - COP1 WT ( green ) , the indicated TRIB1 - HA constructs ( red ) , and empty vector or 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Cell Signaling ) , anti - HA ( Sigma ) , anti - rabbit Alexa Fluor 680 , and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

(B) Representative images of COS7 cells transfected with GFP-COP1 WT (green), the indicated TRIB1-HA constructs (red), and empty vector or 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Cell Signaling), anti-HA (Sigma), anti-rabbit Alexa Fluor 680, and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( C ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( A ) and ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant ; * p < 0 . 01 ; * * * * p < 0 . 00001 ) .

O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (A) and (B). Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; *p < 0.01; ****p < 0.00001).


( E ) Representative images of COS7 cells transiently transfected with the indicated GFP - COP1 constructs ( green ) and 3xFLAG - CRM1 ( grayscale ) . Cells were stained with anti - FLAG ( Sigma ) and anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O

(E) Representative images of COS7 cells transiently transfected with the indicated GFP-COP1 constructs (green) and 3xFLAG-CRM1 (grayscale). Cells were stained with anti-FLAG (Sigma) and anti-mouse Alexa Fluor 568. Nuclei were counterstained with DAPI (blue). Scale bar, 10 µm.


( F ) Quantification of the average ratio of nuclear / cytoplasmic fluorescence of GFP - COP1 constructs in ( E ) and Figure EV6C . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed . Significance was calculated using the Student t test ( ns , not significant ; * * * * p < 0 . 00001 ) .

O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (E) and Figure EV6C. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed. Significance was calculated using the Student t test (ns, not significant; ****p < 0.00001).


( G ) Quantification of percentage of cells observed in ( E ) and Figure EV6C with the indicated GFP - COP1 construct exhibiting nuclear ( Nuc ) , nuclear and cytoplasmic ( Nuc / Cyt ) , or cytoplasmic ( Cyt ) localization . Mean values ± S . E . M . are shown for three independent experiments where 50 individual cells per experiment were analyzed .

O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Quantification of percentage of cells observed in (E) and Figure EV6C with the indicated GFP-COP1 construct exhibiting nuclear (Nuc), nuclear and cytoplasmic (Nuc/Cyt), or cytoplasmic (Cyt) localization. Mean values ± S.E.M. are shown for three independent experiments where 50 individual cells per experiment were analyzed.


B . Western blot of oocyte lysate ( left ) and biotinylation - purified surface ( right ) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1 / PC2 complex compared to either protein expressed separately . Anti - PC1 C - terminus antibody [ 29 ] recognized both full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 . A higher - glycosylated 130 kDa PC2 ( star ) band was only seen when PC1 is coexpressed .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-GENEPROD O O O

B. Western blot of oocyte lysate (left) and biotinylation-purified surface (right) samples showing the expression of PC1 and PC2 in Xenopus oocytes and enhanced surface trafficking of the PC1/PC2 complex compared to either protein expressed separately. Anti-PC1 C-terminus antibody [29] recognized both full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1. A higher-glycosylated 130 kDa PC2 (star) band was only seen when PC1 is coexpressed.


C . Co - IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes . IP was done with an anti - FLAG antibody . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 are indicated . Both 120 and 130 kDa bands of PC2 were seen in the IPed product .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O

C. Co-IP followed by western blot showing the association between PC1 and PC2 that were expressed in Xenopus oocytes. IP was done with an anti-FLAG antibody. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1 are indicated. Both 120 and 130 kDa bands of PC2 were seen in the IPed product.


D . Representative current - voltage relationship ( I - V ) curves ( left ) and a scatter plot and bar graph ( right ) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording . The current of the GOF PC2 _ F604P is included as a control . Currents at + 60 mV are shown in the bar graph . Each point represents the recorded current from one oocyte . Oocyte numbers for scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD ( * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O 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-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative current-voltage relationship (I-V) curves (left) and a scatter plot and bar graph (right) showing coexpression of WT PC1 and PC2 produced no current in TEVC recording. The current of the GOF PC2_F604P is included as a control. Currents at +60 mV are shown in the bar graph. Each point represents the recorded current from one oocyte. Oocyte numbers for scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD (***P < 0.001, Student's t-test).


B . Representative I - V curves ( left ) and a scatter plot and bar graph ( right ) showing that the PC2 _ AA is a GOF mutant and gave rise to a larger current than PC2 _ F604P . Scatter plot and bar graph shows the average current sizes at + 60 mV . The cations included in the bath solution , 100 mM Na + and 2 mM Ca2 + in this case , are indicated by the thick - lined boxes here and in all the following figures . Oocyte numbers for bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O 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. Representative I-V curves (left) and a scatter plot and bar graph (right) showing that the PC2_AA is a GOF mutant and gave rise to a larger current than PC2_F604P. Scatter plot and bar graph shows the average current sizes at +60 mV. The cations included in the bath solution, 100 mM Na+ and 2 mM Ca2+ in this case, are indicated by the thick-lined boxes here and in all the following figures. Oocyte numbers for bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


C . Representative currents of PC2 _ F604P and PC2 _ AA mutants in the divalent ion - free bath solution , which contains 100 mM Na + .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

C. Representative currents of PC2_F604P and PC2_AA mutants in the divalent ion-free bath solution, which contains 100 mM Na+.


D . Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2 + .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

D. Representative currents of indicated WT and mutants of PC2 in a bath solution containing 70 mM Ca2+.


E . Calcium - activated chloride channel ( CaCC ) blocker MONNA ( 10 μM ) or niflumic acid ( NFA ) ( 1 mM ) partially blocked the current recorded from the PC2 _ AA - injected oocytes in the 70 mM Ca2 + bath solution .

O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O

E. Calcium-activated chloride channel (CaCC) blocker MONNA (10 μM) or niflumic acid (NFA) (1 mM) partially blocked the current recorded from the PC2_AA-injected oocytes in the 70 mM Ca2+ bath solution.


A , B . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 100 mM Na + ( A ) , and 100 mM Na + and 2 mM Ca2 + ( B ) . Scatter plot and bar graphs show currents at + 80 mV and - 80 ( or - 70 ) mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 100 mM Na+ (A), and 100 mM Na+ and 2 mM Ca2+ (B). Scatter plot and bar graphs show currents at +80 mV and -80 (or -70) mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, ***P < 0.001, Student's t-test).


C . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 100 mM Na + and 2 mM Ca2 + solution in the absence or presence of 10 μM MONNA .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O

C. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 100 mM Na+ and 2 mM Ca2+ solution in the absence or presence of 10 μM MONNA.


D . Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , and PC1 with WT PC2 , in bath solutions containing 70 mM Ca2 + . Scatter plot and bar graphs show currents at + 80 mV and - 70 mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative I-V curves (left) and scatter plot and bar graphs (right) showing the currents from oocytes expressing PC2_AA alone, PC1 with PC2_AA, and PC1 with WT PC2, in bath solutions containing 70 mM Ca2+. Scatter plot and bar graphs show currents at +80 mV and -70 mV. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


E . Representative I - V curves of PC1 / PC2 _ AA recorded in a bath solution containing 70 mM Ca2 + solution in the absence or presence of 10 μM MONNA .

O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O

E. Representative I-V curves of PC1/PC2_AA recorded in a bath solution containing 70 mM Ca2+ solution in the absence or presence of 10 μM MONNA.


F . Representative currents of PC2 _ AA ( left ) and PC1 / PC2 _ AA ( right ) in solutions containing the indicated Ca2 + concentrations . All solutions contain 20 μM of MONNA to block CaCC current . Corresponding concentrations of NMDG + were added to compensate the osmolarity . See Fig EV2 for currents of the full voltage scale and the details of solutions used .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

F. Representative currents of PC2_AA (left) and PC1/PC2_AA (right) in solutions containing the indicated Ca2+ concentrations. All solutions contain 20 μM of MONNA to block CaCC current. Corresponding concentrations of NMDG+ were added to compensate the osmolarity. See Fig EV2 for currents of the full voltage scale and the details of solutions used.


G . Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1 / PC2 _ AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone . For oocytes injected with PC2 alone , five times more concentrated PC2 RNA was injected to increase its surface expression . The purple dashed line indicates the background 45Ca uptake set by the measurement with the water - injected oocytes . Data were averaged from three independent experiments . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Scatter plot and bar graph showing the higher radiolabeled 45Ca uptake rate of oocytes expressing the PC1/PC2_AA complex channel compared to that of oocytes expressing PC1 alone or PC2 alone. For oocytes injected with PC2 alone, five times more concentrated PC2 RNA was injected to increase its surface expression. The purple dashed line indicates the background 45Ca uptake set by the measurement with the water-injected oocytes. Data were averaged from three independent experiments. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


H . Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O O O O O

H. Surface biotinylation followed by western blot showing the expression levels of the indicated proteins in lysate and plasma membrane of oocytes in 45Ca uptake experiments.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + ( B ) , Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY 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-CELL_TYPE O O O O O O

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ (B), Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( C ) Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY 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-CELL_TYPE O O O O O O

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (C) Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


Representative I - V curves ( left ) and scatter plot and bar graphs ( right ) showing the comparison between the currents of full - length PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 70 mM Ca2 + ( D ) . Currents at both + 80 mV and - 80 ( or - 70 ) mV are displayed in the scatter plot and bar graphs . Oocyte numbers are indicated in parentheses .

Representative I-V curves (left) and scatter plot and bar graphs (right) showing the comparison between the currents of full-length PC1/PC2_AA with that of PC1-CTF/PC2_AA in bath solutions containing 70 mM Ca2+ (D). Currents at both +80 mV and -80 (or -70) mV are displayed in the scatter plot and bar graphs. Oocyte numbers are indicated in parentheses.


E . Scatter plot and bar graph showing that the current of PC1 - CTF / PC2 _ AA is completely abolished when WT PC2 was used , or after introducing R4090W mutation in the putative pore region of PC1 - CTF . Inserted are western blot images showing the expression of the corresponding proteins . Top : anti - PC1 . Bottom : anti - PC2 . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + and 2 mM Ca2 + ( F ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + ( G ) , , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ (G), , showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2 + ( H ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

O O O O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


I . Co - IP followed by western blot showing the association between PC2 _ AA and the indicated full - length or fragments of PC1 . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O 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-GENEPROD O O O

I. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


J . Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2 _ AA and full - length PC1 , PC1 - CTF , or PC1 - TLD at the oocyte surface . Besides the blots shown in the figure , surface samples were also blotted with an anti - PC1 N - terminus antibody , and the cleaved NTF fragment was found associated to the plasma membrane in the full - length sample ( shown in Appendix Fig S4 ) . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O

J. Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2_AA and full-length PC1, PC1-CTF, or PC1-TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti-PC1 N-terminus antibody, and the cleaved NTF fragment was found associated to the plasma membrane in the full-length sample (shown in Appendix Fig S4). Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


A . Representative I - V curves of the PC2 _ AA and PC1 - CTF / PC2 _ AA channels in divalent ion - free bath solutions containing 100 mM of indicated ions , showing the differences in reversal potential .

O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.


B . Bar graphs showing the markedly different reversal potentials of PC2 _ AA channel and the PC1 - CTF / PC2 _ AA channel in bath solutions containing 100 mM of the indicated ions . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * * P < 0 . 001 , Student ' s t - test ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O 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. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test).


Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( C ) These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( D ) . These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions ( B ) , indicating that these amino acids are essential for ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others ( C ) , indicating a less important role of these amino acids in ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( D ) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( E ) . Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( B ) Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 70 mM Ca2 + ( C ) . Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s t - test ) .

O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


D . Surface biotinylation followed by western blot showing the surface expression of the PC1 _ T3041V / PC2 _ AA complex , but not the PC1 _ L3040H / PC2 _ AA complex , in Xenopus oocytes . PC2 _ AA is completely absent from the surface sample when PC1 _ L3040H was coexpressed , indicating that , in our experimental condition , almost all PC2 _ AA were in the complex with PC1 _ L3040H and trapped in the process of cell surface trafficking . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 , and 130 kDa PC2 ( star ) are indicated .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O B-GENEPROD O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O

D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.


( A ) UHMK1 levels in tumor tissues ( T ) and corresponding nontumor tissues ( N ) from five GC patients were analyzed using Western blotting and qRT - PCR . The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates . Pairwise , two - tailed statistical significance was assessed by Student ' s t ‐ test . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) UHMK1 levels in tumor tissues (T) and corresponding nontumor tissues (N) from five GC patients were analyzed using Western blotting and qRT-PCR. The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates. Pairwise, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.


( B ) Immunoblotting and qRT - PCR were used to measure the levels of UHMK1 in GES - 1 and GC cell lines . The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates . Pairwise , two - tailed statistical significance was assessed by Student ' s t ‐ test . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-DISEASE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblotting and qRT-PCR were used to measure the levels of UHMK1 in GES-1 and GC cell lines. The relative mRNA expression of UHMK1 was presented as mean ± standard deviation from three replicates. Pairwise, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.


( C ) IHC analysis of UHMK1 expression in a GC patient tissue array ( n = 102 ) . Representative images are shown . Scale bars = 100μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-DISEASE B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O

(C) IHC analysis of UHMK1 expression in a GC patient tissue array (n=102). Representative images are shown. Scale bars=100μm.


( D ) The IHC signals were scored using the Allred score Data were shown as mean ± standard deviation from three independent experiments . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) The IHC signals were scored using the Allred score Data were shown as mean ± standard deviation from three independent experiments. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.


( E ) UHMK1 expression in 100 GC patients was analyzed based on the following parameters : tumor stage , tumor status , and lymph node invasion status . Fisher ' s test was applied to calculate the exact P value . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O B-GENEPROD B-EXP_ASSAY O O B-DISEASE B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) UHMK1 expression in 100 GC patients was analyzed based on the following parameters: tumor stage, tumor status, and lymph node invasion status. Fisher's test was applied to calculate the exact P value. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.


( F ) Patients with GC were stratified by the UHMK1 level ( n = 100 ) ; OS and DFS were assessed by Kaplan - Meier analysis . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O B-ORGANISM O B-DISEASE O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Patients with GC were stratified by the UHMK1 level (n=100); OS and DFS were assessed by Kaplan-Meier analysis. Data information: *P< 0.05, **P< 0.01, ***P< 0.001.


A ) GC cells ( SGC7901 and MGC803 ) were transfected with ( A ) shUHMK1 - # 1 , - # 2 , - # 3 , or control shRNA lentiviral vector , or ( B ) with a shRNA - resistant expression construct , UHMK1Δ . Western blotting was used to measure the levels of UHMK1 . NIH ImageJ software was used to quantify the band intensity . Western blotting assay were conducted for three replicates . SGC7901 cells were transfected with or without shUHMK1 - # 1 or the shRNA - resistant expression construct UHMK1Δ . CCK - 8 assays were conducted . Scale bars = 5mm . Data information : * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O B-DISEASE O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) GC cells (SGC7901 and MGC803) were transfected with (A) shUHMK1-#1,-#2, -#3, or control shRNA lentiviral vector, or (B) with a shRNA-resistant expression construct, UHMK1Δ. Western blotting was used to measure the levels of UHMK1. NIH ImageJ software was used to quantify the band intensity. Western blotting assay were conducted for three replicates. SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1Δ. CCK-8 assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance.


SGC7901 cells were transfected with or without shUHMK1 - # 1 or the shRNA - resistant expression construct UHMK1 colony formation assays were conducted . Scale bars = 5mm . Data information : * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SGC7901 cells were transfected with or without shUHMK1-#1 or the shRNA-resistant expression construct UHMK1 colony formation assays were conducted. Scale bars=5mm. Data information: **P< 0.01, ***P< 0.001, # marked no significance.


( D and E ) The volumes of subcutaneous gastric tumors derived from SGC7901 cells in NOD / SCID mice were determined at different time points . Tumors and representative bioluminescence images are also shown . UHMK1 silencing in the mouse model was confirmed by western blotting ( three biological replicates ) . Data were presented as mean ± standard deviation from shcontrol and shUHMK1 # 1 mice ( n = 10 mice / group ) . Unpaired , two - tailed statistical significance was assessed by Student ' s t ‐ test . Scale bars = 1cm . . Data information : * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O O O O B-EXP_ASSAY O B-DISEASE I-DISEASE I-DISEASE O O B-CELL_LINE O O O O B-DISEASE B-ORGANISM O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D and E) The volumes of subcutaneous gastric tumors derived from SGC7901 cells in NOD/SCID mice were determined at different time points. Tumors and representative bioluminescence images are also shown. UHMK1 silencing in the mouse model was confirmed by western blotting (three biological replicates). Data were presented as mean ± standard deviation from shcontrol and shUHMK1#1 mice (n = 10 mice/group). Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Scale bars=1cm.. Data information: **P< 0.01, ***P< 0.001, # marked no significance.


( F , G ) UHMK1 silencing ( F ) and overexpression ( G ) affected GC cell abdominal metastasis . The quantitative number of metastatic nodules ( left panel ) in nude mice ( n = 10 mice / group ) . Representative images of abdominal metastases are also shown ( right panel ) . Scale bars = 1cm . Data information : * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O O O B-GENEPROD O O O O O O O O O O B-DISEASE O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O B-EXP_ASSAY O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) UHMK1 silencing (F) and overexpression (G) affected GC cell abdominal metastasis. The quantitative number of metastatic nodules (left panel) in nude mice (n = 10 mice/group). Representative images of abdominal metastases are also shown (right panel). Scale bars=1cm. Data information: **P< 0.01, ***P< 0.001, # marked no significance.


( H ) Heatmap of the top 500 genes upregulated in GC patients with high UHMK1 expression .

O O O O O O O O O O O B-DISEASE B-ORGANISM O O B-GENEPROD O O

(H) Heatmap of the top 500 genes upregulated in GC patients with high UHMK1 expression.


( B ) LC - MS / MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown . The data are shown in the heatmap .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O

(B) LC-MS/MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown. The data are shown in the heatmap.


( C ) ( upper panel ) LC - MS / MS analysis was performed to measure 15N - glutamine - labeled purine synthesis intermediates in BGC823 cells transfected with or without the WT - UHMK1 or UHMK1 - K54R constructs . ( lower panel ) LC - MS / MS was used to analyze metabolites labeled with 13C - glycine in BGC823 cells transfected with or without the WT - UHMK1 or UHMK1 - K54R constructs ( three biological replicates ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) (upper panel) LC-MS/MS analysis was performed to measure 15N-glutamine-labeled purine synthesis intermediates in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs. (lower panel) LC-MS/MS was used to analyze metabolites labeled with 13C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs(three biological replicates). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.


( D ) RNA and DNA with incorporated 14C - glycine in BGC823 cells transfected with or without the WT - UHMK1 or UHMK1 - K54R constructs were examined using LC - MS / MS ( three biological replicates ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) RNA and DNA with incorporated 14C-glycine in BGC823 cells transfected with or without the WT-UHMK1 or UHMK1-K54R constructs were examined using LC-MS/MS(three biological replicates). Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.