Figure 4A . Typical fitted photon counting histograms of GFP and GFP - GFP in live yeast cells . B . The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP - GFP dimer as the brightness standard . Cells of strain yIO664 ( SCC1 - GFP ) were analyzed throughout the cell cycle by PCH . To examine the effects of Wpl1 , Pds5 , and Eco1 on cohesin dimerization , the PCH analysis was repeated in strain yEB005 cells ( SCC1 - GFP WPL1 ∆ ) , strain yEB002 cells ( SCC1 - GFP PDS5 - AID ) , and yAM946 ( SCC1 - GFP ECO1 - AID ) in which Pds5 - AID / Eco1 - AID was depleted with auxin . C . PCH analysis of strain yAM085 ( SMC3 - GFP ) throughout the cell cycle . D . PCH analysis of strains yAM953 ( SCC1 - GFP SMC3 - GFP ) and yAP941 ( SCC1 - GFP SCC3 - GFP ) containing two GFP tagged subunits . Data shown as boxplot ( + data overlap ) with values of minimum , 5 % percentile , 25 % percentile , median , 75 % percentile , maximum , and mean ( center square marks ) from at least three independent experiments .

O B-PANEL_START 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 B-PANEL_START 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

Figure 4A. Typical fitted photon counting histograms of GFP and GFP-GFP in live yeast cells.B. The molecular brightness of different GFP tagged constructs was obtained with GFP monomer and GFP-GFP dimer as the brightness standard. Cells of strain yIO664 (SCC1-GFP) were analyzed throughout the cell cycle by PCH. To examine the effects of Wpl1, Pds5, and Eco1 on cohesin dimerization, the PCH analysis was repeated in strain yEB005 cells (SCC1-GFP WPL1∆), strain yEB002 cells (SCC1-GFP PDS5-AID), and yAM946 (SCC1-GFP ECO1-AID) in which Pds5-AID/Eco1-AID was depleted with auxin.C. PCH analysis of strain yAM085 (SMC3-GFP) throughout the cell cycle.D. PCH analysis of strains yAM953 (SCC1-GFP SMC3-GFP) and yAP941 (SCC1-GFP SCC3-GFP) containing two GFP tagged subunits. Data shown as boxplot (+data overlap) with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) from at least three independent experiments.


Figure 1Fluorescent and histological analysis of liver morphology in WT and yap - / - mutant larvae on a lf : CFP background at 5 dpf . The blue colour overlay represents the fluorescent liver tissue . Scale bar : 200μm ( bright field , BF ) and 50μm ( histology , H + E ) Quantitative analysis of fluorescent liver area in WT and lf : Yap larvae at 5 dpf . n > 30 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± standard error of the mean ( s . e . m . )

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

Figure 1Fluorescent and histological analysis of liver morphology in WT and yap-/- mutant larvae on a lf:CFP background at 5 dpf. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 200μm (bright field, BF) and 50μm (histology, H+E) Quantitative analysis of fluorescent liver area in WT and lf:Yap larvae at 5 dpf. n>30, ****p<0.0001, two-sided Student's t-test; values represent the mean ± standard error of the mean (s.e.m.)


Figure 2Whole - mount in situ hybridization ( WISH ) analysis of progenitor marker prox1 , hhex , and foxa3 expression in WT and yap - / - mutant embryos at 36 hpf . Arrow and bracket highlight the hepatic bud . Scale bar : 200μmqPCR analysis of hhex and prox1 gene expression in WT and yap - / - mutant embryos at 48 hpf . n > 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . mWISH analysis of prox1 , hhex and foxa3 expression in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf . Arrow and bracket highlight the hepatic bud . Scale bar : 200μmQuantitative analysis of hepatic bud area ( prox1 staining ) in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf . n > 10 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Figure 2Whole-mount in situ hybridization (WISH) analysis of progenitor marker prox1, hhex, and foxa3 expression in WT and yap-/- mutant embryos at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μmqPCR analysis of hhex and prox1 gene expression in WT and yap-/- mutant embryos at 48 hpf. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.mWISH analysis of prox1, hhex and foxa3 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. Arrow and bracket highlight the hepatic bud. Scale bar: 200μmQuantitative analysis of hepatic bud area (prox1 staining) in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and fixed at 36 hpf. n>10, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Figure 3Fluorescent and histological analysis of liver morphology in dissected WT and yap - / - mutant adults on a lf : CFP background . The blue colour overlay represents the fluorescent liver tissue . Scale bar : 2mm ( brightfield , BF ) and 50μm ( histology , H + E ) Quantitative determination of body mass in WT and yap - / - adults . n = 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Quantitative determination of liver size in WT and yap - / - adults as determined by measuring liver mass as a percentage of body mass . n = 12 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . mqPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap - / - fish . n > 3 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean ± s . e . m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap - / - mutant fish . n > 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 3Fluorescent and histological analysis of liver morphology in dissected WT and yap-/- mutant adults on a lf:CFP background. The blue colour overlay represents the fluorescent liver tissue. Scale bar: 2mm (brightfield, BF) and 50μm (histology, H+E)Quantitative determination of body mass in WT and yap-/- adults. n=8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Quantitative determination of liver size in WT and yap-/- adults as determined by measuring liver mass as a percentage of body mass. n=12, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.mqPCR analysis of the hhex expression in adult liver tissue isolated from WT and yap-/- fish. n>3, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m qPCR analysis of the prox1 expression in adult liver tissue isolated from WT and yap-/- mutant fish. n>3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Figure 4Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap - / - mutant larvae at 3 dpf . Blue indicates decreased expression , while red demonstrates increased expressionHeatmap analysis of RNAseq data highlights the reduction of glucose transporters ( glut1 and glut2 ) in yap - / - mutant larvae at 3 dpfqPCR analysis of glut1 and glut2 expression in WT and yap - / - mutant larvae at 3 dpf . n > 3 , * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ' s t - test ; values represent the mean ± s . e . mqPCR analysis of glut1 and glut2 expression in WT , hs : dnYap and hs : Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf . n > 3 , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e . mqPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT , yap - / - mutant and lf : Yap transgenic adults . n > 3 , * p < 0 . 05 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Figure 4Heatmap of RNAseq analysis illustrating statistically significant differential gene expression between WT and yap-/- mutant larvae at 3 dpf. Blue indicates decreased expression, while red demonstrates increased expressionHeatmap analysis of RNAseq data highlights the reduction of glucose transporters (glut1 and glut2) in yap-/- mutant larvae at 3 dpfqPCR analysis of glut1 and glut2 expression in WT and yap-/- mutant larvae at 3 dpf. n>3, *p<0.05, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.mqPCR analysis of glut1 and glut2 expression in WT, hs:dnYap and hs:Yap transgenic embryos heat shocked at 24 hpf and isolated at 30 hpf. n>3, *p<0.05, **p<0.01, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.mqPCR analysis of glut1 and glut2 expression in liver tissue dissected from WT, yap-/- mutant and lf:Yap transgenic adults. n>3, *p<0.05, **p<0.01, two-sided Student's t-test; values represent the mean ± s.e.m


Figure 5Glucose tolerance test in 3 - month old WT and yap - / - mutant zebrafish . Blood glucose was measured 0 , 30 , 60 , 120 , 180 and 240 min post glucose injection . n > 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Glucose tolerance test in 12 - month old WT and yap - / - mutant zebrafish . Blood glucose was measured 0 , 30 , 60 , 120 , 180 and 240 min post glucose injection . n > 8 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . mClustergram analysis of polar metabolite abundance from WT and yap - / - mutant larvae at 3 dpf as determined by LC - MS / MS via selected reaction monitoring ( SRM ) analysis . n = 4 , p < 0 . 05 , two - sided Student ' s t - testSteady state abundance of glycolytic intermediates in WT and yap - / - mutant larvae as determined by SRM analysis . n = 4 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Steady state abundance of nucleotides in WT and yap - / - mutant larvae as determined by SRM analysis . n = 4 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e . mRelative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap - / - mutant larvae in [ U13C6 ] - glucose as determined by SRM analysis . n = 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e . m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap - / - larvae in [ U13C6 ] - glucose as determined by SRM analysis . n = 3 , * p < 0 . 05 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Figure 5Glucose tolerance test in 3-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Glucose tolerance test in 12-month old WT and yap-/- mutant zebrafish. Blood glucose was measured 0, 30, 60, 120, 180 and 240 min post glucose injection. n>8, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.mClustergram analysis of polar metabolite abundance from WT and yap-/- mutant larvae at 3 dpf as determined by LC-MS/MS via selected reaction monitoring (SRM) analysis. n=4, p<0.05, two-sided Student's t-testSteady state abundance of glycolytic intermediates in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m Steady state abundance of nucleotides in WT and yap-/- mutant larvae as determined by SRM analysis. n=4, *p<0.05, ***p<0.001, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.mRelative isotopic enrichment of 13C in glycolytic intermediates following incubation of WT and yap-/- mutant larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m Relative isotopic enrichment of 13C in nucleotide precursors following incubation of WT and yap-/- larvae in [U13C6]-glucose as determined by SRM analysis. n=3, *p<0.05, two-sided Student's t-test; values represent the mean ± s.e.m


Figure 6Effect of nucleoside ( NS ) cocktail treatment from 1 - 3 dpf on the incidence of cardiac edema in WT and yap - / - mutant larvae . Each point represents the percentage of affected embryos in each independent clutch . n > 7 , * * p < 0 . 01 , two - sided Student ' s t - test ; values represent the mean s . e . mEffect of WZB - 117 ( WZB , 10 μM ) treatment from 3 - 5 dpf in WT and lf : Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf : GFP reporters . Scale bar : 200μmQuantitative analysis of the effect of WZB treatment from 3 - 5 dpf on liver size as determined by fluorescence microscopy . n > 21 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e . mQuantitative analysis of the effect of Trehalose ( 100 μM ) treatment from 3 - 5 dpf on liver size as determined by fluorescence microscopy . n > 70 , * * * * p < 0 . 0001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Figure 6Effect of nucleoside (NS) cocktail treatment from 1-3 dpf on the incidence of cardiac edema in WT and yap-/- mutant larvae. Each point represents the percentage of affected embryos in each independent clutch. n>7, **p<0.01, two-sided Student's t-test; values represent the mean s.e.mEffect of WZB-117 (WZB, 10 μM) treatment from 3-5 dpf in WT and lf:Yap larvae on liver size as determined by fluorescence microscopy at 5 dpf in lf:GFP reporters. Scale bar: 200μmQuantitative analysis of the effect of WZB treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>21, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.mQuantitative analysis of the effect of Trehalose (100 μM) treatment from 3-5 dpf on liver size as determined by fluorescence microscopy. n>70, ****p<0.0001, two-sided Student's t-test; values represent the mean ± s.e.m


Figure 7Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia ( CCLE , 967 lines ) . Pearson coefficient is 0 . 46 Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia ( CCLE , 967 lines ) . Pearson coefficient is 0 . 45ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts ( LATS2 mutant MSTO211H cancer cells and HA - YAP5SA overexpressing HuCCT1 liver cancer cells ) Gross morphology of the liver from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Scale bar : 1 cmImmunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Scale bar : 50μmFDG PET imaging of coronal views from WT and TetONYap transgenic mice , treated with doxycycline for 10 days . Arrow is pointing to the liverScintillation analysis of FDG uptake ( fraction of injected dose per gram of liver tissue , % ID / g ) in dissected liver tissue from WT and TetON Yap transgenic mice , treated with doxycycline for 10 days . n = 4 , * * * p < 0 . 001 , two - sided Student ' s t - test ; values represent the mean ± s . e .

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

Figure 7Dot plots showing the correlation between GLUT1 and YAP1 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.46 Dot plots showing the correlation between GLUT1 and AMOTL2 mRNA expression in the cancer cell line encyclopedia (CCLE, 967 lines). Pearson coefficient is 0.45ChIPseq tracks at the GLUT1 locus highlighting YAP and TEAD enrichment overlaying H3K27Ac marks at an intragenic enhancer element in different contexts (LATS2 mutant MSTO211H cancer cells and HA-YAP5SA overexpressing HuCCT1 liver cancer cells)Gross morphology of the liver from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 1 cmImmunohistochemical analysis of GLUT1 expression in liver tissue from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Scale bar: 50μmFDG PET imaging of coronal views from WT and TetONYap transgenic mice, treated with doxycycline for 10 days. Arrow is pointing to the liverScintillation analysis of FDG uptake (fraction of injected dose per gram of liver tissue, %ID/g) in dissected liver tissue from WT and TetON Yap transgenic mice, treated with doxycycline for 10 days. n=4, ***p<0.001, two-sided Student's t-test; values represent the mean ± s.e.m


Figure 2A . Fura2 340 / 380 time traces from M - CSF - differentiated macrophages derived from conditionally - immortalised macrophage precursor cell lines ( M - MØP ) of Plcg2P522 mice ( P522 , blue ) and Plcg2R522 mice ( R522 , red ) . One set of cell lines , generated from male mice , is shown . Cells were exposed to 5µg / ml anti - FcγRII / III along with 20µM EGTA and 2µM Ionomycin as indicated . Data shows the mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc tests ( * * = p < 0 . 01 ; * * * * = p < 0 . 0001 ) . B . Fura2 340 / 380 time traces from primary microglia derived from the cortex of Plcg2R522 mice ( blue : P522 ) and Plcg2R522 mice ( red : R522 ) with or without pre - exposure for 2 hours with Edelfosine ( 10µM ) . Cells were exposed to 5µg / ml anti - FcγRII / III along with EGTA and 2µM Ionomycin . Data shows the mean ± SD of 3 independent experiments analysed by two - way ANOVA with Sidak post - hoc tests ( * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ) . C

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

Figure 2A. Fura2 340/380 time traces from M-CSF-differentiated macrophages derived from conditionally-immortalised macrophage precursor cell lines (M-MØP) of Plcg2P522 mice (P522, blue) and Plcg2R522 mice (R522, red). One set of cell lines, generated from male mice, is shown. Cells were exposed to 5µg/ml anti-FcγRII/III along with 20µM EGTA and 2µM Ionomycin as indicated. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; **** = p< 0.0001). B. Fura2 340/380 time traces from primary microglia derived from the cortex of Plcg2R522 mice (blue: P522) and Plcg2R522 mice (red: R522) with or without pre-exposure for 2 hours with Edelfosine (10µM). Cells were exposed to 5µg/ml anti-FcγRII/III along with EGTA and 2µM Ionomycin. Data shows the mean±SD of 3 independent experiments analysed by two-way ANOVA with Sidak post-hoc tests (** = p<0.01; *** = p<0.001; **** = p< 0.0001). C


Figure 3C - E . M - MOPS ( C ) , primary mouse microglia ( D ) and hIPSC ( E ) ( blue : P522 and red : R522 ) were loaded with Fluo - 8 Ca2 + indicator and examined for peak changes in fluoresce after exposure to DOPS - liposomes ( 25 μg / ml ) . Data information : Data shown as the mean ± SD of 3 independent experiments . The data were analysed by two - way ANOVA with Sidak ' s post - tests ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 ) .

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Figure 3C-E. M-MOPS (C), primary mouse microglia (D) and hIPSC (E) (blue: P522 and red: R522) were loaded with Fluo-8 Ca2+ indicator and examined for peak changes in fluoresce after exposure to DOPS-liposomes (25 μg/ml). Data information: Data shown as the mean±SD of 3 independent experiments. The data were analysed by two-way ANOVA with Sidak's post-tests (*=p<0.05, **=p<0.01, ***=p<0.001).


Figure 4A - F . Phagocytotic activity of R522 and P522 M - MOP and microglia was assessed using pHrodo Red E . coli and zymosan BioParticles . Phagocytosis arbitrary units ( A . U ) describes the amount of bioparticle cellular fluorescence emission at each time point . E . coli uptake in M - MOP ( A ) , primary mouse microglia ( B ) , hIPSC - derived microglia ( C ) . Zymosan uptake in M - MOP ( D ) , primary mouse microglia ( E ) and hIPSC - derived microglia ( F ) . For hIPSC - derived microglia , 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments . All microglia and M - MOP data shows the mean ± SD of 3 independent experiments were analysed by two - way ANOVA using Sidak multiple comparison test . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 )

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Figure 4A-F. Phagocytotic activity of R522 and P522 M-MOP and microglia was assessed using pHrodo Red E. coli and zymosan BioParticles. Phagocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. E.coli uptake in M-MOP (A), primary mouse microglia (B), hIPSC-derived microglia (C). Zymosan uptake in M-MOP (D), primary mouse microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)


Figure 5A - F . Endocytic activity of R522 and P522 M - MOP and microglia was assessed using FITC soluble Aβ1 - 42 oligomers and pHrodo Red Dexran ( 10 , 000 MW ) . Endocytosis arbitrary units ( A . U ) describes the amount of bioparticle cellular fluorescence emission at each time point . Aβ1 - 42 oligomer uptake in M - MOP ( A ) , primary microglia ( B ) , and hIPSC - derived microglia ( C ) . Dextran uptake in M - MOP ( D ) , primary microglia ( E ) and hIPSC - derived microglia ( F ) . For hIPSC - derived microglia , 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments . All microglia and M - MOP data shows the mean ± SD of 3 independent experiments were analysed by two - way ANOVA using Sidak ' s multiple comparison test . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 . ( blue : P522 ; red : R522 )

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

Figure 5A-F. Endocytic activity of R522 and P522 M-MOP and microglia was assessed using FITC soluble Aβ1-42 oligomers and pHrodo Red Dexran (10,000 MW). Endocytosis arbitrary units (A.U) describes the amount of bioparticle cellular fluorescence emission at each time point. Aβ1-42 oligomer uptake in M-MOP (A), primary microglia (B), and hIPSC-derived microglia (C). Dextran uptake in M-MOP (D), primary microglia (E) and hIPSC-derived microglia (F). For hIPSC-derived microglia, 3 isogenic P522 and 3 isogenic R522 clones were examined with at least 6 wells in 3 independent experiments. All microglia and M-MOP data shows the mean±SD of 3 independent experiments were analysed by two-way ANOVA using Sidak's multiple comparison test. *=p<0.05, **=p<0.01, ***=p<0.001. (blue: P522; red: R522)


Figure 6level of PIP2 in M - MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg / ml anti - FcγRII / III ( 2 . 4G2 ) , 50 ng / ml LPS or oligomers of 40 µM Aβ1 - 42 Data shows the mean ± SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak ' s multiple comparison * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 ) .

Figure 6level of PIP2 in M-MOPS was examined by measuring immunofluorescence from images at set time points after exposure after exposure to 5µg/ml anti-FcγRII/III (2.4G2), 50 ng/ml LPS or oligomers of 40 µM Aβ1-42 Data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA with Sidak's multiple comparison *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522).


Figure 7A - C . A mass ELISA was used to detect specific PIP species after exposure to anti - FcγRII / III ( 2 . 4G2 ) with or without 3 - a - aminocholestane ( SHIP 1 inhibitor ) , SF1670 ( PTEN inhibitor ) and LY294002 ( PI - 3K inhibitor ) . In M - MOP cells ( left panel ) and primary microglia ( right panel ) PI ( 4 , 5 ) P2 ( A ) , PI ( 3 , 4 ) P2 ( B ) and PIP3 ( C ) were detected . All data shows the mean ± SD of 3 independent experiments were analysed by 2 way ANOVA ( log - transformed data was used in C ) with Sidak ' s multiple comparison tests performed * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . ( blue : P522 ; red : R522 )

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

Figure 7A-C. A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P2 (A), PI(3,4)P2 (B) and PIP3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak's multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)


Figure 8A - C . Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell . The average fluorescence was calculated above background in the cortex and hippocampus at 2 ( A ) , 6 ( B ) and 9 ( C ) months . All data shows the mean ± SD of 3 independent experiments analysed using 2 way ANOVA with Sidak ' s multiple comparison tests performed * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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Figure 8A-C. Iba1 was used as a marker for microglia and PIP2 levels were detected by measuring the intensity of florescence per cell. The average fluorescence was calculated above background in the cortex and hippocampus at 2 (A), 6 (B) and 9 (C) months. All data shows the mean±SD of 3 independent experiments analysed using 2 way ANOVA with Sidak's multiple comparison tests performed **=p<0.01, ****=p<0.0001.


Figure 1Cell migration assays were performed using Transwell migration chambers . HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays ( n = 3 ) . CALIC is the lincRNA targeted by si - LINC00920 . Migration of HCT116 cells transfected with the indicated siRNAs or negative control . ( n = 4 ) Migration of colon cancer cells treated with an siRNA targeting CALIC . ( n = 3 ) Subcellular localization analysis of CALIC . RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT - PCR ( n = 3 ) . Nuclear controls : U6 , MALAT1 ; Cytoplasmic control : GAPDH . CALIC expression levels in cancer tissues . N , normal mucosa ; T , tumor . Expression of CALIC in primary skin tumors ( P ) and their metastases ( M ) . Expression of CALIC in primary skin tumors of patients without ( w / o ) or with ( w ) metastases . qRT - PCR analysis of CALIC expression in early ( I / II ) and late ( III / IV ) stage colon cancers . [ Normal ( N ) n = 8 , stage I / II n = 14 , stage III / IV n = 26 ] Kaplan - Meier curves for disease - free survival of colon cancer patients whose primary tumors expressed low ( blue , n = 113 ) or high ( red , n = 250 ) levels of CALIC .

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Figure 1Cell migration assays were performed using Transwell migration chambers. HCT116 cells were transfected with siRNAs targeting 11 candidate lincRNAs and cell migration activities were evaluated by Transwell migration assays (n = 3). CALIC is the lincRNA targeted by si-LINC00920.Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)Migration of colon cancer cells treated with an siRNA targeting CALIC. (n = 3)Subcellular localization analysis of CALIC. RNAs were isolated from the nuclear and cytoplasmic fractions of HCT116 cells and quantified by qRT-PCR (n = 3). Nuclear controls: U6, MALAT1; Cytoplasmic control: GAPDH.CALIC expression levels in cancer tissues. N, normal mucosa; T, tumor.Expression of CALIC in primary skin tumors (P) and their metastases (M). Expression of CALIC in primary skin tumors of patients without (w/o) or with (w) metastases.qRT-PCR analysis of CALIC expression in early (I/II) and late (III/IV) stage colon cancers. [Normal (N) n = 8, stage I/II n = 14, stage III/IV n = 26]Kaplan-Meier curves for disease-free survival of colon cancer patients whose primary tumors expressed low (blue, n = 113) or high (red, n = 250) levels of CALIC.


Figure 2Biotinylated full - length CALIC or antisense CALIC ( negative control ) generated in vitro were incubated with lysates from DLD - 1 colon cancer cells and precipitated with streptavidin beads . Precipitated proteins were resolved by SDS - PAGE followed by silver staining . The protein band indicated by the arrow was excised and subjected to mass spectrometry . ( Top ) schematic of the full - length and mutants of CALIC used for the precipitation of hnRNP - L from HCT116 cell lysates . The mutated elements are indicated by the cross marks . Se , sense transcript ; AS , antisense transcript . + , detectable binding activity ; - , no detectable activity . ( Bottom ) precipitated hnRNP - L and biotin - labeled fragments of CALIC are shown . RIP analysis was performed using control rabbit IgG or anti - hnRNP - L antibody . HPRT1 and the lncRNA MALAT1 were used as negative controls . Immunoblotting analysis of proteins precipitated by RIP assays using anti - hnRNP - L antibody are shown ( D ) . ChIRP - immunoblotting analysis of hnRNP - L association with endogeneous CALIC in HCT116 cells . Migration of HCT116 cells transfected with the indicated siRNAs or negative control . ( n = 4 )

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

Figure 2Biotinylated full-length CALIC or antisense CALIC (negative control) generated in vitro were incubated with lysates from DLD-1 colon cancer cells and precipitated with streptavidin beads. Precipitated proteins were resolved by SDS-PAGE followed by silver staining. The protein band indicated by the arrow was excised and subjected to mass spectrometry.(Top) schematic of the full-length and mutants of CALIC used for the precipitation of hnRNP-L from HCT116 cell lysates. The mutated elements are indicated by the cross marks. Se, sense transcript; AS, antisense transcript. +, detectable binding activity; -, no detectable activity. (Bottom) precipitated hnRNP-L and biotin-labeled fragments of CALIC are shown.RIP analysis was performed using control rabbit IgG or anti-hnRNP-L antibody. HPRT1 and the lncRNA MALAT1 were used as negative controls.Immunoblotting analysis of proteins precipitated by RIP assays using anti-hnRNP-L antibody are shown (D).ChIRP-immunoblotting analysis of hnRNP-L association with endogeneous CALIC in HCT116 cells.Migration of HCT116 cells transfected with the indicated siRNAs or negative control. (n = 4)


Figure 3RNA - Seq analysis of hnRNP - L - and CALIC - regulated genes in HCT116 cells . Venn diagram showing the overlap of genes differentially expressed by hnRNP - L ( blue ) or CALIC ( purple ) knockdown ( B , top ) . Expression levels of 59 genes regulated in common in both hnRNP - L and CALIC knockdown cells relative to control siRNA experiments ( B , bottom ) . Two biological replicates ( R1 and R2 ) were included for each siRNA treatment . The number of genes within each category is indicated . Scatterplots showing correlations between CALIC - and hnRNP - L - regulated genes in ( B ) . Left , co - regulated 59 genes ; Right , all differentially regulated genes ( DEGs ) . The correlation was calculated using the data except for the gene indicated by the asterisk . Genes induced by CALIC are significantly enriched in those upregulated by hnRNP - L . GSEA comparing the genes repressed by CALIC and hnRNP - L knockdown . NES , normalized enrichment score . Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP - L in colon and rectal cancers . Samples are ordered based on the expression levels of AXL .

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 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 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 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

Figure 3RNA-Seq analysis of hnRNP-L- and CALIC-regulated genes in HCT116 cells. Venn diagram showing the overlap of genes differentially expressed by hnRNP-L (blue) or CALIC (purple) knockdown (B, top). Expression levels of 59 genes regulated in common in both hnRNP-L and CALIC knockdown cells relative to control siRNA experiments (B, bottom). Two biological replicates (R1 and R2) were included for each siRNA treatment. The number of genes within each category is indicated.Scatterplots showing correlations between CALIC- and hnRNP-L-regulated genes in (B). Left, co-regulated 59 genes; Right, all differentially regulated genes (DEGs). The correlation was calculated using the data except for the gene indicated by the asterisk.Genes induced by CALIC are significantly enriched in those upregulated by hnRNP-L. GSEA comparing the genes repressed by CALIC and hnRNP-L knockdown. NES, normalized enrichment score.Heatmap showing the expression levels of genes positively regulated in common by both CALIC and hnRNP-L in colon and rectal cancers. Samples are ordered based on the expression levels of AXL.


Figure 4qRT - PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICimmunoblotting ( B , D ) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICqRT - PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP - L . immunoblotting ( B , D ) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP - L . qRT - PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild - type CALIC ( CALIC - full ) , CALIC - 296 - 913 or CALIC - mutHCT116 cells that had been treated with an siRNA targeting hnRNP - L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays . ChIP assays were performed using anti - hnRNP - L antibody or control mouse IgG . The promoter regions of GAPDH and Egr1 were used as negative and positive controls , respectively . Immunoblotting analysis of proteins precipitated by ChIP assays using anti - hnRNP - L antibodyChIP analysis of hnRNP - L association with the AXL promoter region in CALIC knockdown HCT116 cells . ChIP analysis of hnRNP - L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC - full . ChIRP analysis of CALIC binding to the AXL promoter . Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ . Migratory activity of AXL knockdown HCT116 cells . Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC . Immunoblotting and qRT - PCR analyses of HA - tagged AXL and CALIC expression , respectively ( left ) . α - Tubulin was used as a loading control . Representative images of gross specimens ( top ) and H & E stained sections ( bottom ) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells . Tumors were marked with yellow arrows . The dashed lines depict the boundary between normal and tumor tissues . ( Right ) Percentage of mice with lung metastases . Representative images of gross specimens of primary cecal tumors ( top ) and liver metastatic lesions ( bottom ) in mice orthotopically ( cecal ) injected with CALIC knockdown HCT116 cells . Tumors are marked with yellow arrows . ( Right ) Percentage of mice with primary tumors and liver metastases .

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

Figure 4qRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICimmunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting CALICqRT-PCR analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.immunoblotting (B, D) analyses of AXL in HCT116 and LS180 cells transfected with an siRNAs targeting hnRNP-L.qRT-PCR analyses of AXL in HCT116 cells infected with a lentivirus expressing wild-type CALIC (CALIC-full), CALIC-296-913 or CALIC-mutHCT116 cells that had been treated with an siRNA targeting hnRNP-L or CALIC were transfected with an AXL reporter construct and subjected to luciferase assays.ChIP assays were performed using anti-hnRNP-L antibody or control mouse IgG. The promoter regions of GAPDH and Egr1 were used as negative and positive controls, respectively.Immunoblotting analysis of proteins precipitated by ChIP assays using anti-hnRNP-L antibodyChIP analysis of hnRNP-L association with the AXL promoter region in CALIC knockdown HCT116 cells.ChIP analysis of hnRNP-L association with the AXL promoter region in HCT116 cells expressing lentiviral CALIC-full.ChIRP analysis of CALIC binding to the AXL promoter. Binding of CALIC to the promoter regions of the indicated genes was compared with that of LacZ.Migratory activity of AXL knockdown HCT116 cells.Lentiviral expression of AXL restores the migration activity of HCT116 cells transfected with an siRNA targeting CALIC. Immunoblotting and qRT-PCR analyses of HA-tagged AXL and CALIC expression, respectively (left). α-Tubulin was used as a loading control.Representative images of gross specimens (top) and H&E stained sections (bottom) of lung metastatic lesions in mice injected with CALIC or AXL knockdown HCT116 cells. Tumors were marked with yellow arrows. The dashed lines depict the boundary between normal and tumor tissues. (Right) Percentage of mice with lung metastases.Representative images of gross specimens of primary cecal tumors (top) and liver metastatic lesions (bottom) in mice orthotopically (cecal) injected with CALIC knockdown HCT116 cells. Tumors are marked with yellow arrows. (Right) Percentage of mice with primary tumors and liver metastases.


Figure 2Cloning and characterization of APG9 . A , Wild - type ( WT ; SEY6210 ) , apg9 ( THY154 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ; pAPG9 ( 414 ) ) or multicopy ( 2μ ; pAPG9 ( 424 ) ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods . The positions of precursor and mature API are indicated . The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant . B , WT ( SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with pAPG9 ( 414 ) centromeric plasmid were grown in SMD and transferred to SD ( − N ) as described in Materials and Methods . Aliquots were removed at the indicated times and spread onto YPD plates in triplicate . Numbers of viable colonies were determined after two to three days . The APG9 gene complements the starvation - sensitive phenotype of the apg9Δ mutant .

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

Figure 2Cloning and characterization of APG9. A, Wild-type (WT; SEY6210), apg9 (THY154), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN; pAPG9(414)) or multicopy (2μ; pAPG9(424)) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to API as described in Materials and Methods. The positions of precursor and mature API are indicated. The APG9 gene complements the prAPI accumulation phenotype of the apg9Δ mutant.B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.


Figure 3Apg9p is an unglycosylated , integral membrane protein . A , Wild - type ( WT ; SEY6210 ) , apg9Δ ( JKY007 ) , and the apg9Δ strain transformed with single copy ( CEN ) or multicopy ( 2μ ) plasmids encoding APG9 were grown to log phase in SMD . Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p . Apg9p is detected as an ∼ 125 - kD protein . . B , Hydrophobicity analysis of Apg9p based on the Kyte - Doolittle algorithm ( Kyte and Doolittle 1982 ) predicts six to eight transmembrane domains . C , Cells of wild - type strain YW5 - 1B were grown in YPD , converted to spheroplasts , and osmotically lysed as described in Materials and Methods . After a preclearing step to remove cell debris , the lysates were incubated with or without 1 % Triton X - 100 on ice for 30 min , as indicated . Centrifugation at 10 , 000 g for 1 h separated the lysate into supernatant ( S ) and pellet ( P ) fractions , which were analyzed by immunoblots with antiserum to Apg9p . Apg9p is found in the low - speed pellet in the absence of Triton X - 100 . Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fractionD , Strain CTD1 harboring a multicopy 3 × HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods . A crude lysate was prepared and treated with or without endo H as indicated overnight at 37 ° C and analyzed by immunoblots with anti - HA antibodies or anti - CPY serum . Endo H treatment removes the N - linked glycosyl residues of CPY , causing a shift in its electrophoretic mobility . In contrast , Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment .

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Figure 3Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein.. B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains.C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fractionD, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.


Figure 4Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients . A and B , Strain STY1 ( pep4Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 2 . 5 h at 174 , 000 g as described in Materials and Methods . Fractions were collected from the top ( fraction number 1 ) to the bottom ( fraction number 16 ) . Fractions were subjected to SDS - PAGE and immunoblot with antiserum against Apg9p and : in A , Kex2p ( trans Golgi network ) , Pho8p ( vacuole ) , and Pma1p ( plasma membrane ) ; and in B , Sec12p ( ER ) and Pep12p ( endosome ) . The graphs in panels A and B are from the same gradient , but are presented separately for clarity .

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Figure 4Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.


Figure 5Apg9p does not comigrate with prAPI in a strain that accumulates Cvt / autophagic vesicles . Strain TK415 ( ypt7Δ ) was grown in YPD to log - phase , and osmotically lysed . Lysates were precleared , centrifuged at 100 , 000 g for 1 h , and the pellet fraction resuspended and centrifuged a second time . The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , and centrifuged for 16 h at 174 , 000 g as described in Materials and Methods . 16 fractions were collected starting from the top and subjected to SDS - PAGE and immunoblot with antisera against Apg9p and API . The graph represents an average of two experiments .

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Figure 5Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.


Figure 6Apg9p localizes to large perivacuolar punctate structures . Immunofluorescence microscopy of strains CTD1 ( apg9Δ ) , KSL12C ( vps4Δ ) , and SKD6 - 1D ( apg6Δ ) transformed with the multicopy plasmid 3 × HA APG9 . Cells were grown in YPD to log phase ( vegetative ) , and incubated further in SD ( − N ) medium with 1 mM PMSF for 3 h ( starvation ) . The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods . Anti - HA and FITC - conjugated antibodies mark Apg9p ( left ) , and the vacuole can be visualized by Nomarski optics ( middle ) . An overlay is shown in the right panels .

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Figure 6Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.


Figure 7In vivo examination of the Apg9pGFP fusion protein . Wild - type ( SEY6210 ) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4 - 64 to stain the vacuoles as described in Materials and Methods . The labeled cells were viewed directly with a Leica DM IRB confocal microscope . Large , punctate Apg9pGFP structures appear juxtaposed to the FM 4 - 64 - labeled vacuole membranes . In addition , smaller punctate structures appear throughout the cytoplasm .

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Figure 7In vivo examination of the Apg9pGFP fusion protein. Wild-type (SEY6210) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4-64 to stain the vacuoles as described in Materials and Methods. The labeled cells were viewed directly with a Leica DM IRB confocal microscope. Large, punctate Apg9pGFP structures appear juxtaposed to the FM 4-64-labeled vacuole membranes. In addition, smaller punctate structures appear throughout the cytoplasm.


Figure 8Ultrastructural localization of Apg9p for immunoelectron microscopy . Strain STY1 ( pep4Δ ) expressing 3 × HA Apg9p was grown in YPD to log phase and transferred to SD ( − N ) medium for 2 h . The cells were fixed and stained with anti - HA antibody and 5 - nm colloidal gold - conjugated goat anti - mouse IgG as described in Materials and Methods . A , Section showing autophagic bodies in the vacuole lumen . B , Section showing a cytosolic autophagosome . Apg9p is seen in patches near the vacuole , but not on autophagic bodies or autophagosomes . AB , autophagic body ; AP , autophagosome ; N , nucleus ; V , vacuole ; VM , vacuolar membrane . Arrows point to areas of concentrated Apg9p .

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Figure 8Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti-mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.


Figure 9Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant . A , Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway . Wild - type ( YW5 - 1B ) and apg9Δ ( CTD1 ) cells were grown in YPD ( vegetative ) or transferred to SD ( − N ) ( starvation ) for 3 h in the presence of PMSF ( starvation ) and examined by DIC ( Nomarski ) microscopy ( Zeiss Axioplan ) . PMSF inhibits the degradation of autophagic bodies . Under starvation conditions , wild - type cells accumulate autophagic bodies when vesicle breakdown is blocked . However , under the same conditions , apg9Δ cells do not accumulate autophagic bodies , indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen . The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild - type ( data not shown ) . B , The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature . Wild - type ( SEY6210 ) and apg9Δ ( JKY007 ) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38 ° C for 5 min , pulse - labeled for 10 min , and then subjected to nonradioactive chase reactions for the indicated times . Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS - PAGE as described in Materials and Methods . API - immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph . The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point .

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

Figure 9Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.


Figure 10Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step . A , The nature of prAPI binding and pelleting in the apg9ts strain . Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38 ° C . The spheroplasts were osmotically lysed in a buffer containing no salt ( 20 mM Pipes , pH 6 . 8 ) or a physiological concentration of salt ( 100 mM KOAc , 50 mM KCl , 5 mM MgCl2 , 20 mM Pipes , pH 6 . 8 ) and pelleted at 5 , 000 g . All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK . apg9ts retains the salt dependent binding and pelleting of prAPI . B , prAPI in the apg9ts strain accumulates in both a membrane - associated state and a large pelletable complex . Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into supernatant ( S ) and pellet ( P ) fractions by centrifugation at 5 , 000 g for 5 min . An aliquot was removed for the total lysate control ( T ) . The pellet fraction ( P ) was resuspended in 15 % Ficoll - 400 in gradient buffer ( 20 mM Pipes , 5 mM MgCl2 , complete EDTA - free protease inhibitor cocktail ) in the presence or absence of Triton X - 100 and overlaid with 13 and 2 % Ficoll - 400 in gradient buffer . The step gradients were centrifuged at 13 , 000 g for 10 min . Membrane - containing float ( F ) , nonfloat ( NF ) , and pellet ( P2 ) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods . The position of prAPI is indicated . C , prAPI in the apg9ts strain is protease accessible . Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse - labeled for 10 min and chased for 30 min at 38 ° C . The labeled spheroplasts were then osmotically lysed and separated into low - speed supernatant ( S ) and pellet ( P ) fractions after a 5 , 000 g centrifugation step . The pellet fractions were subjected to protease treatment in the absence or presence of 0 . 2 % Triton X - 100 as described in Materials and Methods . The resulting samples were immunoprecipitated with antiserum to API and PGK . The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager . The percent protease - protected prAPI was determined by dividing the value for prAPI in the protease - treated sample by the value for total prAPI before protease treatment . The percent PGK was determined by dividing the supernatant ( S ) or pellet ( P ) value over the sum of the S and P values .

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

Figure 10Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.


Figure 1A Macroscopic seedling phenotype of 12 - day - old b16b18CRISPR , quadriCRISPR F33 # 1 / # 6 , pentaCRISPR and amiR - 2572 , compared to WT ( Col - 0 ) . E Macroscopic seedling phenotype of 12 - day - old quadriCRISPR B64 compared to WT ( Col - 0 ) and amiR - 2572 . Scale bars = 1 cm . F , G Boxplots showing the quantification of primary root length ( F ) and LR density ( G ) in seedlings depicted in E . n = 12 ( Col - 0 ) , 13 ( quadriCRISPR B64 ) , 11 ( amiR - 2572 ) .

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Figure 1A Macroscopic seedling phenotype of 12-day-old b16b18CRISPR, quadriCRISPR F33#1/#6, pentaCRISPR and amiR-2572, compared to WT (Col-0).E Macroscopic seedling phenotype of 12-day-old quadriCRISPR B64 compared to WT (Col-0) and amiR-2572. Scale bars = 1 cm. F, G Boxplots showing the quantification of primary root length (F) and LR density (G) in seedlings depicted in E. n = 12 (Col-0), 13 (quadriCRISPR B64), 11 (amiR-2572). D


Figure 2C Confocal images of root tips in 5 - day - old Col - 0 , amiR - 2572 and syn - tasi - 1522A # 1 seedlings . LRC cells were indicated by white asterisks . The distal LRC cells were indicated by white arrowsG Macroview stereo microscopic view of DR5 : VENUS expression in root tips of 3 - day - old WT , amiR - 2572 and syn - tasi - 1522A # 1 seedlings , red arrows indicate DR5 stripes in the lateral root cap .

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Figure 2C Confocal images of root tips in 5-day-old Col-0, amiR-2572 and syn-tasi-1522A#1 seedlings. LRC cells were indicated by white asterisks. The distal LRC cells were indicated by white arrowsG Macroview stereo microscopic view of DR5:VENUS expression in root tips of 3-day-old WT, amiR-2572 and syn-tasi-1522A#1 seedlings, red arrows indicate DR5 stripes in the lateral root cap.


Figure 3A Co - localisation of YFP - ABCB15 with Propidium Iodide ( PI ) in root epidermal cells of 3 - day - old seedlings . A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks . C IAA export assay . Export of [ 3H ] - IAA , assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1 , ABCB15 - 22 and ABCB17P980G against vector control . mean ± SE ; n = 26 ( vector control ) , 44 ( ABCB1 ) , 5 ( ABCB15 ) , 4 ( ABCB16 ) , 9 ( ABCB17 ) , 5 ( ABCB18 ) , 9 ( ABCB22 ) and 8 ( ABCB17P980G ) , transport experiments generated from independent tobacco transfections .

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Figure 3A Co-localisation of YFP-ABCB15 with Propidium Iodide (PI) in root epidermal cells of 3-day-old seedlings. A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks.C IAA export assay. Export of [3H]-IAA, assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1, ABCB15-22 and ABCB17P980G against vector control. mean ± SE; n = 26 (vector control), 44 (ABCB1), 5 (ABCB15), 4 (ABCB16), 9 (ABCB17), 5 (ABCB18), 9 (ABCB22) and 8 (ABCB17P980G),transport experiments generated from independent tobacco transfections.


Figure 4B Analysis of DR5 : : VENUS expression in the root elongation zone of 4 - day - old pWOX5 : XVE > > YUC1 - 2A - TAA1 , in Control and amiR - 2572 treated with β - estradiol ( 5 µM ) for 0 , 7 . 5 and 9h . Images are composed of several tiles generated in a single snap with automatic assembly , PI in gray . The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5 : : VENUS signal in the elongation zone .

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Figure 4B Analysis of DR5::VENUS expression in the root elongation zone of 4-day-old pWOX5:XVE>>YUC1-2A-TAA1, in Control and amiR-2572 treated with β-estradiol (5 µM) for 0, 7.5 and 9h. Images are composed of several tiles generated in a single snap with automatic assembly, PI in gray. The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5::VENUS signal in the elongation zone.


Figure 5A , B Expression pattern of proABCB15 : NLS - GFP - GUS , proABCB16 : NLS - GFP - GUS , proABCB17 : NLS - GFP - GUS , proABCB18 : NLS - GFP - GUS , and proABCB22 : NLS - GFP - GUS expression in roots of 3 - day - old seedlings , using confocal microscopy showing longitudinal overview pictures ( Propidium iodide in magenta ) , and images zoomed on the epidermis and LRC corresponding to yellow squares ( A ) ; cytological sections of GUS stained roots of 3 - day - old seedlings , counterstained with ruthenium red ( B ) . Scale bars represent 20 µm for both graphs .

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Figure 5A, B Expression pattern of proABCB15:NLS-GFP-GUS, proABCB16:NLS-GFP-GUS, proABCB17:NLS-GFP-GUS, proABCB18:NLS-GFP-GUS, and proABCB22:NLS-GFP-GUS expression in roots of 3-day-old seedlings, using confocal microscopy showing longitudinal overview pictures (Propidium iodide in magenta), and images zoomed on the epidermis and LRC corresponding to yellow squares (A); cytological sections of GUS stained roots of 3-day-old seedlings, counterstained with ruthenium red (B). Scale bars represent 20 µm for both graphs.


Figure 6A Fluorescence of YFP - ABCB17 in the root meristem and leaf in 3 - day - old F1 crosses with WT , syn - tasi - 1522A # 1 and amiR - 2572 , propidium iodide ( PI ) in magenta . Red arrows highlight the position of the epidermis . Scale bars = 50 µm . B Schematic representation of root geometry with the indication of regions of interest for YFP - ABCB17 signal quantification . Red ( Stele ) , and black ( epidermis / LRC ) . C , D Quantification of YFP - ABCB17 fluorescence intensity in the stele ( C ) and epidermis / LRC ( D ) of A , measured at regions of interest corresponding tG , H Quantification of the oscillation period ( G ) and amplitude ( H ) of DR5 : : LUC in 3 - day - old WT and syn - tasi - 1522A # 1 , n = 27 ( WT ) and 23 ( syn - tasi - 1522A # 1 ) .

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Figure 6A Fluorescence of YFP-ABCB17 in the root meristem and leaf in 3-day-old F1 crosses with WT, syn-tasi-1522A#1 and amiR-2572, propidium iodide (PI) in magenta. Red arrows highlight the position of the epidermis . Scale bars = 50 µm. B Schematic representation of root geometry with the indication of regions of interest for YFP-ABCB17 signal quantification. Red (Stele), and black (epidermis/LRC). C, D Quantification of YFP-ABCB17 fluorescence intensity in the stele (C) and epidermis/LRC (D) of A, measured at regions of interest corresponding tG,H Quantification of the oscillation period (G) and amplitude (H) of DR5::LUC in 3-day-old WT and syn-tasi-1522A#1, n= 27 (WT) and 23 (syn-tasi-1522A#1).


figf1 ( b ) N2a cells coexpressing YFP - PrP and HLA - A2 - CFP were subjected to 6 mM trypsin for 100 s . Images were taken before and after trypsin treatment as indicated . Note that the membrane signal of YFP - PrP was destroyed by the protease , whereas HLA - A2 - CFP ' s membrane signal remained unaffected . ( d ) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP . N2a cells expressing GFP were treated with 20 μM digitonin . Images were taken before and after digitonin application at the indicated time points . Scale bars , 10 μm .

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figf1(b) N2a cells coexpressing YFP-PrP and HLA-A2-CFP were subjected to 6 mM trypsin for 100 s. Images were taken before and after trypsin treatment as indicated. Note that the membrane signal of YFP-PrP was destroyed by the protease, whereas HLA-A2-CFP's membrane signal remained unaffected.(d) Digitonin treatment permeabilizes the plasma membrane and releases first cytosolic and later nucleoplasmicGFP. N2a cells expressing GFP were treated with 20 μM digitonin. Images were taken before and after digitonin application at the indicated time points. Scale bars, 10 μm.


figf2 ( b ) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ - CFP . Images were taken before and after 1 - min treatment with 20 μM digitonin and 4 mM trypsin as indicated . Scale bar , 10 μm .

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figf2(b) FPP assay of N2a cells expressing RFP and the type I membrane protein CD3δ-CFP. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.


figf3 ( b ) NRK cells expressing CD3δ - CFP and YFP - CD3δ were subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 4 mM trypsin as indicated . Scale bar , 10 μm . c ) Biochemical protease protection assay of NRK cells expressing YFP - CD3δ , CD3δ - CFP and GFP . Transfected cells were subjected to SDS - PAGE and Western blot analysis before ( lane 1 ) and after separation of the cytosolic ( lane 2 ) and membrane ( lane 3 ) fractions by ultracentrifugation . Equal amounts of the membrane fraction were treated with trypsin in the absence ( lane 4 ) or presence ( lane 5 ) of Triton X - 100 . Protein extracts were analyzed by immunoblotting with monoclonal anti - GFP antibody JL - 8 .

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figf3(b) NRK cells expressing CD3δ-CFP and YFP-CD3δ were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 4 mM trypsin as indicated. Scale bar, 10 μm.c) Biochemical protease protection assay of NRK cells expressing YFP-CD3δ, CD3δ-CFP and GFP. Transfected cells were subjected to SDS-PAGE and Western blot analysis before (lane 1) and after separation of the cytosolic (lane 2) and membrane (lane 3) fractions by ultracentrifugation. Equal amounts of the membrane fraction were treated with trypsin in the absence (lane 4) or presence (lane 5) of Triton X-100. Protein extracts were analyzed by immunoblotting with monoclonal anti-GFP antibody JL-8.


figf4 ( a ) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT - YFP ( red ) and ER - bound CD3δ - CFP ( green ) , and the position of the fluorescent protein tag relative to its respective membrane . ( b ) N2a cells coexpressing GalT - YFP and CD3δ - CFP were subjected to the FPP assay , as in Figure 2bc ) Cartoon illustrating localization of mitochondrial EYFP - Mito ( red ) and Cd3δ - CFP ( green ) . ( d ) N2a cells coexpressing EYFP - Mito and CD3δ - CFP were subjected to the FPP assay , as in b . Scale bars , 10 μm .

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figf4(a) Cartoon of the FPP assay illustrating the cellular localization of the Golgi marker GalT-YFP (red) and ER-bound CD3δ-CFP (green), and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing GalT-YFP and CD3δ-CFP were subjected to the FPP assay, as in Figure 2bc) Cartoon illustrating localization of mitochondrial EYFP-Mito (red) and Cd3δ-CFP (green). (d) N2a cells coexpressing EYFP-Mito and CD3δ-CFP were subjected to the FPP assay, as in b. Scale bars, 10 μm.


figf5 ( a ) Cartoon of the FPP assay illustrating the cellular localization of RFP - SKL ( red ) and Pex3p - GFP ( green ) and the position of the fluorescent protein tag relative to its respective membrane . ( b ) N2a cells coexpressing RFP - SKL and Pex3p - GFP were subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 100 - s treatment with 4 mM trypsin as indicated . Scale bar , 10 μm .

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figf5(a) Cartoon of the FPP assay illustrating the cellular localization of RFP-SKL (red) and Pex3p-GFP (green) and the position of the fluorescent protein tag relative to its respective membrane. (b) N2a cells coexpressing RFP-SKL and Pex3p-GFP were subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin as indicated. Scale bar, 10 μm.


figf6 ( a ) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane . GAPDH - RFP is shown in red and CFP - LC3 is shown in green . ( b ) NRK cells stably expressing CFP - LC3 were transiently transfected to express GAPDH - RFP and subjected to the FPP assay . Images were taken before and after 1 - min treatment with 20 μM digitonin and 100 - s treatment with 4 mM trypsin . The inset shows the overlap of the GAPDH - RFP and CFP - LC3 signals ( 4 × magnification of the central area ( outlined by a box ) of the micrographs . Scale bar , 10 μm .

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figf6(a) Cartoon of the FPP assay illustrating the cellular localization of the proteins and the position of the fluorescent protein tag relative to its respective membrane. GAPDH-RFP is shown in red and CFP-LC3 is shown in green. (b) NRK cells stably expressing CFP-LC3 were transiently transfected to express GAPDH-RFP and subjected to the FPP assay. Images were taken before and after 1-min treatment with 20 μM digitonin and 100-s treatment with 4 mM trypsin. The inset shows the overlap of the GAPDH-RFP and CFP-LC3 signals (4 × magnification of the central area (outlined by a box) of the micrographs. Scale bar, 10 μm.


Figure 1Monocyte derived macrophages ( MDM ) were treated with 5μM etoposide ( ETO ) for 18h . Cells were stained for γH2AX and 53BP1 nuclear foci , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired and analysed . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; paired t ‐ test ) . Scale bars , 10μm . Monocyte derived macrophages ( MDM ) were treated with 5μM etoposide ( ETO ) for 18h . Cells were stained for γH2AX and 53BP1 nuclear foci , acquired and analysed using the automated cell ‐ imaging system Hermes WiScan and ImageJ . On average 104 cells were acquired and analysed . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 ; paired t ‐ test ) . Scale bars , 10μm . MDM were treated with increasing concentrations of ETO and CTH for 66h . Cells were stained to distinguish between live and dead cells using LIVE / DEAD fixable Dead cell stain protocol . Percentages of live / dead cells were determined using automated cell ‐ imaging system Hermes WiScan and ImageJ . Addition of 20 % ethanol for 10min into cells treated with ETO was used as positive control . ( n = 3 , mean ± s . e . m ) . MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV - G pseudotyped HIV - 1 GFP viruses wild type ( wt ) HIV - 1 capsid mutants ( N74D or P90A ) , HIV - 2 , SIV sooty mangabey ( SIVsm E543 ) , replication competent Adenovirus type 5 AdV ( AdV ) , Semliki Forest Virus ( SFV ) and HSV - 1 . The percentages of infected cells were determined using an automated cell ‐ imaging system Hermes WiScan and ImageJ and normalized to untreated control ~ 100 % . ( n = 3 , mean ± s . e . m . ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 ; ( ns ) non ‐ significant , paired t ‐ test ) . Cells from a representative donor were used for immunoblotting . MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV - G pseudotyped HIV - 1 GFP . 104 cells were recorded and analysed for GFP expression 48h post - infection using an automated cell ‐ imaging system Hermes WiScan and ImageJ . ( n = 4 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full - length replication competent macrophage tropic HIV - 1 virus BaL . Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . Late viral RT products . AZT : MDM treated with 20μM AZT , a reverse - transcriptase inhibitor , were used as control . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) . G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . 2LTR circles . ( n = 3 , mean ± s . e . m . ; * P ‐ value ≤ 0 . 05 , paired t ‐ test ) . G - I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV - 1 BaL and DNA isolated 18 h post ‐ infection for qPCR . Integrated copies of viral DNA , Alu - Gag qPCR . ( n = 3 , mean ± s . e . m . ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h and infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / cell ) . Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post ‐ infection by FACS ( n = 3 , mean ± s . e . m ; * * P ‐ value ≤ 0 . 01 ; * * * P ‐ value ≤ 0 . 001 , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL ( 3 , 6 , 12 ffu / cell ) and DNA isolated 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) . MDM were treated with 5μM ETO for 18h , infected with HIV - 1 BaL and DNA isolated 4 , 6 , and 18 h post ‐ infection for qPCR . ( n = 3 , mean ± s . e . m . ; ( ns ) non ‐ significant , paired t ‐ test ) .

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Figure 1Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.MDM were treated with increasing concentrations of ETO and CTH for 66h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell‐imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10min into cells treated with ETO was used as positive control. (n = 3, mean ± s.e.m).MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent Adenovirus type 5 AdV (AdV), Semliki Forest Virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell‐imaging system Hermes WiScan and ImageJ and normalized to untreated control ~100%. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; ***P‐value ≤ 0.001; (ns) non‐significant, paired t‐test). Cells from a representative donor were used for immunoblotting.MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 4, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test). G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.  Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).  G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.2LTR circles. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05, paired t‐test).G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; ***P‐value ≤ 0.001, paired t‐test).MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).