( 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 were fixed with 0 . 5 % paraformaldehyde tested with 20 - 4 / A4 in a GM ‐ CSF release . EBV ‐ B1 . 11 transfected with pINCO ‐ NeoR ( B ) or pINCO ‐ Tyrosinase ( C ) were tested likewise . Both substances abrogated recognition of neoR ‐ transfected , while HLA ‐ A2 ‐ restricted presentation of 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 were incubated with 200 ng / ml of recombinant NeoR 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 transfected with pINCO ‐ NeoR were harvested 6 96 h post transfection NeoR localization within various fractions determined by Western blot . In addition , supernatant of NeoR ‐ pINCO transfected 293T ( 96 h post transfection ) was included to for release of NeoR into culture supernatant . At 6 h post transfection , NeoR becomes detectable in cytoplasm , while at 96 h post transfection a substantial amount of NeoR is also localized in endosomal / lysosomal fraction . For further characterization of various fractions , blots were stripped re ‐ probed with antibodies specific for LDH , c ‐ myc , early endosomes ( Rab ‐ 5 ) , late endosomes lysosomes ( Lamp ‐ 1 ) 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 96 h post transfection as in ( A ) . To prevent lysosomal degradation of NeoR , half of were treated with leupeptin for last 48 h . These fractions were separated on a 27 % Percoll gradient eight different fractions collected . Successful separation into fractions with increasing density was verified by Western blot with antibodies directed against Lamp ‐ 1 , Rab ‐ 5 cytochrome c . Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR in late endosomal / lysosomal fractions 7 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 vacuolar system , 293T were transfected with pINCO ‐ NeoR , treated with leupeptin to prevent NeoR degradation , endosomal / lysosomal fractions prepared at various time points post transfection separated by Percoll gradient centrifugation . Starting at 16 h post transfection , NeoR becomes detectable in Rab ‐ 5 ‐ positive fraction 5 , then spreads into late endosomal / lysosomal fractions . At 26 h post transfection an almost identical NeoR distribution pattern is observed in 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 NeoR ‐ GFP expression construct subcellular localization of fusion monitored by UV . As compared to untreated , treated with 3 ‐ MA show a much brighter UV .

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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 of of transfected with NeoR ‐ GFP fusion construct . Compared to untreated , treated for 24 h with 7 . 5 mM 3 ‐ MA show an about threefold increase in mean .

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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 of neoR ‐ transfected . Treatment of with 7 . 5 mM 3 ‐ MA causes a dramatic increase in NeoR in only 12 h . ER ‐ resident 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 in 3 ‐ MA ‐ treated is associated with an increase in , a decrease in endosomal / lysosomal NeoR .

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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 of whole lysates prepared from neoR ‐ transfected . Inhibition of synthesis by cycloheximide treatment leads to a rapid decrease in NeoR . Simultaneous treatment of 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 ) , RCC1 . 24 to which NeoR had been added ( exogenous ) , were treated with different concentrations of 3 ‐ MA surface presentation of NeoR ‐ derived peptide on HLA ‐ DP3 monitored with T clone 20 - 4 / A4 ‐ specific T 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 .

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(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 transfected with pINCO ‐ NeoR ( endogenous ) , or incubated with recombinant NeoR ( exogenous ) , were treated with inhibitors of autophagy , wortmannin 3 ‐ MA , HLA ‐ DP3 ‐ restricted presentation of 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 HLA ‐ A2 ‐ restricted tyrosinase peptide in EBV ‐ B1 . 11 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 of HLA ‐ class I HLA ‐ DP expression on RCC1 . 24 ( D ) EBV ‐ B1 . 11 ( E ) As compared to untreated ( thick ‐ lined histogram ) , treatment of 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 ) tetraploid ( RBY18 ) 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 was cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h . indicate maximum Z projections for GFP RFP channels , merged of GFP / RFP 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 RBY1 tetraploid RBY18 were cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 7 days 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 stained 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.


of - seq data showing global gene expression changes in diploid ( SC5314 , 2n ) tetraploid ( RBY18 , 4n ) on YPD 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.


of - seq data for glycolysis oxidative stress genes in diploid ( 2n ) tetraploid ( 4n ) 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 results in diploid tetraploid grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively . were grown for 24 h at which point a TTC overlay solution was applied 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 tetraploid strains were spot inoculated onto YPD or PRE - SPO medium supplemented with pH indicator bromocresol purple 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.


of metabolic flux in diploid tetraploid 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.


mean MitoTracker Green signal in diploid tetraploid grown on YPD medium for 24 h at 30 ° C ( n = 100 n = 5 for volume 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 tetraploid were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h stained with CellROX Green . indicate calcofluor ( wall ; blue ) , GFP , a merged image of GFP / DAPI / DIC channels . Scale bar = 10 μm . Diploid tetraploid were grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , for 24 h , stained with CellROX Green , signal analyzed via flow cytometry .

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


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


of GAL1 loss ( 2 - DOG resistant colonies ) in tetraploid 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 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 . were analyzed to determine of viable 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.


to monitor for loss of GAL1 from tetraploid RBY18 overexpressing indicated genes . were grown for 7 d at 37 ° C on PRE - SPO medium in 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 ) tetraploid ( 4n ) strains expressing a Gam - GFP reporter were cultured on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , in presence ( Gam - GFP ON ) or absence ( Gam - GFP OFF ) of doxycycline ( DOX ) for 24 h . indicate calcofluor ( wall ; blue ) , GFP , 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 of tetraploid Gam - GFP reporter grown on YPD or PRE - SPO medium at 30 ° C or 37 ° C , respectively , in 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 of tetraploid Gam - GFP reporter 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 tetraploid were spot inoculated onto SCD medium with or without - damaging agents hydroxyurea ( HU , 20 mM ) or methyl methanesulfonate ( MMS , 0 . 01 % ) . were grown for 48 h at 30 ° C or 37 ° C 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.


to monitor for loss of GAL1 ( 2 - DOG resistant ) arising from tetraploid 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 for five seconds a day for 7 days .

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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 were grown on SCD medium with or without 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 stained with CellROX Green . indicate calcofluor ( wall ; blue ) , GFP , 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 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 . 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 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 analyzed via microscopy . 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 of tetraploid Gam - GFP reporter 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 to monitor for loss of GAL1 function from tetraploid 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 .

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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 of COS7 expressing GFP - COP1 ( green ) co - transfected with empty vector or indicated TRIB1 - FLAG construct ( red ) . were stained with anti - FLAG ( Sigma ) anti - mouse Alexa Fluor 568 . Nuclei were counterstained with DAPI ( blue ) . Scale bar , 10 µm . See also Appendix Fig S1B for western blots showing expression of 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 ) of average of / of GFP - COP1 in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using Student t ( * * * * 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 of COS7 showing GFP - COP1 ( green ) TRIB1 - FLAG ( red ) colocalizing in punctate structures . Insets show enlarged of these . were stained with anti - FLAG 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 of COS7 showing that GFP - COP1 ( green ) TRIB1 - HA ( red ) are diffuse in nucleoplasm when coexpressed with C / EBPα ( cyan ) . were stained with anti - HA ( Santa Cruz ) , anti - C / EBPα ( Signaling ) , anti - mouse Alexa Fluor 405 , anti - rabbit Alexa Fluor 680 . Scale bar , 10 µm . See also Fig EV2C for of 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 of COS7 expressing COP1 - 3xFLAG ( green ) co - transfected with empty vector , GFP , or indicated GFP - TRIB1 constructs ( red ) . were stained with anti - FLAG ( Sigma ) 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 ) of average of / of COP1 - 3xFLAG in ( A ) . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using Student t ( * 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 of COS7 expressing GFP - COP1 ( green ) co - transfected with empty vector , TRIB1 - FLAG , TRIB2 - FLAG , or TRIB3 - FLAG ( red ) . were stained with anti - FLAG ( Sigma ) 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 ) of average of / of GFP - COP1 in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance relative to GFP - COP1 with empty vector was calculated using Student t ( 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 transiently transfected with indicated GFP - COP1 constructs 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 of COS7 transiently transfected with 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 ) of average of / of GFP - COP1 constructs in ( C ) . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using Student t ( * * * 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 of COS7 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 ) of average of / of GFP - COP1 in ( B ) . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance relative to GFP - COP1 WT was calculated using Student t ( * * * * 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 ) polarization competition measuring ability of unlabeled COP1 PSL , COP1 PSL 4A , TRIB1 tail peptides to compete with FITC - TRIB1 tail for binding to COP1 WD40 domain ( 376 - 731 ) . COP1 WD40 domain was preincubated with FITC - TRIB1 tail before addition of indicated concentrations of unlabeled peptide . change in polarization is plotted as a function of unlabeled peptide . 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 of COS7 transiently transfected with indicated GFP - COP1 constructs ( green ) empty vector or TRIB1 - FLAG ( red ) . were stained with anti - FLAG ( Sigma ) 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 ) of average of / of GFP - COP1 constructs in ( F ) . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance was calculated using Student t ( ns , not significant , * * * p < 0 . 0001 ; * * * * p < 0 . 00001 ) .

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

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

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

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

O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-GENEPROD 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 ) of average of / of GFP - COP1 constructs in ( E ) Figure EV6C . Mean values ± S . E . M . are shown for three independent experiments where 50 per experiment were analyzed . Significance was calculated using Student t ( ns , not significant ; * * * * p < 0 . 00001 ) .

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

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

(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 ) biotinylation - purified surface ( right ) samples showing expression of PC1 PC2 in Xenopus oocytes enhanced surface trafficking of PC1 / PC2 compared to either expressed separately . Anti - PC1 C - terminus antibody [ 29 ] recognized both full - ( asterisk ) GPS - 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 B-GENEPROD I-GENEPROD O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD 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 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 association between PC1 PC2 that were expressed in Xenopus oocytes . IP was done with an anti - FLAG antibody . Bands of full - ( asterisk ) GPS - CTF ( open circle ) of PC1 are indicated . Both 120 130 kDa bands of PC2 were seen in IPed product .

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

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

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 ) a scatter plot bar graph ( right ) showing that PC2 _ AA is a GOF mutant gave rise to a larger current than PC2 _ F604P . Scatter plot bar graph shows average current sizes at + 60 mV . cations included in bath solution , 100 mM Na + 2 mM Ca2 + in this case , are indicated by thick - lined boxes here in 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 - ) .

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

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 PC2 _ AA mutants in divalent ion - free bath solution , which contains 100 mM Na + .

O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD 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 mutants of PC2 in a bath solution containing 70 mM Ca2 + .

O O O B-EXP_ASSAY 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 current recorded from PC2 _ AA - injected oocytes in 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 B-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_TYPE 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 ) scatter plot bar graphs ( right ) showing currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , PC1 with WT PC2 , in bath solutions containing 100 mM Na + ( A ) , 100 mM Na + 2 mM Ca2 + ( B ) . Scatter plot bar graphs show currents at + 80 mV - 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 - ) .

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

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 + 2 mM Ca2 + solution in 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 B-SMALL_MOLECULE I-SMALL_MOLECULE 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 ) scatter plot bar graphs ( right ) showing currents from oocytes expressing PC2 _ AA alone , PC1 with PC2 _ AA , PC1 with WT PC2 , in bath solutions containing 70 mM Ca2 + . Scatter plot bar graphs show currents at + 80 mV - 70 mV . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s t - ) .

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 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 B-EXP_ASSAY 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

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

O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY 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 B-EXP_ASSAY 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 bar graph showing higher radiolabeled 45Ca uptake rate of oocytes expressing PC1 / PC2 _ AA channel compared to that of oocytes expressing PC1 alone or PC2 alone . For oocytes injected with PC2 alone , five times more concentrated PC2 was injected to increase its surface expression . purple dashed indicates background 45Ca uptake set by measurement with 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 - ) .

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

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 expression of indicated in lysate plasma of oocytes in 45Ca uptake experiments .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-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 ) scatter plot bar graphs ( right ) showing comparison between currents of full - PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + ( B ) , Currents at both + 80 mV - 80 ( or - 70 ) mV are displayed in scatter plot 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 B-EXP_ASSAY 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 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 ) scatter plot bar graphs ( right ) showing comparison between currents of full - PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 100 mM Na + 2 mM Ca2 + ( C ) Currents at both + 80 mV - 80 ( or - 70 ) mV are displayed in scatter plot 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 B-EXP_ASSAY 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 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 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 ) scatter plot bar graphs ( right ) showing comparison between currents of full - PC1 / PC2 _ AA with that of PC1 - CTF / PC2 _ AA in bath solutions containing 70 mM Ca2 + ( D ) . Currents at both + 80 mV - 80 ( or - 70 ) mV are displayed in scatter plot 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 bar graph showing that current of PC1 - CTF / PC2 _ AA is completely abolished when WT PC2 was used , or after introducing R4090W mutation in putative pore region of PC1 - CTF . Inserted are western blot showing expression of corresponding . 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 - ) .

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

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 indicated RNAs in a bath solution containing 100 mM Na + 2 mM Ca2 + ( F ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - PC1 / PC2 _ AA channel .

O O O O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 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 - PC1 / PC2 _ AA channel .

O O O O O O B-CELL_TYPE 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 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 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 - PC1 / PC2 _ AA channel .

O O O O O O B-CELL_TYPE 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 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 association between PC2 _ AA indicated full - or fragments of PC1 . Bands of full - ( asterisk ) , GPS - CTF or expressed CTF fragment ( open circle ) , 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 B-EXP_ASSAY O B-GENEPROD 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 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 expression of complexes formed between PC2 _ AA full - PC1 , PC1 - CTF , or PC1 - TLD at oocyte surface . Besides blots shown in figure , surface samples were also blotted with an anti - PC1 N - terminus antibody , NTF fragment was found associated to plasma in full - sample ( shown in Appendix Fig S4 ) . Bands of full - ( asterisk ) , GPS - CTF or expressed CTF fragment ( open circle ) , 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 B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE B-SUBCELLULAR O O B-EXP_ASSAY 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 B-EXP_ASSAY O B-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 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 PC2 _ AA PC1 - CTF / PC2 _ AA channels in divalent ion - free bath solutions containing 100 mM of indicated ions , showing differences in reversal potential .

O O O O O O O O B-GENEPROD 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

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

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY 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

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

O O O B-EXP_ASSAY I-EXP_ASSAY 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

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 reversal potentials of PC1 - CTF / PC2 _ AA indicated mutants tested in bath solutions containing 100 mM of indicated ions . Two PC1 two PC2 mutations caused dramatic changes in reversal potential for almost tested ions ( B ) , indicating that these are essential for ion permeability . Statistical significance between reversal potentials of pore mutants that of 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 - ) .

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

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 reversal potentials of PC1 - CTF / PC2 _ AA indicated mutants tested in bath solutions containing 100 mM of indicated ions . Another two PC1 mutations 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 in ion permeability . Statistical significance between reversal potentials of pore mutants that of 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 - ) .

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 B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY 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

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

O O O B-EXP_ASSAY I-EXP_ASSAY 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 I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 permeability ratios of indicated ions to that of NMDG + ( Px : PNMDG ) ( E ) . Pore mutations in both PC1 PC2 lead to significant changes in permeability ratios in most of cases .

O O O B-EXP_ASSAY I-EXP_ASSAY 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 I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 a scatter plot bar graph showing currents of full - WT mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + 2 mM Ca2 + ( B ) Currents were normalized to average current of PC1 / PC2 _ AA recorded from same batch of oocytes . Oocyte numbers in scatter plot 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 - ) .

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

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 a scatter plot bar graph showing currents of full - WT mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + 70 mM Ca2 + ( C ) . Currents were normalized to average current of PC1 / PC2 _ AA recorded from same batch of oocytes . Oocyte numbers in scatter plot 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 - ) .

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

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 surface expression of PC1 _ T3041V / PC2 _ AA , but not PC1 _ L3040H / PC2 _ AA , in Xenopus oocytes . PC2 _ AA is completely absent from surface sample when PC1 _ L3040H was coexpressed , indicating that , in our experimental condition , almost PC2 _ AA were in with PC1 _ L3040H trapped in process of surface trafficking . Bands of full - ( asterisk ) GPS - CTF ( open circle ) of PC1 , 130 kDa PC2 ( star ) are indicated .

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

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

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

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

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

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

O O O O B-GENEPROD B-EXP_ASSAY O O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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 ) signals were scored using 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 O O O O O O O O O O O O O O O O O O O O O O O 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 was analyzed on following parameters : stage , status , lymph node invasion status . Fisher ' s was applied to calculate 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 O O O O O O O O B-EXP_ASSAY 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

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

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

(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 ( SGC7901 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 of UHMK1 . NIH ImageJ software was used to quantify band . Western blotting were conducted for three replicates . SGC7901 were transfected with or without shUHMK1 - # 1 or 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 B-CELL_LINE I-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-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE 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 were transfected with or without shUHMK1 - # 1 or 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 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 E ) volumes of subcutaneous gastric tumors derived from SGC7901 in NOD / SCID mice were determined at different time points . Tumors representative bioluminescence are also shown . UHMK1 silencing in mouse model was confirmed by western blotting ( three biological replicates ) . Data were presented as mean ± standard deviation from shcontrol shUHMK1 # 1 mice ( n = 10 mice / group ) . Unpaired , two - statistical significance was assessed by Student ' s t ‐ . Scale bars = 1cm . . Data information : * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

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

(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 ) overexpression ( G ) affected GC abdominal metastasis . quantitative number of metastatic nodules ( left panel ) in nude mice ( n = 10 mice / group ) . Representative 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 B-DISEASE B-TISSUE B-EXP_ASSAY 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 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 ) of top 500 genes upregulated in GC with high UHMK1 expression .

O O O O O O O O O B-DISEASE 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 metabolites in SGC7901 with or without UHMK1 knockdown . data are shown in .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O B-GENEPROD 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 was performed to measure 15N - glutamine - labeled purine synthesis intermediates in BGC823 transfected with or without WT - UHMK1 or UHMK1 - K54R constructs . ( lower panel ) LC - MS / MS was used to analyze metabolites labeled with 13C - glycine in BGC823 transfected with or without 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 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 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 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 ) with incorporated 14C - glycine in BGC823 transfected with or without 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 O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE 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.


( E ) qRT - PCR assays were used to analyze effects of WT - UHMK1 or UHMK1 - K54R on genes controlling purine metabolism . Data were presented as mean ± standard deviation from three replicates . Unpaired , two - statistical significance was assessed by Student ' s t ‐ . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 O O 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) qRT-PCR assays were used to analyze the effects of WT-UHMK1 or UHMK1-K54R on the genes controlling purine metabolism. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.


( F ) UHMK1 silencing significantly decreased SGC7901 proliferation , while ATIC overexpression or purine supplementation markedly reversed this inhibition . Data were presented as mean ± standard deviation from three replicates . Unpaired , two - statistical significance was assessed by Student ' s t ‐ . . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O B-GENEPROD O O O B-CELL_LINE B-EXP_ASSAY O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O 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) UHMK1 silencing significantly decreased SGC7901 cell proliferation, while ATIC overexpression or purine supplementation markedly reversed this inhibition. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. . Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.


( G ) Treatment with ATIC inhibitor significantly reversed proliferation of BGC823 induced by UHMK1 overexpression . Data were presented as mean ± standard deviation from three replicates . Unpaired , two - statistical significance was assessed by Student ' s t ‐ . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O B-CELL_LINE 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 O O O O O O O O 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) Treatment with the ATIC inhibitor significantly reversed the proliferation of BGC823 cells induced by UHMK1 overexpression. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.


( A ) ( Ai ) Western blot was used to confirm UHMK1 overexpression ATF4 knockdown in BGC823 . ( Aii ) Silencing ATF4 significantly decreased of indicated metabolites in BGC823 overexpressing UHMK1 ( three biological replicates ) . ( Aiii ) Silencing ATF4 significantly decreased of containing U - 14C - glycine in BGC823 overexpressing UHMK1 ( three biological replicates ) . ( B ) LC - MS / MS was used to examine metabolites in SGC7901 with or without UHMK1 knockdown or ATF4 reintroduction . data are shown in . ( C ) Silencing ATF4 significantly decreased of indicated genes in BGC823 overexpressing UHMK1 . Data were presented as mean ± standard deviation from three replicates . Unpaired , two - statistical significance was assessed by Student ' s t ‐ . ( D ) ( Di ) qRT - PCR was used to measure expression of ATF4 in BGC823 overexpressing UHMK1 . ( Dii ) UHMK1 overexpression enhanced translocation of ATF4 in BGC823 . ( Diii ) ChIP assays on ATIC PPAT promoters were performed in BGC823 with indicated antibody . Data were presented as mean ± standard deviation from three replicates . Unpaired , two - statistical significance was assessed by Student ' s t ‐ . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , # marked no significance .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE O B-GENEPROD O 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 B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O 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 O O O O O O O O O O O O O O O O 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) (Ai) Western blot analysis was used to confirm UHMK1 overexpression and ATF4 knockdown in BGC823 cells. (Aii) Silencing ATF4 significantly decreased the levels of the indicated metabolites in BGC823 cells overexpressing UHMK1(three biological replicates). (Aiii) Silencing ATF4 significantly decreased the levels of RNA and DNA containing U-14C-glycine in BGC823 cells overexpressing UHMK1(three biological replicates). (B) LC-MS/MS was used to examine the metabolites in SGC7901 cells with or without UHMK1 knockdown or ATF4 reintroduction. The data are shown in the heatmap. (C) Silencing ATF4 significantly decreased the levels of the indicated genes in BGC823 cells overexpressing UHMK1. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. (D) (Di) qRT-PCR was used to measure the expression of ATF4 in BGC823 cells overexpressing UHMK1. (Dii) UHMK1 overexpression enhanced the nuclear translocation of ATF4 in BGC823 cells. (Diii) ChIP assays on the ATIC and PPAT promoters were performed in BGC823 cells with the indicated antibody. Data were presented as mean ± standard deviation from three replicates. Unpaired, two-tailed statistical significance was assessed by Student's t‐test. Data information: *P< 0.05, **P< 0.01, ***P< 0.001, # marked no significance.