Figure 2 ( A ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of Cdc42 and borg4 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( B ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the Cdc42 - borg4 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * * P < 0 . 0001 . ( C ) Representative immunofluorescence confocal microscopy images of young , aged and aged + CASIN ( 5µM ) treated LT - HSCs for the proximity ligation assay ( red signal ) , testing the extent of close physical interaction of borg4 and septin 7 . A red fluorescent signal indicates close proximity of the two proteins tested . Nuclei were stained with DAPI ( blue ) , scale bar = 5µm . ( D ) Quantification of the level of fluorescent signal in young , aged and aged + CASIN ( 5µM ) treated LT - HSCs to quantify the borg4 - septin 7 proximity interaction . Data are normalized to the mean fluorescence signal of aged LT - HSCs . 3 biological repeats , n = at least 17 cells per condition , error bars indicate mean ± SEM , unpaired t - test , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

Figure 2(A) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of Cdc42 and borg4. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (B) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the Cdc42-borg4 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ****P<0.0001. (C) Representative immunofluorescence confocal microscopy images of young, aged and aged + CASIN (5µM) treated LT-HSCs for the proximity ligation assay (red signal), testing the extent of close physical interaction of borg4 and septin 7. A red fluorescent signal indicates close proximity of the two proteins tested. Nuclei were stained with DAPI (blue), scale bar= 5µm. (D) Quantification of the level of fluorescent signal in young, aged and aged + CASIN (5µM) treated LT-HSCs to quantify the borg4-septin 7 proximity interaction. Data are normalized to the mean fluorescence signal of aged LT-HSCs. 3 biological repeats, n=at least 17 cells per condition, error bars indicate mean±SEM, unpaired t-test, ***P<0.001, ****P<0.0001.


Figure 3 ( A , B ) Representative immunofluorescence microscopy images of the distribution of septin 7 ( red ) , Cdc42 ( red ) and tubulin ( green ) in LT - HSCs from borg4fl / fl or borg4 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) , scale bar = 5 µm . ( C ) Percentage of cells with a polar distribution of septin 7 , Cdc42 or tubulin in borg4fl / fl or borg4 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 50 cells were scored per sample . Error bars indicate mean ± SEM , Two - way ANOVA analysis , * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 . ( D , E ) Representative immunofluorescence microscopy images of the distribution of borg4 ( red ) , Cdc42 ( red ) or tubulin ( green ) in LT - HSCs from septin 7fl / fl or septin 7 ∆ / ∆ mice . Nuclei were stained with DAPI ( blue ) . scale bar = 5 µm . ( F ) Percentage of cells with a polar distribution of borg4 , Cdc42 or tubulin in septin 7fl / fl or septin 7 ∆ / ∆ LT - HSCs . At least 3 biological repeats , at least 30 to 50 cells were scored per sample . Error bars indicate mean ± SEM , Two - way ANOVA analysis , * * P < 0 . 01 , * P < 0 . 05 .

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

Figure 3(A,B) Representative immunofluorescence microscopy images of the distribution of septin 7 (red), Cdc42 (red) and tubulin (green) in LT-HSCs from borg4fl/fl or borg4∆/∆ mice. Nuclei were stained with DAPI (blue), scale bar= 5 µm. (C) Percentage of cells with a polar distribution of septin 7, Cdc42 or tubulin in borg4fl/fl or borg4∆/∆ LT-HSCs. At least 3 biological repeats, at least 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, ***P<0.001, **P<0.01, *P<0.05. (D,E) Representative immunofluorescence microscopy images of the distribution of borg4 (red), Cdc42 (red) or tubulin (green) in LT-HSCs from septin 7fl/fl or septin 7∆/∆ mice. Nuclei were stained with DAPI (blue). scale bar= 5 µm. (F) Percentage of cells with a polar distribution of borg4, Cdc42 or tubulin in septin 7fl/fl or septin 7∆/∆ LT-HSCs. At least 3 biological repeats, at least 30 to 50 cells were scored per sample. Error bars indicate mean±SEM, Two-way ANOVA analysis, **P<0.01, *P<0.05.


Figure 4 ( J - O ) Percentage of donor derived ( borg4fl / fl or borg4 ∆ / ∆ or septin 7fl / fl or septin 7 ∆ / ∆ ) B - cells ( B220 positive cells ) , T - cells ( CD3 positive cells ) and myeloid cells ( Mac - 1 + or Gr + positive cells ) among BM cells , percentage of donor derived LT - HSCs , ST - HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin - Sca1C - kit low cells in animals at post - transplantation ( n = 12 ) . Error bars indicate mean ± SD , * * * * P < 0 . 0001 , * * P < 0 . 01 and * P < 0 . 05 , Two - way ANOVA analysis and 2 tailed unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(J-O) Percentage of donor derived (borg4fl/fl or borg4∆/∆ or septin 7fl/fl or septin 7∆/∆) B-cells (B220 positive cells), T-cells (CD3 positive cells) and myeloid cells (Mac-1+ or Gr+ positive cells) among BM cells, percentage of donor derived LT-HSCs, ST-HSCs and LMPPs in LSK cells and percentage of donor derived CLPs in lin-Sca1C-kit low cells in animals at post- transplantation (n=12). Error bars indicate mean±SD, ****P<0.0001, **P<0.01 and *P<0.05, Two-way ANOVA analysis and 2 tailed unpaired t-test.


Figure 5 ( A ) Percentage of single LT - HSCs from septin 7fl / fl of septin 7 ∆ / ∆ mice that completed first and second division up to 48 hours post initiation of culture . ( 192 sorted HSCs cells were analysed , error bars indicate mean ± SD ) .

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Figure 5(A) Percentage of single LT-HSCs from septin 7fl/fl of septin 7∆/∆ mice that completed first and second division up to 48 hours post initiation of culture. (192 sorted HSCs cells were analysed, error bars indicate mean±SD).


Figure 1 ( A ) Picrosirius red ( PSR ) staining of collagens ( red ) in PDAC tissue following surgical resection . Scale bar represents 200 µm . ( B ) Quantification of PSR in low - grade ( 1 - 2 ) and high - grade ( 3 ) tumors indicated in panel A as percentage of area . n = 15 primary PDAC samples . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Quantification of tumor cell percentage as scored by pathologists in low - and high - grade PDAC indicated in panel A . n = 15 primary PDAC samples . Data information : data are represented as mean ± SD , Student ' s t - test . ( D ) PSR and α - SMA staining of indicated PDAC cell lines which were injected in NSG mice . Scale bar represents 200 µm for PSR and 100 µm for α - SMA images . ( E ) Quantification of stainings indicated in panel D . Three tumor ROIs of a tumor derived from epithelial cell lines ( Capan - 2 and AsPC - 1 ) and mesenchymal cell lines ( MIA PaCa - 2 and PANC - 1 ) are displayed and averages were pooled for analysis . Data information : data are represented as mean ± SD , Student ' s t - test . ( F ) IHC staining of α - SMA in 3D organotypic co - cultures of PS - 1 cells and indicated PDAC cell lines . Scale bar represents 100 µm .

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 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 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 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

Figure 1(A) Picrosirius red (PSR) staining of collagens (red) in PDAC tissue following surgical resection. Scale bar represents 200 µm. (B) Quantification of PSR in low-grade (1-2) and high-grade (3) tumors indicated in panel A as percentage of area. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.(C) Quantification of tumor cell percentage as scored by pathologists in low- and high-grade PDAC indicated in panel A. n=15 primary PDAC samples. Data information: data are represented as mean±SD, Student's t-test.(D) PSR and α-SMA staining of indicated PDAC cell lines which were injected in NSG mice. Scale bar represents 200 µm for PSR and 100 µm for α-SMA images. (E) Quantification of stainings indicated in panel D. Three tumor ROIs of a tumor derived from epithelial cell lines (Capan-2 and AsPC-1) and mesenchymal cell lines (MIA PaCa-2 and PANC-1) are displayed and averages were pooled for analysis. Data information: data are represented as mean±SD, Student's t-test.(F) IHC staining of α-SMA in 3D organotypic co-cultures of PS-1 cells and indicated PDAC cell lines. Scale bar represents 100 µm.


Figure 2 ( A ) Brightfield images of PS - 1 cells which were exposed for 72 hrs to CM from control ( CM of PS - 1 cells ) , epithelial ( blue ) or mesenchymal ( red ) cell lines . Scale bar represents 100 µm . ( B ) Quantification of bead - calibrated cell count of PS - 1 cells after treatment using flowcytometry . n = 6 per group , 3 biological replicates for each cell line . ( C ) EdU assay performed after treatment showing percentage of PS - 1 cells in G0 / G1 phase , G2 / M phase and S phase . n = 6 per group , 3 biological replicates for each cell line . .

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Figure 2(A) Brightfield images of PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) cell lines. Scale bar represents 100 µm.(B) Quantification of bead-calibrated cell count of PS-1 cells after treatment using flowcytometry. n=6 per group, 3 biological replicates for each cell line.(C) EdU assay performed after treatment showing percentage of PS-1 cells in G0/G1 phase, G2/M phase and S phase. n=6 per group, 3 biological replicates for each cell line..


Figure 3 ( A ) Gene expression level of stromal activation markers COL1A1 , ACTA2 , SPARC , FN1 and FAP in PS - 1 cells which were exposed for 72 hrs to CM from control ( CM of PS - 1 cells ) , epithelial ( blue ) or mesenchymal ( red ) PDAC cell lines using qPCR . Values were normalized to control and n = 6 per group , 3 biological replicates for each cell line . Data information : data are represented as mean ± SD , Student ' s t - test . ( B ) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS - 1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m . Values were Log2 transformed and normalized to Suit - 2 expression . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Brightfield images of PS - 1 cells exposed to 10 days of treatment with 1 and 10 µM all - trans retinoic acid ( ATRA ) . Scale bar represents 100 µm . ( D ) Gene expression levels of COL1A1 and ACTA2 in PS - 1 cells after treatment using qPCR , values were normalized to control . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test . ( E ) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control ( CM of primary CAFs ) or mesenchymal PDAC cell lines . Scale bar represents 100 µm . ( F ) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR , values were normalized to control . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 3(A) Gene expression level of stromal activation markers COL1A1, ACTA2, SPARC, FN1 and FAP in PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) PDAC cell lines using qPCR. Values were normalized to control and n=6 per group, 3 biological replicates for each cell line. Data information: data are represented as mean±SD, Student's t-test.(B) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS-1 cells after treatment using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m. Values were Log2 transformed and normalized to Suit-2 expression. Data information: data are represented as mean±SD, Student's t-test.(C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm.(D) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.(E) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control (CM of primary CAFs) or mesenchymal PDAC cell lines. Scale bar represents 100 µm.(F) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment using qPCR, values were normalized to control. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.


Figure 4 ( A ) Brightfield images of PS - 1 cells after 72 hrs of treatment with unfiltered , < 100 kDa or < 30 kDa filtered CM of indicated cell lines . Scale bar represents 100 µm . ( B ) Gene expression levels of COL1A1 and ACTA2 in PS - 1 cells after treatment using qPCR . Data was normalized against control CM . Statistical significance was tested against unfiltered CM samples . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Graphical representation of 64 cyto - and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward - phase protein array . Significantly differential expression of proteins ( triangles ) and genes ( green triangles ) are identified between epithelial and mesenchymal cell lines . Dotted line indicates P = 0 . 05 . Data information : data are represented as mean ± SD , Student ' s t - test . ( D ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or epithelial PDAC cells supplemented with recombinant human CSF - 1 . Scale bar represents 100 µm . ( E ) Gene expression levels of COL1A1 in PS - 1 cells after treatment using qPCR . n = 3 biological replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test . ( F ) PS - 1 cells were starved and stimulated with 500 ng / ml rh - CSF - 1 at indicated time points . Activation of pathways involved in CSF1R signaling was analyzed using western blot . Samples were derived from the same experiment and blots were processed in parallel .

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

Figure 4(A) Brightfield images of PS-1 cells after 72 hrs of treatment with unfiltered, <100 kDa or <30 kDa filtered CM of indicated cell lines. Scale bar represents 100 µm.(B) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment using qPCR. Data was normalized against control CM. Statistical significance was tested against unfiltered CM samples. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.(C) Graphical representation of 64 cyto- and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward-phase protein array. Significantly differential expression of proteins (triangles) and genes (green triangles) are identified between epithelial and mesenchymal cell lines. Dotted line indicates P=0.05. Data information: data are represented as mean±SD, Student's t-test.(D) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or epithelial PDAC cells supplemented with recombinant human CSF-1. Scale bar represents 100 µm.(E) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. n=3 biological replicates per group. Data information: data are represented as mean±SD, Student's t-test.(F) PS-1 cells were starved and stimulated with 500 ng/ml rh-CSF-1 at indicated time points. Activation of pathways involved in CSF1R signaling was analyzed using western blot. Samples were derived from the same experiment and blots were processed in parallel.


Figure 5 ( A ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or mesenchymal PDAC cell lines supplemented with anti - CSF - 1 . Scale bar represents 100 µm . ( B ) Gene expression levels of COL1A1 in PS - 1 cells after treatment using qPCR . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , Student ' s t - test . ( C ) Brightfield images of PS - 1 cells after 72 hrs of treatment with PANC - 1 CM of pLKOctrl or shCSF1 ( # 560 - 562 ) . Scale bar represents 100 µm . ( D ) Correlation graph of CSF1 expression in PANC - 1 pLKOctrl or shCSF1 ( # 560 - 562 ) and how these various CM affect COL1A1 expression in PS - 1 cells using qPCR , values were normalized for RPLPO expression . n = 3 biological replicates for all individual experiments , error bars indicated SD . R2 and P - value were analyzed with linear regression . ( E ) Brightfield images of PS - 1 cells after 72 hrs of treatment with CM of Capan - 2 cells with control vector or Capan - 2 cells overexpressing CSF1 . Scale bar represents 100 µm . ( F ) Gene expression level of CSF1 in Capan - 2 cells after transduction with control vector or overexpressing CSF1 . n = 3 technical replicates per group . Data information : data are represented as mean ± SD , Student ' s t - test . ( G ) Gene expression levels of COL1A1 in PS - 1 cells after treatment indicated using qPCR . Graph shows n = 3 technical replicates of an example experiment that was performed three times . Data information : data are represented as mean ± SD , 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 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 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 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 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

Figure 5(A) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or mesenchymal PDAC cell lines supplemented with anti-CSF-1. Scale bar represents 100 µm.(B) Gene expression levels of COL1A1 in PS-1 cells after treatment using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.(C) Brightfield images of PS-1 cells after 72 hrs of treatment with PANC-1 CM of pLKOctrl or shCSF1 (#560-562). Scale bar represents 100 µm.(D) Correlation graph of CSF1 expression in PANC-1 pLKOctrl or shCSF1 (#560-562) and how these various CM affect COL1A1 expression in PS-1 cells using qPCR, values were normalized for RPLPO expression. n=3 biological replicates for all individual experiments, error bars indicated SD. R2 and P-value were analyzed with linear regression.(E) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of Capan-2 cells with control vector or Capan-2 cells overexpressing CSF1. Scale bar represents 100 µm.(F) Gene expression level of CSF1 in Capan-2 cells after transduction with control vector or overexpressing CSF1. n=3 technical replicates per group. Data information: data are represented as mean±SD, Student's t-test.(G) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times. Data information: data are represented as mean±SD, Student's t-test.


Figure 6 ( A ) IHC staining of CSF - 1 in PDAC tissue following surgical resection representative image of CSF - 1 expression in grade 1 and grade 3 PDAC is depicted . Scale bar represents 100 µm . ( B ) For each tumor of panel A , CSF - 1 expression in ductal adenocarcinoma cells was quantified . Student ' s t - test . Shown is mean ± SD . n = 15 primary PDAC samples . ( C ) A validation cohort of PDAC patients ( n = 21 ) that underwent direct resection was stained for CSF - 1 ( scale bar represents 100 µm ) , PSR ( scale bar represents 500 µm ) and α - SMA ( scale bar represents 200 µm ) . OK8 is low in CSF - 1 expression and high in PSR and α - SMA expression , while the opposite accounts for OK7 . ( D ) Correlation of % of PSR in the tumor and CSF - 1 expressing tumor cells , as indicated in panel C . R2 and P - value were analyzed with linear regression . n = 21 primary PDAC samples ( E ) Correlation of % of α - SMA in the tumor and CSF - 1 expressing tumor cells , as indicated in panel C , R2 and P - value were analyzed with linear regression . n = 21 primary PDAC samples

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Figure 6(A) IHC staining of CSF-1 in PDAC tissue following surgical resection representative image of CSF-1 expression in grade 1 and grade 3 PDAC is depicted. Scale bar represents 100 µm. (B) For each tumor of panel A, CSF-1 expression in ductal adenocarcinoma cells was quantified. Student's t-test. Shown is mean±SD. n=15 primary PDAC samples.(C) A validation cohort of PDAC patients (n=21) that underwent direct resection was stained for CSF-1 (scale bar represents 100 µm), PSR (scale bar represents 500 µm) and α-SMA (scale bar represents 200 µm). OK8 is low in CSF-1 expression and high in PSR and α-SMA expression, while the opposite accounts for OK7. (D) Correlation of % of PSR in the tumor and CSF-1 expressing tumor cells, as indicated in panel C. R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples (E) Correlation of % of α-SMA in the tumor and CSF-1 expressing tumor cells, as indicated in panel C, R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples


Figure 1Immunoblot analysis of mouse intestinal crypts treated with Wnt3a ( 60 ng / mL ) for indicated time . The relative protein level was quantified and shown under each band . Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min . The right panels show the distributions of Ythdf1 and Actb in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) . Immunoblot analysis of SW620 cells with or without APC overexpression . The relative protein level was quantified and shown under each band . Polysome profiles of SW620 cells with or without APC overexpression . The right panels show the distributions of YTHDF1 and ACTB in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) . Dual - Luciferase assay wih a construct bearing the 5 ' UTR of YTHDF1 in SW620 cells with or without APC overexpression . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) . YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc + / + and Apcmin / + mice . N , normal tissue . T , tumor tissue . A , tumor adjacent tissue . YTHDF1 staining in matched samples of human CRC tissue and adjacent non - tumor tissue . Scale bar , 450 μm . The right panel shows the statistics of IHC scores . Chi - square test ; n = 75 for each group .

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Figure 1Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band.Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band.Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue.YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.


Figure 2The representative jejunum from wildtype ( Ythdf1CTL ) and Ythdf1cKO mice . Left : hematoxylin and eosin staining ( H & E ) ; middle : BrdU staining ; right : quantification of crypt height and BrdU + cells . Scale bar , 50 μm . Data are represented as mean ± SEM . ( 9 biological replicates , t - test ) . Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR . YTHDF1 ( left ) and Ki67 ( right ) staining of small intestine from wildtype mice 72 hr after 12 Gy IR . Scale bar , 50 μm . Red arrows indicate the proliferating cells . Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR . Left : H & E staining ; middle : Ki67 staining ; right : quantification of crypt height and Ki67 + cells . Scale bar , 50 μm . Vertical bars indicate the length of the crypts . Data are represented as mean ± SEM . * * * * p < 0 . 0001 ( 9 biological replicates , t - test ) . RT - qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 5 biological replicates , t - test ) .

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Figure 2The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR.YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells.Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test).RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).


Figure 3Small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . H & E staining of small intestines from 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . Scale bar , 1 mm . The black arrows indicate the tumors . Tumor number ( C ) and size distribution ( D ) from the small intestines of 24 - week - old Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice . Data are shown as mean ± SEM ( 6 mice for each group ) . * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) . Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM / DSS induction . H & E staining of colons from Apcmin / + ; Ythdf1CTL and Apcmin / + ; Ythdf1cKO mice after AOM / DSS induction . Scale bar , 200 μm . Colon tumor number ( H ) and size distribution ( I ) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction . Data are shown as mean ± SEM ( 6 mice for each group ) . * * p < 0 . 01 ( t - test ) .

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Figure 3Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice.H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors.Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01, ***p<0. 001 (t-test).Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01 (t-test).


Figure 4Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines . Scale bar , 50 μm . Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells . Flowcytometry analysis of YTHDF1 protein expression in GFPhigh / low / neg populations from Lgr5 - GFP - IRES - creERT2 mice . Morphology of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant . Scale bar , 250 μm . Quantification of differentiated versus undifferentiated organoids from ( D ) . Data are represented as mean ± SEM . * * p < 0 . 01 ( 4 biological replicates , t - test ) . Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in ( D ) . Data are represented as mean ± SEM . 4 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( t - test ) .

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Figure 4Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm.Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells.Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **p<0. 01 (4 biological replicates, t-test).Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).


Figure 5Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells . The upregulated ( red ) and downregulated ( blue ) genes in translational efficiency ( TE ) are highlighted . The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown . POI : peak over input . Down , genes significantly downregulated in YTHDF1 knockdown cells ( < 0 . 5 fold ) . Unchanged , genes not significantly changed . Up , genes significantly upregulated ( > 2 fold ) . The upper and lower quartiles and the median are shown for each group . Mann - Whitney test . * * p < 0 . 01 . * * * p < 0 . 001 . Violin plots showing TE change between control and YTHDF1 knockdown cells for not - methylated ( non - m6A ) and methylated ( m6A ) transcripts . The upper and lower quartiles and the median are indicated for each group . Mann - Whitney test . Cumulative distributions of TE change for non - m6A and m6A transcripts as in ( C ) . Kolmogorov - Smirnov test . Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells . The values show the fold change of the indicated color of the heatmap .

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Figure 5Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted.The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.


Figure 6Immunoblot analysis of HCT116 cells with YTHDF1 knockdown . Two major bands were detected for human TCF7L2 due to alternative splicing . Polysome profiles of HCT116 cells with or without YTHDF1 knockdown . The right panels show the distributions of TCF7L2 , and ACTB in polysome fractions . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 ( 3 biological replicates , t - test ) . Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice . Multiple bands were detected for mouse TCF7L2 representing different isoforms . Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min . MeRIP - qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a . Data are represented as mean ± SEM . * p < 0 . 05 ( 3 biological replicates , t - test ) . RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA . The enrichment was measured by qPCR and normalized to input . Data are represented as mean ± SEM . * p < 0 . 05 ( 3 biological replicates , t - test ) . β - catenin / TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2 . Data are represented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - test ) . Morphology of organoids from Lgr5 - creERT2 : Ythdf1fl / fl mice in Wnt3a - conditioned medium without or with 4 - OHT induction and infected with lentivirus expressing TCF7L2 . Scale bar , 250 μm . Quantification of differentiated versus undifferentiated organoids from ( H ) . Data are represented as mean ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 ( 3 biological replicates , t - test ) .

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Figure 6Immunoblot analysis of HCT116 cells with YTHDF1 knockdown. Two major bands were detected for human TCF7L2 due to alternative splicing.Polysome profiles of HCT116 cells with or without YTHDF1 knockdown. The right panels show the distributions of TCF7L2, and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice. Multiple bands were detected for mouse TCF7L2 representing different isoforms.Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min.MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA. The enrichment was measured by qPCR and normalized to input. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).β-catenin/TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01, ***p<0.001 (3 biological replicates, t-test).Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing TCF7L2. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).


Figure 7Colons from mice in ( A ) treated with or without TAM on day 80 of AOM / DSS induction . Colon tumor number from mice treated with or without TAM on day 80 of AOM / DSS induction . Data are shown as mean ± SEM ( 7 mice for each group ) . * * * * p < 0 . 0001 ( t - test ) . Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM . Kaplan - Meier survival plots for Apcmin / + ; Lgr5 - creERT2 : Ythdf1fl / fl mice ( 8 mice for each group ) treated with or without TAM . Log - rank ( Mantel Cox ) test . * * p < 0 . 01 ( t - test ) .

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Figure 7Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).


Figure 1 ( A ) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Sec63 were transformed with a centromeric plasmid expressing BFP - Ape1 . Cells were grown in SGC - LEU , shifted to SG - N for the indicated time , and analyzed by fluorescence microscopy . Images are shown as single planes . Scale bar , 5 µm . Arrows indicate Ykt6 dots colocalizing with Ape1 . ( B ) Percentage of Ykt6 puncta simultaneously co - localizing with both Ape1 and Sec63 . The data were quantified from ( A ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 50 ) were quantified . ( C ) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Snx41 ( endosomes ) , Sec63 ( ER ) , Vac8 ( vacuole ) , or Mnn9 ( Golgi ) were were grown in SGC - LEU , then shifted to SG - N for 2 h , and analyzed by fluorescence microscopy . Single imaging planes are shown . Scale bar , 5 µm . Arrows indicate Ykt6 dots that colocalize with marker proteins .

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Figure 1(A) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Sec63 were transformed with a centromeric plasmid expressing BFP-Ape1. Cells were grown in SGC-LEU, shifted to SG-N for the indicated time, and analyzed by fluorescence microscopy. Images are shown as single planes. Scale bar, 5 µm. Arrows indicate Ykt6 dots colocalizing with Ape1. (B) Percentage of Ykt6 puncta simultaneously co-localizing with both Ape1 and Sec63. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥50) were quantified.(C) Localization of Ykt6 relative to different organelle marker proteins during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Snx41 (endosomes), Sec63 (ER), Vac8 (vacuole), or Mnn9 (Golgi) were were grown in SGC-LEU, then shifted to SG-N for 2 h, and analyzed by fluorescence microscopy. Single imaging planes are shown. Scale bar, 5 µm. Arrows indicate Ykt6 dots that colocalize with marker proteins.


Figure 2Localization of Ykt6 in multiple atg mutants . ( A ) Wild - type or the respective mutant cells expressing mCherry - tagged Ape1 and GFP - tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1 - promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 ( A ) ( B ) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 70 ) were quantified . ( C ) Localization of Atg9 relative to Ykt6 . Cells expressing GFP - tagged Ykt6 and mScarlet - tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9 . Cells ( n ≥ 200 ) and Atg9 dots ( n ≥ 80 ) were quantified . Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 ( C ) Ykt6 localizes to the isolation membrane during starvation . ( D ) Cells expressing mCherry - tagged Atg8 and GFP - Ykt6 carrying the Ape1 encoding plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SGC medium containing CuSO4 and starved for 1 h , before to be analyzed by microscopy . Scale bar , 5 µm . ( E ) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view . Cells expressing mCherry - tagged Atg8 and GFP - Ykt6 carrying plasmid pRS315 - CUP1pr - BFP - APE1 , cells , images were analyzed by Imaris . Scale bar , 5 µm . Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 ( D , E ) . ( F ) The only partial time points ( 90 s to 600 s ) of images were assembled , and analyzed by Imaris and image J . Scale bar , 5 µm . 500 - 1000 cells were analyzed in each independent experiment . The 3D stacks were cropped by Imaris and different channels were split by Image J .

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

Figure 2Localization of Ykt6 in multiple atg mutants. (A) Wild-type or the respective mutant cells expressing mCherry-tagged Ape1 and GFP-tagged Ykt6 were grown in SGC to allow for Ykt6 expression from the GAL1-promoter and analyzed Arrows in the images indicate dots of Ykt6 colocalizing with Ape1 (A)(B) Quantification of colocalization of Ykt6 relative to Ape1 as a PAS marker. Cells (n≥250) and Ape1 dots (n≥70) were quantified.(C) Localization of Atg9 relative to Ykt6. Cells expressing GFP-tagged Ykt6 and mScarlet-tagged Atg9 were grown and analyzed The left is the quantification of colocalization of Ykt6 relative to Atg9. Cells (n≥200) and Atg9 dots (n≥80) were quantified. Arrows in the images indicate dots of Ykt6 colocalizing with Atg9 (C)Ykt6 localizes to the isolation membrane during starvation. (D) Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying the Ape1 encoding plasmid pRS315-CUP1pr-BFP-APE1 were grown in SGC medium containing CuSO4 and starved for 1 h, before to be analyzed by microscopy. Scale bar, 5 µm. (E) Volume rendering of LLSM image from a single time point after 3D deconvolution from 3D view. Cells expressing mCherry-tagged Atg8 and GFP-Ykt6 carrying plasmid pRS315-CUP1pr-BFP-APE1, cells , images were analyzed by Imaris. Scale bar, 5 µm. Arrows in the images indicate dots of Ykt6 colocalizing with Atg8 (D,E).(F) The only partial time points (90 s to 600 s) of images were assembled, and analyzed by Imaris and image J. Scale bar, 5 µm. 500-1000 cells were analyzed in each independent experiment. The 3D stacks were cropped by Imaris and different channels were split by Image J.


Figure 3 ( A ) Effect of ykt6 and dsl1 complex ts mutants on autophagosome - vacuole fusion . Cells carrying a CEN plasmid expressing GFP - Atg8 were grown at 24 ° C in SGC and then shifted to SG - N for 2 h at 24 ° C or 37 ° C . Vacuoles were stained by FM4 - 64 and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . ( B ) Analysis of autophagy over time . Cells were grown at 24 ° C and shifted to starvation medium at 24 ° C or 37 ° C for the indicated time periods . The cells were harvested and proteins were extracted from cells by TCA precipitation . The extracted proteins were analyzed by SDS - PAGE and Western blotting against GFP . ( C ) Protease protection assay of ykt6 - 11 and dsl3 mutants . Lysates of atg1 ∆ , ypt7 ∆ , ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP - Atg8 were grown at 37 ° C and subjected to the protease ( PK ) - protection ( D ) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12 - 1 ts mutant . Cells carrying a CEN plasmid expressing GFP - Atg8 and encoding 3xmCherry - tagged Atg1 were grown at 24 ° C in SDC and then shifted to SD - N for 2 h at 24 ° C or 37 ° C , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Arrows in images indicate dots colocalization of Atg1 with Atg8 ( D ) ( E ) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions . Cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry - tagged Dsl1 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . ( F ) Percentage of Ykt6 puncta co - localizing with Dsl1 . The data was quantified from ( E ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Dsl1 dots ( n ≥ 50 ) were quantified . Arrows in images indicate dots colocalization of Ykt6 with Dsl1 ( E ) . ( G ) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation . Cells expressing GFP - tagged Dsl3 and mCherry - tagged Atg8 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Scale bar , 5 µm .

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 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 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 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 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 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

Figure 3(A) Effect of ykt6 and dsl1 complex ts mutants on autophagosome-vacuole fusion. Cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 24°C in SGC and then shifted to SG-N for 2 h at 24°C or 37°C. Vacuoles were stained by FM4-64 and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset is 0.6 µm.(B) Analysis of autophagy over time. Cells were grown at 24°C and shifted to starvation medium at 24°C or 37°C for the indicated time periods. The cells were harvested and proteins were extracted from cells by TCA precipitation. The extracted proteins were analyzed by SDS-PAGE and Western blotting against GFP.(C) Protease protection assay of ykt6-11 and dsl3 mutants. Lysates of atg1∆, ypt7∆, ykt6ts and dsl3ts cells carrying a CEN plasmid expressing GFP-Atg8 were grown at 37°C and subjected to the protease (PK)-protection(D) Localization of Atg8 relative to Atg1 during nitrogen starvation condition in sec12-1 ts mutant. Cells carrying a CEN plasmid expressing GFP-Atg8 and encoding 3xmCherry-tagged Atg1 were grown at 24°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Arrows in images indicate dots colocalization of Atg1 with Atg8 (D)(E) Localization of Ykt6 relative to Dsl1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Dsl1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (F) Percentage of Ykt6 puncta co-localizing with Dsl1. The data was quantified from (E). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Dsl1 dots (n≥50) were quantified. Arrows in images indicate dots colocalization of Ykt6 with Dsl1 (E).(G) Localization of Dsl3 relative to Atg8 during growth and nitrogen starvation. Cells expressing GFP-tagged Dsl3 and mCherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Scale bar, 5 µm.


Figure 4 ( A ) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes . Wild - type and selected ts strains ( sec12 - 1 , tip20 - 5 and dsl3 - 2 ) expressing GFP - tagged Ykt6 and mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 37 ° C . Vacuoles were stained by CMAC and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar in inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 and Atg8 ( A ) Wild - type and selected ts strains ( sec12 - 1 , tip20 - 5 and dsl3 - 2 ) expressing GFP - tagged Ykt6 and mCherry - tagged Atg8 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 37 ° C . ( B ) Percentage of Ykt6 puncta co - localizing with Atg8 . Cells ( n ≥ 150 ) and Atg8 dots ( n ≥ 100 ) were quantified . ( C ) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions . tip20 - 5 cells expressing GFP - tagged Ykt6 and 3xmCherry - tagged Sec63 or Sec13 were grown at 24 ° C in SGC for 8 h and then shifted to SG - N for 2 h at 24 ° C or 37 ° C , and analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar of inset , 0 . 7 µm . Arrows indicate dot colocalization of Ykt6 with Sec63 ( C ) . ( D ) Effect of dsl mutation on autophagosome - vacuole fusion in vitro . The vam3 ∆ or dsl ts strain expressing Atg9 - 3xFLAG and GFP - Atg8 were starved for 3 h at 30 ° C before purifying autophagosomes , which was sufficient to induce the ts phenotype . Vacuoles were isolated from pep4 ∆ cells expressing Vac8 - 3xmCherry and then incubated with autophagosomes at 26 ° C for 1 h with or without ATP ( E ) Quantification of ( D ) . Error bars represent standard deviation of 3 independent experiments . Fluorescence intensity of GFP - Atg8 in the vacuolar lumen was quantified by image J using the ROI ( region of interest ) manager tool . Vacuoles ( n ≥ 40 ) for each experiment were quantified .

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Figure 4(A) Effect of Dsl and COPII ts mutants on Ykt6 localization to autophagosomes. Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. Vacuoles were stained by CMAC and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar in inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 and Atg8 (A)Wild-type and selected ts strains (sec12-1, tip20-5 and dsl3-2) expressing GFP-tagged Ykt6 and mCherry-tagged Atg8 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 37°C. (B) Percentage of Ykt6 puncta co-localizing with Atg8. Cells (n≥150) and Atg8 dots (n≥100) were quantified.(C) Localization of Ykt6 relative to Sec63 and Sec13 during growth and nitrogen starvation conditions. tip20-5 cells expressing GFP-tagged Ykt6 and 3xmCherry-tagged Sec63 or Sec13 were grown at 24°C in SGC for 8 h and then shifted to SG-N for 2 h at 24°C or 37°C, and analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar of inset, 0.7 µm. Arrows indicate dot colocalization of Ykt6 with Sec63 (C).(D) Effect of dsl mutation on autophagosome-vacuole fusion in vitro. The vam3∆ or dsl ts strain expressing Atg9-3xFLAG and GFP-Atg8 were starved for 3 h at 30°C before purifying autophagosomes, which was sufficient to induce the ts phenotype. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.


Figure 5 ( A - B ) Quantification of size and number of autophagosomes in the indicated ts mutant strains . Error bars represent standard deviation of 3 independent experiments . GFP - tagged Atg8 in the different mutants were grown at 23 ° C ( ts mutant ) or 30 ° C ( atg1 ∆ ) in SDC and then shifted to SD - N for 2 h at 30 ° C ( atg1 ∆ ) or 37 ° C ( ts mutant ) , and analyzed by fluorescence microscopy . Scale bar , 1 µm . ( C ) Localization of Atg8 relative to Atg9 during nitrogen starvation . dsl3 cells expressing mCherry - tagged Atg9 and carrying a CEN plasmid expressing GFP - Atg8 were grown at 23 ° C in SDC and then shifted to SD - N for 2 h at 24 ° C or 37 ° C . Cells were analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm . ( D ) Atg9 localization in dsl3 cells during starvation . dsl3 ts cells expressing mCherry - tagged Atg9 , and carrying a CEN plasmid expressing GFP - Atg8 and plasmid pRS315 - CUP1pr - BFP - APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure , and starved for 1 h . Cells were analyzed by fluorescence microscopy . Scale bar , 5 µm . Scale bar for inset is 0 . 6 µm .

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

Figure 5(A-B) Quantification of size and number of autophagosomes in the indicated ts mutant strains. Error bars represent standard deviation of 3 independent experiments. GFP-tagged Atg8 in the different mutants were grown at 23°C (ts mutant) or 30°C (atg1∆) in SDC and then shifted to SD-N for 2 h at 30°C (atg1∆) or 37°C (ts mutant), and analyzed by fluorescence microscopy. Scale bar, 1 µm.(C) Localization of Atg8 relative to Atg9 during nitrogen starvation. dsl3 cells expressing mCherry-tagged Atg9 and carrying a CEN plasmid expressing GFP-Atg8 were grown at 23°C in SDC and then shifted to SD-N for 2 h at 24°C or 37°C. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.(D) Atg9 localization in dsl3 cells during starvation. dsl3 ts cells expressing mCherry-tagged Atg9, and carrying a CEN plasmid expressing GFP-Atg8 and plasmid pRS315-CUP1pr-BFP-APE1 were grown in SDC medium containing CuSO4 to induce formation of the giant Ape1 structure, and starved for 1 h. Cells were analyzed by fluorescence microscopy. Scale bar, 5 µm. Scale bar for inset is 0.6 µm.


Figure 6 ( B ) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions . Cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry - tagged Atg1 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . Scale bar of inset is 0 . 6 µm . Right , percentage of Ykt6 puncta co - localizing with Atg1 . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 200 ) and Atg1 dots ( n ≥ 100 ) were quantified . Arrows indicate Ykt6 positive dots colocalizing with Atg1 ( B ) ( C ) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex . Purified Ykt6 was incubated with the Atg1 - Atg13 complex in the presence or absence of ATP , and Ykt6 band was cut out and analyzed by mass spectrometry . ( D ) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions . Wild - type and atg1D211A cells expressing GFP - tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry - tagged Atg8 were grown in SDC or SD - N for 2 h , and analyzed by fluorescence microscopy . Individual focal planes are shown . Scale bar , 5 µm . ( E ) Percentage of Ykt6 puncta co - localizing with Atg8 . Numbers in the column indicates the percentage . Cells ( n ≥ 300 ) and Atg8 dots ( n ≥ 60 ) were quantified . Arrows indicate Ykt6 positive dots colocalizing with Atg8 ( D ) .

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 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 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

Figure 6(B) Localization of Ykt6 relative to Atg1 during growth and nitrogen starvation conditions. Cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically 3xmCherry-tagged Atg1 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. Scale bar of inset is 0.6 µm. Right, percentage of Ykt6 puncta co-localizing with Atg1. Error bars represent standard deviation of three independent experiments. Cells (n≥200) and Atg1 dots (n≥100) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg1 (B)(C) In vitro phosphorylation of purified Ykt6 by the Atg1 kinase complex. Purified Ykt6 was incubated with the Atg1-Atg13 complex in the presence or absence of ATP, and Ykt6 band was cut out and analyzed by mass spectrometry.(D) Localization of Ykt6 relative to Atg8 under growth and nitrogen starvation conditions. Wild-type and atg1D211A cells expressing GFP-tagged Ykt6 under the control of the PHO5 promoter and genomically Cherry-tagged Atg8 were grown in SDC or SD-N for 2 h, and analyzed by fluorescence microscopy. Individual focal planes are shown. Scale bar, 5 µm. (E) Percentage of Ykt6 puncta co-localizing with Atg8. Numbers in the column indicates the percentage. Cells (n≥300) and Atg8 dots (n≥60) were quantified. Arrows indicate Ykt6 positive dots colocalizing with Atg8 (D).


Figure 7 ( A ) Growth test of Ykt6 wild - type and mutants . ykt6 ∆ cells carrying a URA3 plasmid coding for wild - type Ykt6 and pRS413 - GAL1pr - YKT6 - GFP ( wild - type or mutant ) were spotted as serial dilutions on SGC - HIS to select for the mutant plasmid or 5 - Fluoroorotic acid ( 5 - FOA ) to force the loss of the plasmid coding for wild - type Ykt6 . ( B ) Effect of Ykt6 phospho - mutants on autophagy . ykt6 ∆ cells expressing wild - type Ykt6 plus a pRS413 plasmid encoding GAL1pr - YKT6 ( wild - type or mutants with S to A mutations [ SA ; S182 , S183A ] , or S to D mutations [ SD ; S182D , S183D ] ) , and a plasmid expressing GFP - Atg8 were grown in SGC and then shifted to SG - N for 2 h . Cells were analyzed by fluorescence microscopy . Individual slices are shown . Scale bar , 5 µm . ( C ) Quantification of Atg8 dots per cell from images in ( B ) . Cells ( n ≥ 100 ) and Atg8 dots ( n ≥ 50 ) were quantified . ( D ) Effect of Atg1 - Atg13 subcomplex on autophagosome - vacuole fusion in vitro . The vam3 ∆ strain carrying pRS413 plasmid encoding GAL1pr - YKT6 ( wild - type or mutant ) , and expressing Atg9 - 3xFLAG and GFP - Atg8 , were starved for 3 h at 30 ° C before purifying autophagosomes . Vacuoles were isolated from pep4 ∆ cells expressing Vac8 - 3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1 - Atg13 subcomplex at 26 ° C for 1 h with or without ATP ( E ) Quantification of ( D ) . Error bars represent standard deviation of 3 independent experiments . Fluorescence intensity of GFP - Atg8 in the vacuolar lumen was quantified by image J using the ROI ( region of interest ) manager tool . Vacuoles ( n ≥ 40 ) for each experiment were quantified .

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Figure 7(A) Growth test of Ykt6 wild-type and mutants. ykt6∆ cells carrying a URA3 plasmid coding for wild-type Ykt6 and pRS413-GAL1pr-YKT6-GFP (wild-type or mutant) were spotted as serial dilutions on SGC-HIS to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to force the loss of the plasmid coding for wild-type Ykt6.(B) Effect of Ykt6 phospho-mutants on autophagy. ykt6∆ cells expressing wild-type Ykt6 plus a pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutants with S to A mutations [SA; S182, S183A], or S to D mutations [SD; S182D, S183D]), and a plasmid expressing GFP-Atg8 were grown in SGC and then shifted to SG-N for 2 h. Cells were analyzed by fluorescence microscopy. Individual slices are shown. Scale bar, 5 µm. (C) Quantification of Atg8 dots per cell from images in (B). Cells (n≥100) and Atg8 dots (n≥50) were quantified.(D) Effect of Atg1-Atg13 subcomplex on autophagosome-vacuole fusion in vitro. The vam3∆ strain carrying pRS413 plasmid encoding GAL1pr-YKT6 (wild-type or mutant), and expressing Atg9-3xFLAG and GFP-Atg8, were starved for 3 h at 30°C before purifying autophagosomes. Vacuoles were isolated from pep4∆ cells expressing Vac8-3xmCherry and then incubated with autophagosomes in the presence or absence of 50 nM Atg1-Atg13 subcomplex at 26°C for 1 h with or without ATP (E) Quantification of (D). Error bars represent standard deviation of 3 independent experiments. Fluorescence intensity of GFP-Atg8 in the vacuolar lumen was quantified by image J using the ROI (region of interest) manager tool. Vacuoles (n≥40) for each experiment were quantified.


Figure 1 ( E ) Western blot analysis with an anti - HA antibody or in - house - generated anti - SMC3 antibody ( indicated to the left of each blot ) of nuclear extracts from synchronous SMC1 - 3HA - dd ( D ) or STAG - 3HA - dd ( E ) late - stage parasites before ( input , " In " ) and after ( flow - through , " FT " ) immunoprecipitation with an antibody against HA ( " 𝛼 - HA IP " ) . An antibody against histone H3 is used as a control . Immunoprecipitation with IgG ( " IgG IP " ) was used as a control in the right panel of ( E ) . Molecular weights are shown to the right . SMC1 - 3HA - dd has a predicted molecular weight of approximately 230 kDa and STAG - 3HA - dd has a predicted molecular weight of approximately 298 kDa ( of which 3 . 3 and 11 . 9 kDa correspond to the 3HA tag and the ddFKBP , respectively ) .

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Figure 1(E) Western blot analysis with an anti-HA antibody or in-house-generated anti-SMC3 antibody (indicated to the left of each blot) of nuclear extracts from synchronous SMC1-3HA-dd (D) or STAG-3HA-dd (E) late-stage parasites before (input, "In") and after (flow-through, "FT") immunoprecipitation with an antibody against HA ("𝛼-HA IP"). An antibody against histone H3 is used as a control. Immunoprecipitation with IgG ("IgG IP") was used as a control in the right panel of (E). Molecular weights are shown to the right. SMC1-3HA-dd has a predicted molecular weight of approximately 230 kDa and STAG-3HA-dd has a predicted molecular weight of approximately 298 kDa (of which 3.3 and 11.9 kDa correspond to the 3HA tag and the ddFKBP, respectively).


Figure 2Western blot analysis of nuclear extracts of ring ( R ) , trophozoite ( T ) , and schizont ( S ) stages from a synchronous population of SMC3 - 3HA - glmS parasites . SMC3 - 3HA is detected with an anti - HA antibody . An antibody against histone H3 is used as a control . Molecular weights are shown to the right . SMC3 - 3HA has a predicted molecular weight of 147 . 3 kDa ( of which 3 . 3 kDa correspond to the 3HA tag ) . ( C ) Immunofluorescence assays of fixed RBCs infected with trophozoite or schizont stage SMC3 - 3HA - glmS parasites . DNA was stained with DAPI ( blue ) and SMC3 - 3HA was detected with anti - HA ( green in B and magenta in C ) antibody . HP1 was detected with anti - HP1 antibody ( green in C ) . DIC , differential interference contrast . Scale bars equal 10 µm ( B ) and 5 µm ( C ) .

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Figure 2Western blot analysis of nuclear extracts of ring (R), trophozoite (T), and schizont (S) stages from a synchronous population of SMC3-3HA-glmS parasites. SMC3-3HA is detected with an anti-HA antibody. An antibody against histone H3 is used as a control. Molecular weights are shown to the right. SMC3-3HA has a predicted molecular weight of 147.3 kDa (of which 3.3 kDa correspond to the 3HA tag).(C) Immunofluorescence assays of fixed RBCs infected with trophozoite or schizont stage SMC3-3HA-glmS parasites. DNA was stained with DAPI (blue) and SMC3-3HA was detected with anti-HA (green in B and magenta in C) antibody. HP1 was detected with anti-HP1 antibody (green in C). DIC, differential interference contrast. Scale bars equal 10 µm (B) and 5 µm (C).


Figure 3Zoomed - in view of SMC3 ChIP - seq data from clone A corresponding to chromosome 4 ( 604 , 345 - 709 , 167 bp ) , including the centromere ( represented with dark red line below the x - axis ) . For 12 ( blue ) , 24 ( coral ) , and 36 ( green ) hpi , the y - axis is enrichment ( ChIP / Input ) , with vertical lines below representing significant peaks obtained from peak calling algorithm macs2 ( present in both clones , q - value < 0 . 05 ) . The x - axis is DNA sequence , with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction .

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Figure 3Zoomed-in view of SMC3 ChIP-seq data from clone A corresponding to chromosome 4 (604,345 - 709,167 bp), including the centromere (represented with dark red line below the x-axis). For 12 (blue), 24 (coral), and 36 (green) hpi, the y-axis is enrichment (ChIP/Input), with vertical lines below representing significant peaks obtained from peak calling algorithm macs2 (present in both clones, q-value < 0.05). The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction.


Figure 4Western blot analysis of nuclear extracts at 12 , 24 , and 36 hpi from a clonal population of SMC3 - 3HA - glmS parasites in the absence ( - ) or presence ( + ) of glucosamine ( GlcN ) . SMC3 - 3HA is detected with an anti - HA antibody . An antibody against histone H3 is used as a control . Molecular weights are shown to the right . Growth curve showing parasite growth rate ( parasitemia at Day X / parasitemia at Day 1 ) over five days for WT and two clones of SMC3 - 3HA - glmS parasites in the absence or presence of glucosamine ( GlcN ) . Glucosamine treatment was started 96 h ( two IDC cycles ) before Day 1 to ensure SMC3 knockdown during the days sampled Uninfected red blood cells ( Blood ) served as reference of background . Error bars indicate standard deviation of three technical replicates ( n = 3 ) . A two - way ANOVA with Tukey post hoc test was used for statistical analysis . No significant differences were found .

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Figure 4Western blot analysis of nuclear extracts at 12, 24, and 36 hpi from a clonal population of SMC3-3HA-glmS parasites in the absence (-) or presence (+) of glucosamine (GlcN). SMC3-3HA is detected with an anti-HA antibody. An antibody against histone H3 is used as a control. Molecular weights are shown to the right.Growth curve showing parasite growth rate (parasitemia at Day X/parasitemia at Day 1) over five days for WT and two clones of SMC3-3HA-glmS parasites in the absence or presence of glucosamine (GlcN). Glucosamine treatment was started 96 h (two IDC cycles) before Day 1 to ensure SMC3 knockdown during the days sampled Uninfected red blood cells (Blood) served as reference of background. Error bars indicate standard deviation of three technical replicates (n = 3). A two-way ANOVA with Tukey post hoc test was used for statistical analysis. No significant differences were found.


Figure 5RNA - seq of an SMC3 - 3HA - glmS clone shows smc3 transcript levels ( FPKM ) at 12 ( blue , q = 8 . 5 x 10 - 3 ) , 24 ( coral , q = 1 . 3 x 10 - 39 ) , and 36 ( green , q = 4 . 1 x 10 - 54 ) hpi in the absence ( filled circles ) or presence ( empty circles ) of glucosamine ( GlcN ) . Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites , for which there were two replicates . P - values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 q = Bonferroni corrected P - value . Asterisks indicate q values < 0 . 5 .

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Figure 5RNA-seq of an SMC3-3HA-glmS clone shows smc3 transcript levels (FPKM) at 12 (blue, q = 8.5 x 10-3), 24 (coral, q = 1.3 x 10-39), and 36 (green, q = 4.1 x 10-54) hpi in the absence (filled circles) or presence (empty circles) of glucosamine (GlcN). Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites, for which there were two replicates. P-values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 q = Bonferroni corrected P-value. Asterisks indicate q values < 0.5.


Figure 6ChIP - seq data showing enrichment of SMC3 ( y - axis = ChIP / Input ) at 12 ( blue ) , 24 ( coral ) , and 36 ( green ) hpi in clonal SMC3 - 3HA - glmS parasites at the rhoptry - associated protein 2 ( rap2 , PF3D7 _ 0501600 ) and the glideosome - associated protein 45 ( gap45 , PF3D7 _ 1222700 ) gene loci . The x - axis is DNA sequence , with the gene represented by a black box with white arrowheads to indicate transcription direction . One replicate ( clone A ) was used for ChIP - seq . ATAC - seq data from closely corresponding time points ( 15 , 25 , and 35 hpi ) are shown in grey , with the y - axis representing ATAC - seq ( RPM ) / gDNA ( RPM ) . RNA - seq of an SMC3 - 3HA - glmS clone shows transcript levels ( FPKM ) for rap2 ( PF3D7 _ 0501600 ) at 12 ( q = 3 x 10 - 2 ) and 24 ( q = 3 . 3 x 10 - 2 ) hpi and gap45 ( PF3D7 _ 1222700 ) at 12 ( q = 8 x 10 - 1 ) and 24 ( q = 1 . 6 x 10 - 2 ) hpi in the absence ( black ) or presence ( grey ) of glucosamine ( GlcN ) . Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites , for which there were two replicates . P - values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 ( Love et al , 2014 ) . q = Bonferroni corrected P - value . Expression values of rap2 ( PF3D7 _ 0501600 ) and gap45 ( PF3D7 _ 1222700 ) genes across the IDC ( indicated on the x - axis by hpi ) from the transcriptomics time course Data corresponding to 12 ( blue ) , 24 ( coral ) , and 36 ( green ) hpi time points are highlighted .

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Figure 6ChIP-seq data showing enrichment of SMC3 (y-axis = ChIP/Input) at 12 (blue), 24 (coral), and 36 (green) hpi in clonal SMC3-3HA-glmS parasites at the rhoptry-associated protein 2 (rap2, PF3D7_0501600) and the glideosome-associated protein 45 (gap45, PF3D7_1222700) gene loci. The x-axis is DNA sequence, with the gene represented by a black box with white arrowheads to indicate transcription direction. One replicate (clone A) was used for ChIP-seq. ATAC-seq data from closely corresponding time points (15, 25, and 35 hpi) are shown in grey, with the y-axis representing ATAC-seq (RPM)/gDNA (RPM).RNA-seq of an SMC3-3HA-glmS clone shows transcript levels (FPKM) for rap2 (PF3D7_0501600) at 12 (q = 3 x 10-2) and 24 (q = 3.3 x 10-2) hpi and gap45 (PF3D7_1222700) at 12 (q = 8 x 10-1) and 24 (q = 1.6 x 10-2) hpi in the absence (black) or presence (grey) of glucosamine (GlcN). Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites, for which there were two replicates. P-values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected P-value.Expression values of rap2 (PF3D7_0501600) and gap45 (PF3D7_1222700) genes across the IDC (indicated on the x-axis by hpi) from the transcriptomics time course Data corresponding to 12 (blue), 24 (coral), and 36 (green) hpi time points are highlighted.


Figure 1A . Representative FISH images showing RAB13 RNA distribution in MDA - MB - 231 cells . Nuclei and cell outlines are shown in blue and green respectively . Arrows points to RAB13 RNA concentrated at protrusive regions . Boxed regions are magnified in the insets . B . Representative immunofluorescence images of RAB13 protein in cells transfected with the indicated siRNAs . Reduction of intensity in RAB13 knockdown cells confirms the specificity of the signal . Arrows point to perinuclear RAB13 protein . Calibration bar shows intensity values . C . Ratios of peripheral / perinuclear intensity calculated from images as shown in A and B . Bars : mean ± s . e . m . . Values within each bar represent number of cells observed in 3 independent experiments . D . Protrusions ( Ps ) and cell bodies ( CB ) of cells induced to migrate towards LPA were isolated and analyzed to detect the indicated proteins ( by Western blot ; left panels ) or RNAs ( by RT - ddPCR ; right panel ) . Ps / CB enrichment ratios from 2 independent experiments are shown . Bars : mean ± s . e . m . . The enrichment of pY397 - FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction .

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Figure 1A. Representative FISH images showing RAB13 RNA distribution in MDA-MB-231 cells. Nuclei and cell outlines are shown in blue and green respectively. Arrows points to RAB13 RNA concentrated at protrusive regions. Boxed regions are magnified in the insets.B. Representative immunofluorescence images of RAB13 protein in cells transfected with the indicated siRNAs. Reduction of intensity in RAB13 knockdown cells confirms the specificity of the signal. Arrows point to perinuclear RAB13 protein. Calibration bar shows intensity values. C. Ratios of peripheral/perinuclear intensity calculated from images as shown in A and B. Bars: mean ± s.e.m..Values within each bar represent number of cells observed in 3 independent experiments. D. Protrusions (Ps) and cell bodies (CB) of cells induced to migrate towards LPA were isolated and analyzed to detect the indicated proteins (by Western blot; left panels) or RNAs (by RT-ddPCR; right panel). Ps/CB enrichment ratios from 2 independent experiments are shown. Bars: mean ± s.e.m..The enrichment of pY397-FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction.


Figure 2B . FISH images of mouse fibroblasts expressing the β - globin coding sequence followed by the indicated UTRs . β - globin RNA is shown in yellow . Nuclei and cell outlines are shown in blue . Arrows points to β - globin RNA concentrated at protrusive regions . ∆ 1 and ∆ 1 + 2 indicate deletions of the regions shown in ( A ) . Scale bars : 10 μm . C . Distribution of β - globin RNA , or of Ddr2 RNA detected in the same cells , quantified by measuring a Peripheral Distribution Index ( PDI ) . N = 35 - 55 cells observed in 3 independent experiments . Bars : mean ± 95 % CI . * * * * : p < 0 . 0001 by analysis of variance with Dunnett ' s multiple comparisons test .

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Figure 2B. FISH images of mouse fibroblasts expressing the β-globin coding sequence followed by the indicated UTRs. β-globin RNA is shown in yellow. Nuclei and cell outlines are shown in blue. Arrows points to β-globin RNA concentrated at protrusive regions. ∆1 and ∆1+2 indicate deletions of the regions shown in (A). Scale bars: 10 μm. C. Distribution of β-globin RNA, or of Ddr2 RNA detected in the same cells, quantified by measuring a Peripheral Distribution Index (PDI). N=35-55 cells observed in 3 independent experiments. Bars: mean ± 95% CI. ****:p<0.0001 by analysis of variance with Dunnett's multiple comparisons test.


Figure 3B . FISH images and corresponding PDI measurements of mouse fibroblast cells treated with the indicated PMOs . Cyb5r3 is an APC - dependent RNA also enriched at protrusions . Arrows : peripheral Rab13 RNA . Arrowheads : perinuclear Rab13 RNA . Boxed regions are magnified in the insets . Note that Rab13 RNA becomes perinuclear in cells treated with PMOs against the GA - rich region . Scale bars : 10 μm . ( 4 μm in insets ) . * * * * : p < 0 . 0001 by analysis of variance with Dunnett ' s multiple comparisons test . N = 40 - 90 cells observed in 3 - 6 independent experiments . Bars : mean ± 95 % CI . C . Protrusion ( Ps ) and cell body ( CB ) fractions were isolated from cells treated with control - PMO or Rab13 - PMO # 2 . The indicated RNAs were detected through nanoString analysis to calculate Ps / CB enrichment ratios ( n = 3 ; bars : mean ± s . e . m . ) . Note that only the distribution of Rab13 RNA is affected . * * : p = 0 . 01 by two - way ANOVA with Bonferroni ' s multiple comparisons test against the corresponding control . D . Levels of the indicated RNAs were determined using nanoString analysis from control - or Rab13 PMO # 2 - treated cells . ( n = 4 ; bars : mean ± s . e . m . ) . No significant differences were detected by two - way ANOVA against the corresponding controls .

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Figure 3B. FISH images and corresponding PDI measurements of mouse fibroblast cells treated with the indicated PMOs. Cyb5r3 is an APC-dependent RNA also enriched at protrusions. Arrows: peripheral Rab13 RNA. Arrowheads: perinuclear Rab13 RNA. Boxed regions are magnified in the insets. Note that Rab13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region. Scale bars: 10 μm. (4 μm in insets). ****: p<0.0001 by analysis of variance with Dunnett's multiple comparisons test. N=40-90 cells observed in 3-6 independent experiments. Bars: mean ± 95% CI.C. Protrusion (Ps) and cell body (CB) fractions were isolated from cells treated with control-PMO or Rab13-PMO #2. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n=3; bars: mean ± s.e.m.). Note that only the distribution of Rab13 RNA is affected. **: p=0.01 by two-way ANOVA with Bonferroni's multiple comparisons test against the corresponding control. D. Levels of the indicated RNAs were determined using nanoString analysis from control- or Rab13 PMO #2-treated cells. (n=4; bars: mean ± s.e.m.). No significant differences were detected by two-way ANOVA against the corresponding controls.


Figure 4A . Schematic showing % GA content and positions along the human RAB13 3 ' UTR targeted by the indicated PMOs . Red rectangles indicate the location of GA - rich motifs . Graphs present PDI measurements of RAB13 RNA ( upper panel ) or NET1 RNA ( another APC - dependent RNA ; bottom panel ) detected in MDA - MB - 231 cells treated with the indicated PMOs . PDI = 1 indicates a diffuse distribution . p - values : * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 by analysis of variance with Dunnett ' s multiple comparisons test . n . s . : non - significant . N = 30 - 73 cells observed in 3 - 5 independent experiments . Bars : mean ± s . e . m . . B . Representative FISH images of cells treated with the indicated PMOs . Arrows point to peripheral RNA . Arrowheads point to perinuclear RNA . Boxed regions are magnified in the insets . Note that RAB13 RNA becomes perinuclear in cells treated with PMOs against the GA - rich region , while NET1 remains localized at protrusions . Scale bars : 10 μm ( 4 μm in insets ) . C . RAB13 protein levels were measured by quantitative Western blot and normalized to total α - Tubulin or GAPDH levels ( for representative blot , see Figure 5E ) . Relative levels in RAB13 PMO - treated compared to control are shown . No significant differences were detected by Kruskal - Wallis test with Dunn ' s multiple comparisons test . N = 3 - 8 . Bars : mean ± SD .

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Figure 4A. Schematic showing %GA content and positions along the human RAB13 3'UTR targeted by the indicated PMOs. Red rectangles indicate the location of GA-rich motifs. Graphs present PDI measurements of RAB13 RNA (upper panel) or NET1 RNA (another APC-dependent RNA; bottom panel) detected in MDA-MB-231 cells treated with the indicated PMOs. PDI=1 indicates a diffuse distribution. p-values: **<0.01, ***<0.001, ****<0.0001 by analysis of variance with Dunnett's multiple comparisons test. n.s.: non-significant. N=30-73 cells observed in 3-5 independent experiments. Bars: mean ± s.e.m..B. Representative FISH images of cells treated with the indicated PMOs. Arrows point to peripheral RNA. Arrowheads point to perinuclear RNA. Boxed regions are magnified in the insets. Note that RAB13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region, while NET1 remains localized at protrusions. Scale bars: 10 μm (4 μm in insets).C. RAB13 protein levels were measured by quantitative Western blot and normalized to total α-Tubulin or GAPDH levels (for representative blot, see Figure 5E). Relative levels in RAB13 PMO-treated compared to control are shown. No significant differences were detected by Kruskal-Wallis test with Dunn's multiple comparisons test. N=3-8. Bars: mean ± SD.