Figure 2A N1E - 115 cells treated with siControl , siINPP5E , or siAtg2a / 2b were cultured in growth medium . The post - nuclear fraction ( PNS ) was separated into LSP , HSP , and HSS fractions , and then analyzed by immunoblots using anti - p62 , anti - LC3 , and GAPDH antibodies . The subfractions were treated with proteinase K ( Pro . K ) with or without Triton X - 100 . Quantitation of protein signal intensities from immunoblots showing LC3 - II ( left ) or p62 ( right ) levels , following normalization to the control protein GAPDH . * * , P < 0 . 01 ; * , P < 0 . 05 . B NIE - 115 cells stably expressing LAMP1 - mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors ( 10 µg / ml Pepstatin A and 10 µg / ml E - 64 - d ) . Because the treatment of protease inhibitors inhibits lysosomal degradation , LC3puncta persist even ifautophagosomes fuse with lysosomes . Cells were fixed and stained with anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . C Percentages of colocalization of LC3 dots with LAMP1 dots ( mean ± s . d . ; n > 20 cells from three independent experiments ) . * * , P < 0 . 01 .

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Figure 2A N1E-115 cells treated with siControl, siINPP5E, or siAtg2a/2b were cultured in growth medium. The post-nuclear fraction (PNS) was separated into LSP, HSP, and HSS fractions, and then analyzed by immunoblots using anti-p62, anti-LC3, and GAPDH antibodies. The subfractions were treated with proteinase K (Pro. K) with or without Triton X-100. Quantitation of protein signal intensities from immunoblots showing LC3-II (left) or p62 (right) levels, following normalization to the control protein GAPDH. **, P < 0.01; *, P < 0.05.B NIE-115 cells stably expressing LAMP1-mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d). Because the treatment of protease inhibitors inhibits lysosomal degradation, LC3puncta persist even ifautophagosomes fuse with lysosomes. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.C Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.


Figure 3B N1E - 115 cells stably expressing mSt - INPP5E ( WT , D477N , ∆ CAAX , or 295 - 644 ) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf . A1 for 2 h , and then analyzed by immunoblot using anti - LC3 and anti - GAPDH antibodies . The mSt - INPP5Es used in this study were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence . C Quantitation of protein signal intensities from immunoblots in B , showing differences in LC3 - II levels in the presence and absence of Baf . A1 following normalization to the control proteinGAPDH . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 ; * , P < 0 . 05 . D NIE - 115 cells stably expressing mSt - INPP5E ( WT , D477N , or ΔCAAX ) were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . The number showing to the right side indicates the colocalization rate of LAMP1 with mSt - INPP5E . Bar , 10 µm .

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Figure 3B N1E-115 cells stably expressing mSt-INPP5E (WT, D477N, ∆CAAX, or 295-644) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and then analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mSt-INPP5Es used in this study were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B, showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.D NIE-115 cells stably expressing mSt-INPP5E (WT, D477N, or ΔCAAX) were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. The number showing to the right side indicates the colocalization rate of LAMP1 with mSt-INPP5E. Bar, 10 µm.


Figure 4A N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg / ml DQ - BSA for 12 h and chased for 2 h . Cells were fixed and stained with anti - LAMP1 antibodies and analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . B Quantitation of signal intensities in A showing colocalization of DQ - BSA with LAMP1 ( means ± s . d . ; n > 50 cells from three independent experiments ) . Baf . A1 was used as a control for inhibition of delivery and degradation of DQ - BSA via the endocytic pathway . * * , P < 0 . 01 ; n . s . , non significant . C N1E - 115 cells treated with siControl , siINPP5Es , or siCHMP5 were stimulated with 100 ng / ml EGF for the indicated time periods , and then analyzed by immunoblot using anti - EGFR and anti - α - tubulin antibodies . Levels of CHMP5 mRNA72 h after transfection of N1E - 115 cells with siControl or siCHMP5 , as analyzed by RT - PCR . D Quantitation of EGFR degradation ratio in C . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 .

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Figure 4A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg/ml DQ-BSA for 12 h and chased for 2 h. Cells were fixed and stained with anti-LAMP1 antibodies and analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.B Quantitation of signal intensities in A showing colocalization of DQ-BSA with LAMP1 (means ± s.d.; n > 50 cells from three independent experiments). Baf.A1 was used as a control for inhibition of delivery and degradation of DQ-BSA via the endocytic pathway. **, P < 0.01; n.s., non significant.C N1E-115 cells treated with siControl, siINPP5Es, or siCHMP5 were stimulated with 100 ng/ml EGF for the indicated time periods, and then analyzed by immunoblot using anti-EGFR and anti-α-tubulin antibodies. Levels of CHMP5 mRNA72 h after transfection of N1E-115 cells with siControl or siCHMP5, as analyzed by RT-PCR.D Quantitation of EGFR degradation ratio in C. Results represent means ± s.d. of three independent experiments. **, P < 0.01.


Figure 5A NIE - 115 cells stably expressing mSt - 2xML1N or - 2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1 . Cells were fixed and stained with anti - LAMP1 or anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . B NIE - 115 cells stably expressing mSt - 2xML1N treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . C Quantitation of signal intensities in B showing mSt - 2xML1N colocalizing with LAMP1 ( means ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 ; * , P < 0 . 05 . D N1E - 115 cells stably expressing GFP - 2xML1N treated with siControl or siINPP5E were transiently transfected with mSt - INPP5E ( WT , D477N ) . Quantitation of signal intensities showing GFP - 2xML1N colocalizing with LAMP1 ( means ± s . d . ; n > 20 cells from three independent experiments ) . * * , P < 0 . 01 ; * , P < 0 . 05 . E NIE - 115 cells stably expressing mCherry - 2xFYVE treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies , and then analyzed by immunofluorescence microscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . F Quantitation of signal intensities in E showing mCherry - 2xFYVE colocalizing with LAMP1 ( means ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 .

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Figure 5A NIE-115 cells stably expressing mSt-2xML1N or -2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1. Cells were fixed and stained with anti-LAMP1 or anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.B NIE-115 cells stably expressing mSt-2xML1N treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.C Quantitation of signal intensities in B showing mSt-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01; *, P < 0.05.D N1E-115 cells stably expressing GFP-2xML1N treated with siControl or siINPP5E were transiently transfected with mSt-INPP5E (WT, D477N). Quantitation of signal intensities showing GFP-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01; *, P < 0.05.E NIE-115 cells stably expressing mCherry-2xFYVE treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.F Quantitation of signal intensities in E showing mCherry-2xFYVE colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.


Figure 6A N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies and Phalloidin , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 ( means ± s . d . ; n > 40 cells from three independent experiments ) . * * , P < 0 . 01 . B N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - phospho - Y421 cortactin and anti - LAMP1 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . Quantitation of signal intensities showing cortactin ( pY421 ) colocalizing with LAMP1 ( means ± s . d . ; n > 40 cells from three independent experiments ) . * * , P < 0 . 01 . C N1E - 115 cells were treated with 0 . 5 µM Latrunculin A ( Lat A ) in growth medium for 6 h . Cells were fixed and stained with anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Bar , 10 µm . Quantitation of the number of LC3puncta per cell . ( mean ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 . D NIE - 115 cells stably expressing LAMP1 - mCherry were treated with DMSO or 0 . 5 µM Lat A in growth medium for 4 h , subsequently cultured with 200 nM Torin1 and protease inhibitors ( 10 µg / ml Pepstatin A and 10 µg / ml E - 64 - d ) for 2 h ( Lat A treatment for total 6h ) . Cells were fixed and stained with anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . Percentages of colocalization of LC3 dots with LAMP1 dots ( mean ± s . d . ; n > 20 cells from three independent experiments ) . * * , P < 0 . 01 .

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Figure 6A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies and Phalloidin, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.B N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-phospho-Y421 cortactin and anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing cortactin (pY421) colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.C N1E-115 cells were treated with 0.5 µM Latrunculin A (Lat A) in growth medium for 6 h. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Bar, 10 µm. Quantitation of the number of LC3puncta per cell. (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.D NIE-115 cells stably expressing LAMP1-mCherry were treated with DMSO or 0.5 µM Lat A in growth medium for 4 h, subsequently cultured with 200 nM Torin1 and protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d) for 2 h (Lat A treatment for total 6h). Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.


Figure 7B N1E - 115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf . A1 for 2 h , and analyzed by immunoblot using anti - LC3 and anti - GAPDH antibodies . The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence . C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3 - II levels in the presence and absence of Baf . A1 following normalization to the control proteinGAPDH . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 ; * , P < 0 . 05 .

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Figure 7B N1E-115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.


Figure 1Viability 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 . 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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Figure 1Viability 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.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.


Figure 2Heatmap 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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Figure 2Heatmap 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.


Figure 3Heatmap 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 . 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 . 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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Figure 3Heatmap 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.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.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.).


Figure 4Diploid 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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Figure 4Diploid 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.


Figure 5Analysis 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 . 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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Figure 5Analysis 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.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).


Figure 6Diploid ( 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 . 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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Figure 6Diploid (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.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.


Figure 710 - 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 .

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Figure 710-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.


Figure 8Tetraploid 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 . 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 . 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 .

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Figure 8Tetraploid 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.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.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.


Figure 1Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( A ) CTGF interacts with LRP4 but not with MuSK . Full - length CTGF ( CTGF - full - myc ) was immunoprecipitated with the ectodomain of LRP4 ( LRP4ect - Flag , lane 6 ) but not with the ectodomain of MuSK ( MuSKect - Flag , lane 3 ) . However , when CTGF - full - myc , LRP4ect , and MuSKect - Flag were expressed together , CTGF - full - myc was immunoprecipitated with MuSKect - Flag ( lane 5 ) . The CT domain of CTGF ( CTGF - CT - myc ) was sufficient to immunoprecipitate LRP4ect - Flag ( lane 7 ) . Predicted positions of full - length and shortened CTGF proteins are indicated by bars and / or asterisks . Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( B ) Deletion of the CT domain of human CTGF ( CTGF - ΔCT _ ΔTSP1 and CTGF - ΔCT ) impairs the interaction between CTGF and LRP4ect - Flag . Predicted positions of full - length and shortened CTGF proteins are indicated by bars and / or asterisks . Representative immunoprecipitation ( IP ) of HEK293 cells transfected with indicated cDNAs . ( C ) Anti - CTGF antibody immunoprecipitated human LRP4 deleted with the 1st , 2nd , and 4th β - propeller domains ( Δ1 , Δ2 , and Δ4 ) but not with the 3rd β - propeller domain ( ∆ 3 ) . Cell - free plate - binding assays . A purified tagged - protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase ( AP ) was overlaid . Bound protein was quantified by measuring AP activity . ( D ) LRP4ect - Flag , but not MuSKect - Flag , bound to overlaid CTGF - full - mycAP and CTGF - CT - mycAP . In contrast , LRP4ect - Flag did not bind to CTGF - ΔCT - mycAP . Rspo2 - mycAP were used as Control - mycAP and control - myc , respectively . Mean and SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for the three panels in ( D ) p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group . Cell - free plate - binding assays . A purified tagged - protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase ( AP ) was overlaid . Bound protein was quantified by measuring AP activity . ( E , upper panel ) LRP4ect - Flag bound to overlaid MuSKect - mycAP in a dose - dependent manner of CTGF - full - myc , but not of CTGF - ΔCT - myc . ( E , lower panel ) LRP4ect - Flag bound to overlaid agrin - mycAP independent of CTGF - full - myc . Rspo2 - mycAP and Rspo2 - myc were used as Control - mycAP and control - myc , respectively . Mean and SD ( n = 9 wells ; 3 wells in 3 independent experiments ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA for the three panels in ( E ) . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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Figure 1Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (A) CTGF interacts with LRP4 but not with MuSK. Full-length CTGF (CTGF-full-myc) was immunoprecipitated with the ectodomain of LRP4 (LRP4ect-Flag, lane 6) but not with the ectodomain of MuSK (MuSKect-Flag, lane 3). However, when CTGF-full-myc, LRP4ect, and MuSKect-Flag were expressed together, CTGF-full-myc was immunoprecipitated with MuSKect-Flag (lane 5). The CT domain of CTGF (CTGF-CT-myc) was sufficient to immunoprecipitate LRP4ect-Flag (lane 7). Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (B) Deletion of the CT domain of human CTGF (CTGF-ΔCT_ΔTSP1 and CTGF-ΔCT) impairs the interaction between CTGF and LRP4ect-Flag. Predicted positions of full-length and shortened CTGF proteins are indicated by bars and/or asterisks.Representative immunoprecipitation (IP) of HEK293 cells transfected with indicated cDNAs. (C) Anti-CTGF antibody immunoprecipitated human LRP4 deleted with the 1st, 2nd, and 4th β-propeller domains (Δ1, Δ2, and Δ4) but not with the 3rd β-propeller domain (∆3).Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (D) LRP4ect-Flag, but not MuSKect-Flag, bound to overlaid CTGF-full-mycAP and CTGF-CT-mycAP. In contrast, LRP4ect-Flag did not bind to CTGF-ΔCT-mycAP. Rspo2-mycAP were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (D) p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.Cell-free plate-binding assays. A purified tagged-protein was attached to a plastic plate and a purified recombinant protein fused to alkaline phosphatase (AP) was overlaid. Bound protein was quantified by measuring AP activity. (E, upper panel) LRP4ect-Flag bound to overlaid MuSKect-mycAP in a dose-dependent manner of CTGF-full-myc, but not of CTGF-ΔCT-myc. (E, lower panel) LRP4ect-Flag bound to overlaid agrin-mycAP independent of CTGF-full-myc. Rspo2-mycAP and Rspo2-myc were used as Control-mycAP and control-myc, respectively. Mean and SD (n = 9 wells; 3 wells in 3 independent experiments) are indicated. p-value < 0.05 by two-way repeated measures ANOVA for the three panels in (E). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


Figure 2 ( A ) Representative immunoblotting of Lrp4 , MuSK , Transferrin Receptor ( TfR ) , CTGF , and β - actin in whole - cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM ( 1 ng / ml ) agrin and / or 250 pM ( 100 ng / ml ) CTGF for 24 h . CTGF increases Lrp4 on the plasma membrane in the presence of agrin . ( B ) C2C12 myotubes were treated with 1 pM agrin and / or 250 pM CTGF for 6 h . Lrp4 was immunoprecipitated ( IP ) with an anti - Lrp4 antibody , and phosphorylated Lrp4 was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . ( C ) Representative immunoblotting of Lrp4 , MuSK , CTGF , and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18 . 5 of wild - type ( WT ) and Ctgf - / - mice . ( D ) Representative immunoblotting of Y755 - phosphorylated MuSK , total MuSK , and β - actin in the diaphragm at E18 . 5 of wild - type ( WT ) , Ctgf - / - , and Ctgf + / - mice .

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Figure 2(A) Representative immunoblotting of Lrp4, MuSK, Transferrin Receptor (TfR), CTGF, and β-actin in whole-cell lysates and the plasma membrane fraction of C2C12 myotubes treated with 1 pM (1 ng/ml) agrin and/or 250 pM (100 ng/ml) CTGF for 24 h. CTGF increases Lrp4 on the plasma membrane in the presence of agrin.(B) C2C12 myotubes were treated with 1 pM agrin and/or 250 pM CTGF for 6 h. Lrp4 was immunoprecipitated (IP) with an anti-Lrp4 antibody, and phosphorylated Lrp4 was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody.(C) Representative immunoblotting of Lrp4, MuSK, CTGF, and TfR in whole tissue lysates and the plasma membrane fraction of lower limb muscles at E18.5 of wild-type (WT) and Ctgf-/- mice.(D) Representative immunoblotting of Y755-phosphorylated MuSK, total MuSK, and β-actin in the diaphragm at E18.5 of wild-type (WT), Ctgf-/-, and Ctgf+/- mice.


Figure 3 ( A ) C2C12 myotubes were treated with indicated concentrations of agrin - mycAP , CTGF - mycAP , and / or CTGF - ∆ CT - mycAP for 3 h . Total MuSK was immunoprecipitated ( IP ) with an anti - MuSK antibody , and phosphorylated MuSK was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . CTGF - mycAP , but not CTGF - ∆ CT - mycAP , enhanced agrin - mediated MuSK phosphorylation . ( B ) C2C12 myotubes were treated with 5 pM agrin - mycAP , 250 pM CTGF - mycAP , and / or 250 pM CTGF - ∆ CT - mycAP for 18 h . BSA was added to the control medium . AChRs and the plasma membrane were visualized with Alexa594 - conjugated α - bungarotoxin ( red ) and CellMask ( green ) , respectively . Scale bar = 20 µm . Arrowheads point to the AChR clusters with an axis length of 4 μm or more . CTGF enhances agrin - mediated AChR clustering . ( C ) C2C12 myotubes were infected with lentivirus expressing shControl , shCtgf - 1 , or shCtgf - 2 . Doxycycline was added for 2 days to induce shRNA expression . BSA or agrin was added to the medium at 10 pM . Total MuSK was immunoprecipitated ( IP ) by an anti - MuSK antibody , and phosphorylated MuSK was immunoblotted with an anti - phosphotyrosine ( p - Tyr ) antibody . Ctgf knockdown suppressed MuSK phosphorylation . ( D ) Ten pM agrin was added to the medium . AChRs were visualized with Alexa594 - conjugated α - bungarotoxin ( red signals ) in the infected myotube ( green GFP signals ) . Ctgf knockdown decreased the number and length of AChR clusters . Scale bar = 10 µm .

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Figure 3(A) C2C12 myotubes were treated with indicated concentrations of agrin-mycAP, CTGF-mycAP, and/or CTGF-∆CT-mycAP for 3 h. Total MuSK was immunoprecipitated (IP) with an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. CTGF-mycAP, but not CTGF-∆CT-mycAP, enhanced agrin-mediated MuSK phosphorylation.(B) C2C12 myotubes were treated with 5 pM agrin-mycAP, 250 pM CTGF-mycAP, and/or 250 pM CTGF-∆CT-mycAP for 18 h. BSA was added to the control medium. AChRs and the plasma membrane were visualized with Alexa594-conjugated α-bungarotoxin (red) and CellMask (green), respectively. Scale bar = 20 µm. Arrowheads point to the AChR clusters with an axis length of 4 μm or more. CTGF enhances agrin-mediated AChR clustering.(C) C2C12 myotubes were infected with lentivirus expressing shControl, shCtgf-1, or shCtgf-2. Doxycycline was added for 2 days to induce shRNA expression. BSA or agrin was added to the medium at 10 pM. Total MuSK was immunoprecipitated (IP) by an anti-MuSK antibody, and phosphorylated MuSK was immunoblotted with an anti-phosphotyrosine (p-Tyr) antibody. Ctgf knockdown suppressed MuSK phosphorylation.(D) Ten pM agrin was added to the medium. AChRs were visualized with Alexa594-conjugated α-bungarotoxin (red signals) in the infected myotube (green GFP signals). Ctgf knockdown decreased the number and length of AChR clusters. Scale bar = 10 µm.


Figure 4 ( A ) Quantitative RT - PCR of Chrne , Musk , Lrp4 , and Ctgf in C2C12 myotubes treated with 10 ng / ml BSA or recombinant human CTGF . ( B ) Quantitative RT - PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng / ml agrin and / or 10 ng / ml neuregulin - 1 . ( C ) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18 . 5 . Co - localization of AChR ( red ) and CTGF ( green ) is indicated in the merged images . Red arrow points to an AChR cluster . Scale bar = 20 μm . ( D ) Quantitative RT - PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse . Mean and SD ( n = 3 mice ) are indicated . p - value < 0 . 05 by two - way repeated measures ANOVA . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group .

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Figure 4(A) Quantitative RT-PCR of Chrne, Musk, Lrp4, and Ctgf in C2C12 myotubes treated with 10 ng/ml BSA or recombinant human CTGF.(B) Quantitative RT-PCR of Ctgf in C2C12 myotubes treated with indicated combinations of 10 ng/ml agrin and/or 10 ng/ml neuregulin-1.(C) Localizations of CTGF and AChR in a representative cross section of the lower limb muscle at E18.5. Co-localization of AChR (red) and CTGF (green) is indicated in the merged images. Red arrow points to an AChR cluster. Scale bar = 20 μm.(D) Quantitative RT-PCR of Ctgf and neural Agrn in the spinal cord and the diaphragm in mouse. Mean and SD (n = 3 mice) are indicated. p-value < 0.05 by two-way repeated measures ANOVA. p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.


Figure 5 ( A ) Representative confocal images of the left diaphragm at E18 . 5 labeled with α - bungarotoxin ( red ) and anti - peripherin antibody ( green ) to visualize AChRs and the motor axon , respectively . The endplates of Ctgf - / - diaphragm were smaller ( lower left panel ) than those of the wild - type diaphragm ( upper left panel ) . In the Ctgf - / - diaphragm , 10 . 0 ± 3 . 2 % ( mean and SD , n = 105 in 5 embryos ) of peripherin - positive axons passed through the AChR - positive areas . In contrast , in the wild - type diaphragm , 2 . 0 ± 1 . 4 % ( mean and SD , n = 102 in 5 embryos ) of peripherin - positive axons passed through the AChR - positive areas . Quantification was performed in a blinded manner . Scale bar = 5 µm . ( B ) Representative confocal images of the left diaphragm at E18 . 5 labeled with α - bungarotoxin ( red ) and anti - synaptophysin antibody ( green ) to visualize AChRs and the nerve terminal , respectively . Synaptophysin signals mostly overlapped with α - bungarotoxin signals in the wild - type diaphragm , whereas synaptophysin signals extended into the axon in the Ctgf - / - diaphragm . Scale bar = 5 µm . ( C - E ) Blinded morphometric analysis of AChR signals ( C ) , synaptophysin signals ( D ) , and the ratio of synaptophysin signals not colocalized with AChR signals ( E ) of confocal images shown in ( A ) and ( B ) . AChR areas ( C ) , but not synaptophysin - positive areas ( D ) , were decreased in Ctgf - / - mice at E18 . 5 . ( E ) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf - / - diaphragm . Mean and SD ( n = 40 - 50 NMJs in 6 left diaphragms ) are indicated . p - value < 0 . 05 by one - way ANOVA for ( C and E ) , but not for ( D ) . p < 0 . 05 by post - hoc Tukey test is indicated by a single letter representing each group . ( F ) Representative electron micrographs of the NMJs in the diaphragm of wild - type and Ctgf - / - mice at E18 . 5 . In Ctgf - / - mice , synaptic vesicles are sparser and the number of mitochondria at the nerve terminals is reduced compared to those in wild - type mice . See Table 1 for morphometric analysis . Regions indicated by rectangles are enlarged in the insets . Arrowheads point to synaptic vesicles ( SV ) . Mit , mitochondria . Blinded morphometric measurements are shown in Table 1 . Scale bar = 500 nm .

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

Figure 5(A) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-peripherin antibody (green) to visualize AChRs and the motor axon, respectively. The endplates of Ctgf-/- diaphragm were smaller (lower left panel) than those of the wild-type diaphragm (upper left panel). In the Ctgf-/- diaphragm, 10.0 ± 3.2% (mean and SD, n = 105 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. In contrast, in the wild-type diaphragm, 2.0 ± 1.4% (mean and SD, n = 102 in 5 embryos) of peripherin-positive axons passed through the AChR-positive areas. Quantification was performed in a blinded manner. Scale bar = 5 µm.(B) Representative confocal images of the left diaphragm at E18.5 labeled with α-bungarotoxin (red) and anti-synaptophysin antibody (green) to visualize AChRs and the nerve terminal, respectively. Synaptophysin signals mostly overlapped with α-bungarotoxin signals in the wild-type diaphragm, whereas synaptophysin signals extended into the axon in the Ctgf-/- diaphragm. Scale bar = 5 µm.(C-E) Blinded morphometric analysis of AChR signals (C), synaptophysin signals (D), and the ratio of synaptophysin signals not colocalized with AChR signals (E) of confocal images shown in (A) and (B). AChR areas (C), but not synaptophysin-positive areas (D), were decreased in Ctgf-/- mice at E18.5. (E) A large fraction of synaptophysin signalswere not colocalized with AChR signals in Ctgf-/- diaphragm. Mean and SD (n = 40-50 NMJs in 6 left diaphragms) are indicated. p-value < 0.05 by one-way ANOVA for (C and E), but not for (D). p < 0.05 by post-hoc Tukey test is indicated by a single letter representing each group.(F) Representative electron micrographs of the NMJs in the diaphragm of wild-type and Ctgf-/- mice at E18.5. In Ctgf-/- mice, synaptic vesicles are sparser and the number of mitochondria at the nerve terminals is reduced compared to those in wild-type mice. See Table 1 for morphometric analysis. Regions indicated by rectangles are enlarged in the insets. Arrowheads point to synaptic vesicles (SV). Mit, mitochondria. Blinded morphometric measurements are shown in Table 1. Scale bar = 500 nm.


Figure 2A ) Time course of biomass concentration . B ) Time course of glucose concentration . Data information : The means for n = 3 biologically independent samples are shown . The error bars represent ± SD . . Note : although no nitrogen source was present in the medium , some minor growth ( especially of the control and wild type strains ) remained ( A ) , which is a known phenomenon within the first hours of cultivation after nitrogen depletionC ) Specific glucose ( Glc ) uptake rate and specific productivity for ethanol ( Eth ) , acetate ( Ace ) , formate ( For ) , lactate ( Lac ) , and succinate ( Suc ) . Data information : The reaction rates in C were calculated from the beginning of cultivation until the last sampling time point where glucose was still present in the medium . The means and individual data for n = 3 biologically independent samples are shown . The error bars represent ± SD . .

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Figure 2A) Time course of biomass concentration. B) Time course of glucose concentration. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.. Note: although no nitrogen source was present in the medium, some minor growth (especially of the control and wild type strains) remained (A), which is a known phenomenon within the first hours of cultivation after nitrogen depletionC) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). Data information: The reaction rates in C were calculated from the beginning of cultivation until the last sampling time point where glucose was still present in the medium. The means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD..


Figure 3For all growth regimes tested , the glucose uptake rate shows a biphasic response curve to increasing ATPase levels . Data information : The means for n = 3 biologically independent samples are shown . The error bars represent ± SD .

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Figure 3For all growth regimes tested, the glucose uptake rate shows a biphasic response curve to increasing ATPase levels. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.


Figure 4Simulation of the PFK rate with varying ADP / ( ATP + ADP ) ratios . Data information : The PFK flux was simulated as single ( isolated ) reaction with the following fixed metabolite concentrations relevant for the kinetic rate law of the PFK PEP : 0 . 27 µmol / gDW ; F6P : 0 . 91 µmol / gDW ; FBP : 9 . 74 µmol / gDW ; total concentration ATP + ADP : 2 . 67 µmol / gDW .

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Figure 4Simulation of the PFK rate with varying ADP/(ATP+ADP) ratios. Data information: The PFK flux was simulated as single (isolated) reaction with the following fixed metabolite concentrations relevant for the kinetic rate law of the PFK PEP: 0.27 µmol/gDW; F6P: 0.91 µmol/gDW; FBP: 9.74 µmol/gDW ; total concentration ATP+ADP: 2.67 µmol/gDW.


Figure 5A ) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured exchange fluxes . Data information : For the measurements , the means for n = 3 biologically independent samples are shown and the error bars represent ± SD . B ) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured metabolite concentrations . Data information : For the measurements , the means for n = 3 biologically independent samples are shown and the error bars represent ± SD .

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Figure 5A) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured exchange fluxes. Data information: For the measurements, the means for n = 3 biologically independent samples are shown and the error bars represent ± SD.B) Comparison of the simulations of the kinetic model version 1 and version 2 with the measured metabolite concentrations. Data information: For the measurements, the means for n = 3 biologically independent samples are shown and the error bars represent ± SD.


Figure 7A ) Time course of biomass concentration . B ) Time course of glucose concentration . C Data information : The means for n = 3 biologically independent samples are shown . The error bars represent ± SD . C ) Specific glucose ( Glc ) uptake rate and specific productivity for ethanol ( Eth ) , acetate ( Ace ) , formate ( For ) , lactate ( Lac ) , and succinate ( Suc ) . D ) Yield of Eth , Ace , For , Lac , and Suc . Data information : The reaction rates in C were calculated for the exponential phase under assumption of quasi - steady state . The means ( and the means and individual data for n = 3 biologically independent samples are shown . The error bars represent ± SD .

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Figure 7A) Time course of biomass concentration. B) Time course of glucose concentration. C Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD. C) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). D) Yield of Eth, Ace, For, Lac, and Suc. Data information: The reaction rates in C were calculated for the exponential phase under assumption of quasi-steady state. The means ( and the means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD.


Figure 1C . Autoradiograph of hTrmt13 - RNA complexes in the MDA - MB - 231 cells immunoprecipitated with an antibody against hTrmt13 ( right lane ) and IgG as a negative control ( left lane ) . * RNA residing in the regions between marked lines was recovered for sequencing . H . Mass chromatograms of the nucleosides , Um ( Q1 / Q3 = 259 . 1 / 113 . 1 ) , Cm ( Q1 / Q3 = 258 . 1 / 112 . 1 ) , and G ( Q1 / Q3 = 284 . 1 / 152 . 2 ) of HctRNAGly ( GCC ) , HctRNAGly ( CCC ) , HctRNAGly ( UCC ) , and HctRNAPros isolated from WT and hTrmt13 KO cells . Um , 2 ′ - O - methyluridine , Cm , 2 ′ - O - methylcytidine . Q1 / Q3 : the mass of the precursor ion and the mass of the product ion . These endogenous tRNAs were purified separately from total cellular RNAs using sequence - specific probes . I . Quantification of the Um / G and Cm / G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells . Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for three independent experiments .

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Figure 1C. Autoradiograph of hTrmt13-RNA complexes in the MDA-MB-231 cells immunoprecipitated with an antibody against hTrmt13 (right lane) and IgG as a negative control (left lane). * RNA residing in the regions between marked lines was recovered for sequencing.H. Mass chromatograms of the nucleosides, Um (Q1/Q3 = 259.1/113.1), Cm (Q1/Q3 = 258.1/112.1), and G (Q1/Q3 = 284.1/152.2) of HctRNAGly(GCC), HctRNAGly(CCC), HctRNAGly(UCC), and HctRNAPros isolated from WT and hTrmt13 KO cells. Um, 2′-O-methyluridine, Cm, 2′-O-methylcytidine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. These endogenous tRNAs were purified separately from total cellular RNAs using sequence-specific probes.I. Quantification of the Um/G and Cm/G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.


Figure 2A . De novo protein synthesis measured by puromycin incorporation in shSCR , hTrmt13 knockdown cells ( sh - 1 and sh - 2 ) , and sh - 1 with stable expression of wild type hTrmt13 ( sh - 1 + hTrmt13 ) or enzymatically - inactive hTrmt13 ( sh - 1 + E463A ) . Puromycin - labeled proteins were detected by immunoblotting . Representative images ( left ) and statistical analysis ( right ) are shown . Knockdown and overexpression efficiency of hTrmt13 in MDA - MB - 231 verified by Western blots . Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments . C . The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot . The samples were also deacylated ( DA ) by incubating with alkaline buffer ( pH 9 . 0 ) to obtain fully uncharged tRNA control . D . RT - qPCR using a stem - loop primer to quantify the tRFs levels in shSCR , sh - 1 , and sh - 2 . Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments . G . Quantification of de novo protein synthesis in cells treated with different concentration of tRFs H . Quantification of de novo protein synthesis in cells treated with different concentration of tRNAGly ( CCC ) Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments .

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Figure 2A. De novo protein synthesis measured by puromycin incorporation in shSCR, hTrmt13 knockdown cells (sh-1 and sh-2), and sh-1 with stable expression of wild type hTrmt13 (sh-1+hTrmt13) or enzymatically-inactive hTrmt13 (sh-1+E463A). Puromycin-labeled proteins were detected by immunoblotting. Representative images (left) and statistical analysis (right) are shown. Knockdown and overexpression efficiency of hTrmt13 in MDA-MB-231 verified by Western blots. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.C. The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot. The samples were also deacylated (DA) by incubating with alkaline buffer (pH 9.0) to obtain fully uncharged tRNA control.D. RT-qPCR using a stem-loop primer to quantify the tRFs levels in shSCR, sh-1, and sh-2. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.G. Quantification of de novo protein synthesis in cells treated with different concentration of tRFs H. Quantification of de novo protein synthesis in cells treated with different concentration of tRNAGly(CCC) Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.


Figure 3A , B . Representative images showing endogenous hTrmt13 ( A ) and HA - tagged hTrmt13 ( HA - hTrmt13 , B ) subcellular localization by confocal microscopy analysis . DAPI staining was included to visualize the cell nucleus ( Blue ) . C . Representative confocal microscopy images showing the subcellular localization of hTrmt13 in MDA - MB - 231 cells cultured with 10 % FBS ( left ) or no FBS ( right ) . DAPI staining was included to visualize the cell nucleus ( Blue ) . E . Confocal microscopy images of MDA - MB - 231 cells were co - immunolabeled with antibodies against hTrmt13 and histone H3 ( left ) . The images were captured by GE DeltaVision OMX SR . Co - localization of H3 and hTrmt13 was analyzed by ImageJ ( right , n = 20 cells , center line , the median of the data ) . DAPI staining was included to visualize the cell nucleus ( Blue ) . I . The binding profiles of hTrmt13 , H3K4me3 , H3K27Ac , and other transcription factors ( MYC , E2F1 , TP53 , FOSL1 , EZH2 , FOXM1 ) from ChIP - seq and ATAC - seq on representative target genes TGFB1 , MEF2A , and PKN2 . hTrmt13 track were average across 2 biological replicates . N . EMSA of hTrmt13 for synthesized DNA ( 0 . 25 μM ) carrying consensus motif or mutated motif .

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Figure 3A, B. Representative images showing endogenous hTrmt13 (A) and HA-tagged hTrmt13 (HA-hTrmt13, B) subcellular localization by confocal microscopy analysis. DAPI staining was included to visualize the cell nucleus (Blue).C. Representative confocal microscopy images showing the subcellular localization of hTrmt13 in MDA-MB-231 cells cultured with 10% FBS (left) or no FBS (right). DAPI staining was included to visualize the cell nucleus (Blue).E. Confocal microscopy images of MDA-MB-231 cells were co-immunolabeled with antibodies against hTrmt13 and histone H3 (left). The images were captured by GE DeltaVision OMX SR. Co-localization of H3 and hTrmt13 was analyzed by ImageJ (right, n = 20 cells, center line, the median of the data). DAPI staining was included to visualize the cell nucleus (Blue).I. The binding profiles of hTrmt13, H3K4me3, H3K27Ac, and other transcription factors (MYC, E2F1, TP53, FOSL1, EZH2, FOXM1) from ChIP-seq and ATAC-seq on representative target genes TGFB1, MEF2A, and PKN2. hTrmt13 track were average across 2 biological replicates.N. EMSA of hTrmt13 for synthesized DNA (0.25 μM) carrying consensus motif or mutated motif.


Figure 4B . Transwell migration assays in MDA - MB - 231 cells . Control with siRNA scramble ( NC ) , treated with siRNA against hTrmt13 ( si - 1 , si - 2 ) and stably expressing wild - type hTrmt13 ( si - 1 + WT ) or enzymatic inactive hTrmt13 mutant ( si - 1 + E463A ) . Representative images ( left panel ) and statistical analysis ( middle panel ) are shown . Data information : for each group , five different fields were chosen and counted using a microscope with 20 - fold magnification . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for four independent experiments . C . Transwell migration assays in HCC38 cells infected with scrambled siRNA ( NC ) or siRNA against hTrmt13 ( si - 1 , si - 2 ) . Data information : for each group , five different fields were chosen and counted using a microscope with 20 - fold magnification . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for three independent experiments . E . Volcano plot of differential expressed genes analysis results for RNA - seq data comparing hTrmt13 knockdown MDA - MB - 231 cells ( N = 2 ) to control cells ( N = 2 ) , the dots for significant regulated genes ( FDR corrected p - value < 0 . 05 ) were colored red , the dots and labels of interesting genes with oncogenic implications have been colored in blue or red .

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Figure 4B. Transwell migration assays in MDA-MB-231 cells. Control with siRNA scramble (NC), treated with siRNA against hTrmt13 (si-1, si-2) and stably expressing wild-type hTrmt13 (si-1+WT) or enzymatic inactive hTrmt13 mutant (si-1+E463A). Representative images (left panel) and statistical analysis (middle panel) are shown. Data information: for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for four independent experiments.C. Transwell migration assays in HCC38 cells infected with scrambled siRNA (NC) or siRNA against hTrmt13 (si-1, si-2). Data information: for each group, five different fields were chosen and counted using a microscope with 20-fold magnification. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.E. Volcano plot of differential expressed genes analysis results for RNA-seq data comparing hTrmt13 knockdown MDA-MB-231 cells (N=2) to control cells (N=2), the dots for significant regulated genes (FDR corrected p-value < 0.05) were colored red, the dots and labels of interesting genes with oncogenic implications have been colored in blue or red.


Figure 5B . The mRNA level of the indicated genes was measured by RT - qPCR . Data information : statistical analysis was performed using t - tests , and the error bars indicate mean ± SD for three independent experiments .

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Figure 5B. The mRNA level of the indicated genes was measured by RT-qPCR. Data information: statistical analysis was performed using t-tests, and the error bars indicate mean ± SD for three independent experiments.


Figure 6B . Interaction of FLAG - hTrmt13 with endogenous FOSL1 , E2F1 , and USF1 . Whole - cell lysates from MDA - MB - 231 cells stably expressing FLAG - hTrmt13 were prepared and immunoprecipitation was performed with anti - FLAG followed by immunoblotting with antibodies against indicated proteins . F . ChIP - qPCR analysis of the selected promoters was performed using antibodies against indicated proteins in control and hTrmt13 knockdown MDA - MB - 231 cells . Results are represented as fold change over control IgG . Statistical analysis was performed using t - tests . Error bars indicate mean ± SD for three independent experiments .

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Figure 6B. Interaction of FLAG-hTrmt13 with endogenous FOSL1, E2F1, and USF1. Whole-cell lysates from MDA-MB-231 cells stably expressing FLAG-hTrmt13 were prepared and immunoprecipitation was performed with anti-FLAG followed by immunoblotting with antibodies against indicated proteins.F. ChIP-qPCR analysis of the selected promoters was performed using antibodies against indicated proteins in control and hTrmt13 knockdown MDA-MB-231 cells. Results are represented as fold change over control IgG. Statistical analysis was performed using t-tests. Error bars indicate mean ± SD for three independent experiments.


Figure 7C . The binding shift assays of hTrmt13 , USF1 , and their mixture for synthesized DNA from the TGFB1 promoter . In the reaction system , 10 ng / μL DNA was used , and 1 μM hTrmt13 or 1 μM USF1 or a mixture ( 0 . 5 μM hTrmt13 + 0 . 5 μM USF1 ) was applied . D . The binding shift assays of hTrmt13 for HctRNAGly ( GCC ) with the presence of various concentrations of USF1 . In the reaction system , 0 . 3 μM HctRNAGly ( GCC ) and 0 . 5 μM hTrmt13 were used , and a varied amount of USF1 ( 0 , 0 . 5 , 1 , 2 , 3 , 4 μM ) were used . E . The binding shift assays of hTrmt13 ( 0 . 5 μM ) , or USF1 ( 0 . 5 μM ) or the hTrmt13 - USF1 complex ( 0 . 5 μM ) for both tRNA ( 0 . 1 μM ) and DNA ( 15 ng / μL ) in the same reaction .

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Figure 7C. The binding shift assays of hTrmt13, USF1, and their mixture for synthesized DNA from the TGFB1 promoter. In the reaction system, 10 ng/μL DNA was used, and 1 μM hTrmt13 or 1 μM USF1 or a mixture (0.5 μM hTrmt13 + 0.5 μM USF1) was applied. D. The binding shift assays of hTrmt13 for HctRNAGly(GCC) with the presence of various concentrations of USF1. In the reaction system, 0.3 μM HctRNAGly(GCC) and 0.5 μM hTrmt13 were used, and a varied amount of USF1 (0, 0.5, 1, 2, 3, 4 μM) were used. E. The binding shift assays of hTrmt13 (0.5 μM), or USF1 (0.5 μM) or the hTrmt13-USF1 complex (0.5 μM) for both tRNA (0.1 μM) and DNA (15 ng/μL) in the same reaction.


Figure 8A . Volcano plot of differential expressed genes analysis results for RNA - seq data on TCGA comparing hTrmt13 expression low group patients ( first quartile ) versus high group patients ( fourth quartile ) . Significant differentially expressed genes ( FDR corrected p - value < 0 . 05 ) were colored by red , interesting genes with oncogenic implications are labelled in blue . Pseudo values 1e - 15 were added to FDR correlated p - value for better visualization . E . Immunohistochemical staining of tissue arrays containing 24 breast carcinoma samples paired with adjacent normal mammary tissues for hTrmt13 expression . Representative images are shown ( left ) , and the mean staining intensity was calculate using Image - Pro Plus software ( right ) . Statistical analysis was performed using t - tests . F . Kaplan - Meier survival analysis of Kaplan - Meier plotter ( http : / / kmplot . com / analysis / ) for the relationship between the relapse - free survival ( RFS ) or overall survival time ( OS ) of breast cancer patients ( BRCA , n = 349 ( low ) / 205 ( high ) ) , liver cancer patients ( LIHC , n = 270 ( low ) / 100 ( high ) ) , patients with kidney renal clear cell carcinoma ( KRIC , n = 156 ( low ) / 374 ( high ) ) , and patients with kidney renal papillary cell carcinoma ( KIRP , n = 208 ( low ) / 79 ( high ) ) and their expression of hTrmt13 .

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

Figure 8A. Volcano plot of differential expressed genes analysis results for RNA-seq data on TCGA comparing hTrmt13 expression low group patients (first quartile) versus high group patients (fourth quartile). Significant differentially expressed genes (FDR corrected p-value < 0.05) were colored by red, interesting genes with oncogenic implications are labelled in blue. Pseudo values 1e-15 were added to FDR correlated p-value for better visualization.E. Immunohistochemical staining of tissue arrays containing 24 breast carcinoma samples paired with adjacent normal mammary tissues for hTrmt13 expression. Representative images are shown (left), and the mean staining intensity was calculate using Image-Pro Plus software (right). Statistical analysis was performed using t-tests.F. Kaplan-Meier survival analysis of Kaplan-Meier plotter (http://kmplot.com/analysis/) for the relationship between the relapse-free survival (RFS) or overall survival time (OS) of breast cancer patients (BRCA, n=349 (low)/205 (high)), liver cancer patients (LIHC, n=270 (low)/100 (high)), patients with kidney renal clear cell carcinoma (KRIC, n=156 (low)/374 (high)), and patients with kidney renal papillary cell carcinoma (KIRP, n=208 (low)/79 (high)) and their expression of hTrmt13.


Figure 1 ( A ) Schematic of experimental setup depicting MECs grown as monolayers on a rigid rBM ( two - dimensional culture model , 2D ; top left ) or within a compliant rBM to generate multicellular spheroid structures with apical - basal polarity ( three - dimensional culture model , 3D ) top right ) . Heatmap of microarray analysis of gene expression in HMT - 3522 S - 1 MECs cultured either as a 2D monolayer or as spheroids ( Bottom ) . Expression of the top 200 differentially expressed genes between the two experimental conditions ( n = 3 independent biological replicates ; R = replicate ) . ( C ) Samples were stained with antibodies for β4 - integrin or laminin ( green ; yellow arrows ) . The actin and nucleus were counterstained with phalloidin ( red ) and DAPI ( blue ) , respectively . Scale bar , 10 μm . ( D ) HMT - 3522 S - 1 MECs plated as monolayers on a rigid rBM ( 2D ) or as spheroids within rBM ( 3D ) were treated with increasing doses of Paclitaxel ( Pac ) , TRAIL , Doxorubicin ( Doxo ) and irradiation ( IR ) . Percent cell death was quantified by immunofluorescence as percentage of cells stained positive for cleaved caspase - 3 at 48h post - treatment ( mean ± SEM ; n = 3 independent biological replicates ) . * * * * P < 0 . 0001 ( Student ' s t test ) . ( F ) Representative immunofluorescence microscopy images of MCF10A MECs plated in the indicated conditions for 18h and stained with phalloidin to reveal F - actin organization . Cells were plated as single cells on rigid glass coverslips ( 2D glass ; top left ) or fully embedded within rBM ( 3D fully embedded ; bottom left ) . Cell spreading was inhibited by plating cells on either laminin - 111 conjugated , 10 - μm micropatterned glass ( 2D / micropatterned , rigid substrate ; top middle ) or on compliant 75 Pa rBM - laminated polyacrylamide ( PA ) gels ( 2D / PA , soft substrate ; top right ) . The single 2D cells were overlaid with either purified laminin - 111 ( bottom middle ) or rBM ( bottom right ) to create a 3D ECM microenvironment . Images show maximum intensity z - projections of confocal stacks for F - actin phalloidin staining . Scale bar , 10 μ ( G ) Representative immunofluorescence microscopy images of MCF10A MECs stably expressing recombinant mCherry - tagged golgi marker ( mCherry - GalT ; red ; yellow arrows ) ligated with rBM in either 2D or 3D for 18h . The actin cortex and nucleus were counterstained with phalloidin ( green ) and DAPI ( blue ) , respectively . Images show the cross - sectional view of each cell compartment ( dashed lines ; xy plane ) and side view of confocal stacks ( xz plane ) in individual MCF10A MECs . Scale bar , 10 μm . ( H ) Golgi staining was assessed within non - spread MCF10A MECs ligated with rBM in 2D and 3D for 18h and values were plotted as a function of subcellular localization ( 2D , n = 8 ; 3D , n = 9 cells from two independent experiments ) . ( I ) Heatmap of RNA - seq experiment from MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating . Data show the expression of the top 200 genes that are differentially expressed between the 2D and 3D rBM conditions . ( n = 3 independent biological replicates ) .

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

Figure 1(A) Schematic of experimental setup depicting MECs grown as monolayers on a rigid rBM (two-dimensional culture model, 2D; top left) or within a compliant rBM to generate multicellular spheroid structures with apical-basal polarity (three-dimensional culture model, 3D) top right). Heatmap of microarray analysis of gene expression in HMT-3522 S-1 MECs cultured either as a 2D monolayer or as spheroids (Bottom). Expression of the top 200 differentially expressed genes between the two experimental conditions (n=3 independent biological replicates; R=replicate).(C) Samples were stained with antibodies for β4-integrin or laminin (green; yellow arrows). The actin and nucleus were counterstained with phalloidin (red) and DAPI (blue), respectively. Scale bar, 10 μm.(D) HMT-3522 S-1 MECs plated as monolayers on a rigid rBM (2D) or as spheroids within rBM (3D) were treated with increasing doses of Paclitaxel (Pac), TRAIL, Doxorubicin (Doxo) and irradiation (IR). Percent cell death was quantified by immunofluorescence as percentage of cells stained positive for cleaved caspase-3 at 48h post-treatment (mean ± SEM; n=3 independent biological replicates). ****P < 0.0001 (Student's t test).(F) Representative immunofluorescence microscopy images of MCF10A MECs plated in the indicated conditions for 18h and stained with phalloidin to reveal F-actin organization. Cells were plated as single cells on rigid glass coverslips (2D glass; top left) or fully embedded within rBM (3D fully embedded; bottom left). Cell spreading was inhibited by plating cells on either laminin-111 conjugated, 10-μm micropatterned glass (2D/micropatterned, rigid substrate; top middle) or on compliant 75 Pa rBM-laminated polyacrylamide (PA) gels (2D/PA, soft substrate; top right). The single 2D cells were overlaid with either purified laminin-111 (bottom middle) or rBM (bottom right) to create a 3D ECM microenvironment. Images show maximum intensity z-projections of confocal stacks for F-actin phalloidin staining. Scale bar, 10 μ(G) Representative immunofluorescence microscopy images of MCF10A MECs stably expressing recombinant mCherry-tagged golgi marker (mCherry-GalT; red; yellow arrows) ligated with rBM in either 2D or 3D for 18h. The actin cortex and nucleus were counterstained with phalloidin (green) and DAPI (blue), respectively. Images show the cross-sectional view of each cell compartment (dashed lines; xy plane) and side view of confocal stacks (xz plane) in individual MCF10A MECs. Scale bar, 10 μm. (H) Golgi staining was assessed within non-spread MCF10A MECs ligated with rBM in 2D and 3D for 18h and values were plotted as a function of subcellular localization (2D, n=8; 3D, n=9 cells from two independent experiments).(I) Heatmap of RNA-seq experiment from MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating. Data show the expression of the top 200 genes that are differentially expressed between the 2D and 3D rBM conditions. (n=3 independent biological replicates).


Figure 2 ( A ) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD , * * P = 0 . 0022 . ( B ) MCF10A MECs expressing VSVGts045 - EGFP were ligated to rBM in 2D or 3D and incubated at 40 ° C for 16h to trap VSVGts045 - EGFP protein in the ER . Cells were shifted to 32 ° C for 2h to release VSVGts045 - EGFP into the secretory pathway . Representative fluorescence microscopy images of MECs expressing VSVGts045 - EGFP 2h post incubation at 32 ° C . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 2 μm . ( C ) Scatter plot of the secretory trafficking efficiency of VSVGts045 - EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B ( mean ± SEM ; 2D , n = 45 ; 3D , n = 49 cells from three independent experiments ) . * * * * P < 0 . 0001 ( Student ' s t test ) . ( D ) Representative line graph showing the changes in cytosolic [ Ca2 + ] levels of MCF10A MECs under different treatment . Fura - 2 - loaded MECs ligated to 2D ( blue ) or 3D rBM ( red ) were pre - incubated with EGTA to chelate extracellular Ca2 + in the bath solution , challenged with 2μM thapsigargin to induce ER Ca2 + release , and replenished with 4 mM Ca2 + in bath solution at the indicated time . F340 / F380 values for each cell were quantified over the course of imaging ( mean ± SD ; 2D , n = 7 ; 3D , n = 7 cells from one experiment ) . ( E ) Quantification of the amplitude of the Ca2 + response induced by thapsigargin ( ∆ F = F - F0 ) , where F0 is the basal fluorescence before thapsigargin treatment ( mean ± SEM ( 2D , n = 20 ; 3D , n = 21 cells from three independent experiments ) . * * * P = 0 . 0007 ( Student ' s t test ) . ( F ) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 3 independent biological replicates ) . Statistical analysis by one - way ANOVA followed by Uncorrected Fisher ' s LSD , * * P = 0 . 0037 . ( G ) Representative immunoblots of phosphorylated EIF2a ( pEIF2a ) , total EIF2a and alpha - tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data ( mean ± SEM ; n = 6 independent biological replicates ) . Statistical analysis by Student ' s t test , * * P = 0 . 0049 . ( H ) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post - plating ( mean ± SEM ; n = 4 independent biological replicates ) . * P = 0 . 0371 ( Student ' s t test ) . ( I ) Representative fluorescence microscopy images of the ER - plasma membrane contact site reporter GFP - MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating . Scale bar ( whole cell ) , 10 μm ; Scale bar ( magnified ) , 1 μm . ( J ) Graph of the levels of GFP - MAPPER at the plasma membrane relative total cellular GFP - MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I ( mean ± SEM ; 2D , n = 52 ; 3D , n = 57 cells from three independent experiments ) . * * P = 0 . 0032 ( Student ' s t test ) .

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

Figure 2(A) Bar graph showing qPCR quantification of SEC61B level in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0022.(B) MCF10A MECs expressing VSVGts045-EGFP were ligated to rBM in 2D or 3D and incubated at 40°C for 16h to trap VSVGts045-EGFP protein in the ER. Cells were shifted to 32°C for 2h to release VSVGts045-EGFP into the secretory pathway. Representative fluorescence microscopy images of MECs expressing VSVGts045-EGFP 2h post incubation at 32°C. Scale bar (whole cell), 10 μm; Scale bar (magnified), 2 μm. (C) Scatter plot of the secretory trafficking efficiency of VSVGts045-EGFP based on the fluorescence of VSVGts045 at the plasma membrane versus total fluorescence measured in cells from Panel B (mean ± SEM; 2D, n=45; 3D, n=49 cells from three independent experiments). ****P < 0.0001 (Student's t test).(D) Representative line graph showing the changes in cytosolic [Ca2+] levels of MCF10A MECs under different treatment. Fura-2-loaded MECs ligated to 2D (blue) or 3D rBM (red) were pre-incubated with EGTA to chelate extracellular Ca2+ in the bath solution, challenged with 2μM thapsigargin to induce ER Ca2+ release, and replenished with 4 mM Ca2+ in bath solution at the indicated time. F340/F380 values for each cell were quantified over the course of imaging (mean ± SD; 2D, n=7; 3D, n=7 cells from one experiment). (E) Quantification of the amplitude of the Ca2+ response induced by thapsigargin (∆F=F-F0), where F0 is the basal fluorescence before thapsigargin treatment (mean ± SEM (2D, n=20; 3D, n=21 cells from three independent experiments). ***P = 0.0007 (Student's t test).(F) Bar graph showing qPCR of the relative level of ATF3 mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=3 independent biological replicates). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD, **P = 0.0037.(G) Representative immunoblots of phosphorylated EIF2a (pEIF2a), total EIF2a and alpha-tubulin in cell lysate from MCF10A MECs ligated to a rBM in 2D or 3D and corresponding quantification data (mean ± SEM; n=6 independent biological replicates). Statistical analysis by Student's t test, **P = 0.0049.(H) Bar graph of qPCR data measuring the relative levels of CRACR2B mRNA in MCF10A MECs ligated with rBM in 2D and 3D 12h post-plating (mean ± SEM; n=4 independent biological replicates). *P = 0.0371 (Student's t test).(I) Representative fluorescence microscopy images of the ER-plasma membrane contact site reporter GFP-MAPPER in MCF10A MECs ligated with rBM in 2D or 3D 18h post plating. Scale bar (whole cell), 10 μm; Scale bar (magnified), 1 μm. (J) Graph of the levels of GFP-MAPPER at the plasma membrane relative total cellular GFP-MAPPER fluorescence in MCF10A MECs ligated with rBM in 2D or 3D in Panel I (mean ± SEM; 2D, n=52; 3D, n=57 cells from three independent experiments). **P = 0.0032 (Student's t test).