( F ) of α - SMA in 3D organotypic co - cultures of PS - 1 indicated PDAC lines . Scale bar represents 100 µm .

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

(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.


( A ) Brightfield of PS - 1 which were exposed for 72 hrs to CM from control ( CM of PS - 1 ) , epithelial ( blue ) or mesenchymal ( red ) lines . Scale bar represents 100 µm .

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

(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 ) of bead - calibrated of PS - 1 after treatment using flowcytometry . n = 6 per group , 3 biological replicates for each .

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

(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 performed after treatment showing of PS - 1 in G0 / G1 phase , G2 / M phase S phase . n = 6 per group , 3 biological replicates for each . .

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

(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..


( A ) Gene expression level of stromal activation markers COL1A1 , ACTA2 , SPARC , FN1 FAP in PS - 1 which were exposed for 72 hrs to CM from control ( CM of PS - 1 ) , epithelial ( blue ) or mesenchymal ( red ) PDAC lines using qPCR . Values were normalized to control n = 6 per group , 3 biological replicates for each . Data information : data are represented as mean ± SD , Student ' s t - .

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

(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 ) representation of COL1A1 ACTA2 relative gene expression measured with qPCR in PS - 1 after treatment using a large cohort of PDAC lines cultures 67 53m . Values were Log2 transformed normalized to Suit - 2 expression . Data information : data are represented as mean ± SD , Student ' s t - .

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

(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 of PS - 1 exposed to 10 days of treatment with 1 10 µM - trans retinoic acid ( ATRA ) . Scale bar represents 100 µm .

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

(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 of COL1A1 ACTA2 in PS - 1 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 - .

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

(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 of human CAFs exposed to 72 hrs of treatment with CM from control ( CM of CAFs ) or mesenchymal PDAC lines . Scale bar represents 100 µm .

O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE B-DISEASE O O O O O O O O

(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 of COL1A1 ACTA2 in 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 - .

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

(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.


( A ) Brightfield of PS - 1 after 72 hrs of treatment with unfiltered , < 100 kDa or < 30 kDa filtered CM of indicated lines . Scale bar represents 100 µm .

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

(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 of COL1A1 ACTA2 in PS - 1 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 - .

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

(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 - chemokines present in CM of epithelial mesenchymal PDAC lines using forward - phase array . Significantly differential expression of ( triangles ) genes ( green triangles ) are identified between epithelial mesenchymal lines . Dotted indicates P = 0 . 05 . Data information : data are represented as mean ± SD , Student ' s t - .

O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of PS - 1 after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or epithelial PDAC supplemented with recombinant human CSF - 1 . Scale bar represents 100 µm .

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

(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 of COL1A1 in PS - 1 after treatment using qPCR . n = 3 biological replicates per group . Data information : data are represented as mean ± SD , Student ' s t - .

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

(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 were starved 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 same experiment blots were processed in parallel .

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

(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.


( A ) Brightfield of PS - 1 after 72 hrs of treatment with CM of control ( CM of PS - 1s ) or mesenchymal PDAC lines supplemented with anti - CSF - 1 . Scale bar represents 100 µm .

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

(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 of COL1A1 in PS - 1 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 - .

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

(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 of PS - 1 after 72 hrs of treatment with PANC - 1 CM of pLKOctrl or shCSF1 ( # 560 - 562 ) . Scale bar represents 100 µm .

O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O

(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 ) how these various CM affect COL1A1 expression in PS - 1 using qPCR , values were normalized for RPLPO expression . n = 3 biological replicates for experiments , error bars indicated SD . R2 P - value were analyzed with linear regression .

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

(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 of PS - 1 after 72 hrs of treatment with CM of Capan - 2 with control vector or Capan - 2 overexpressing CSF1 . Scale bar represents 100 µm .

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

(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 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 - .

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

(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 of COL1A1 in PS - 1 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 - .

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

(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.


( A ) of CSF - 1 in PDAC tissue following surgical resection representative image of CSF - 1 expression in grade 1 grade 3 PDAC is depicted . Scale bar represents 100 µm . ( B ) For each of panel A , CSF - 1 expression in adenocarcinoma was quantified . Student ' s t - . Shown is mean ± SD . n = 15 PDAC samples .

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

(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 ( n = 21 ) that underwent direct resection was stained for CSF - 1 ( scale bar represents 100 µm ) , PSR ( scale bar represents 500 µm ) α - SMA ( scale bar represents 200 µm ) . OK8 is low in CSF - 1 expression high in PSR α - SMA expression , while opposite accounts for OK7 . ( D ) Correlation of % of PSR in CSF - 1 expressing , as indicated in panel C . R2 P - value were analyzed with linear regression . n = 21 PDAC samples ( E ) Correlation of % of α - SMA in CSF - 1 expressing , as indicated in panel C , R2 P - value were analyzed with linear regression . n = 21 PDAC samples

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

(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


D . Immunoblot detection of indicated in whole extracts ( WCE ) , PrimPol immunoprecipitates ( IP ) , non - precipitated flow - through fractions ( FT ) . IgG shows presence of anti - PrimPol antibody in IP fractions ( lanes 4 - 6 ) .

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

D. Immunoblot detection of the indicated proteins in whole cell extracts (WCE), PrimPol immunoprecipitates (IP), and non-precipitated flow-through fractions (FT). IgG shows the presence of anti-PrimPol antibody in the IP fractions (lanes 4-6).


B . Confocal microscopy of PrimPol γH2AX ( damage control ) immunofluorescence in control ( UVA ) or TMP - UVA - irradiated ( 10 µM TMP , 2 h followed by UVA irradiation ) WT PRIMPOL KO . is counterstained with DAPI . UVA path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show average of PrimPol - positive SD of three assays ( n ≥ 100 per TMP - treated conditions in each replicate ) . Circle dots in each column represent values of replicates . Statistical was conducted with one - way ANOVA followed by Bonferroni post - . p - values of comparisons are indicated .

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

B. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-laser irradiated (10 µM TMP, 2 h followed by UVA laser irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


C . Confocal microscopy of PrimPol γH2AX ( damage control ) immunofluorescence in control ( UVA ) or TMP - UVA - treated following control or RPA2 downregulation . is counterstained with DAPI . UVA path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show average PrimPol - positive SD of three assays ( n ≥ 100 per TMP - treated conditions in each replicate ) . Circle dots in each column represent values of replicates . Statistical was conducted with one - way ANOVA followed by Bonferroni post - . p - values of comparisons are indicated .

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

C. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated cells following control or RPA2 downregulation. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average PrimPol-positive cells and SD of three assays (n ≥100 cells per TMP-treated conditions in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


D . Confocal microscopy of PrimPol γH2AX ( damage control ) immunofluorescence in control ( UVA ) or TMP - UVA - treated PRIMPOL KO stably expressing V5 - tagged WT or RPA binding domain mutant ( RBDm ) PrimPol versions . is counterstained with DAPI . UVA path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show average of PrimPol - positive SD of two assays ( n ≥ 100 per condition in each replicate ) . Circle dots in each column represent values of replicates . Statistical was conducted with one - way ANOVA followed by Bonferroni post - . p - values of comparisons are indicated .

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

D. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated PRIMPOL KO cells stably expressing V5-tagged WT or RPA binding domain mutant (RBDm) PrimPol versions. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of two assays (n ≥100 cells per condition in each replicate). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


E . Confocal microscopy of PrimPol γH2AX ( damage control ) immunofluorescence in control ( UVA ) or TMP - UVA - treated WT BLM KO upon downregulation of TOP3A , RMI1 RMI2 ( siTR ) when indicated . is counterstained with DAPI . UVA path is indicated . Scale bar , 10 µm . Histograms ( right panel ) show average of PrimPol - positive SD of three assays ( n ≥ 250 per condition ) . Circle dots in each column represent values of replicates . Statistical was conducted with one - way ANOVA followed by Bonferroni post - .

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

E. Confocal microscopy images of PrimPol and γH2AX (damage control) immunofluorescence staining in control (UVA) or TMP-UVA-treated WT and BLM KO cells upon downregulation of TOP3A, RMI1 and RMI2 (siTR) when indicated. Nuclear DNA is counterstained with DAPI. UVA laser path is indicated. Scale bar, 10 µm. Histograms (right panel) show the average percentage of PrimPol-positive cells and SD of three assays (n ≥250 cells per condition). Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test.


H . Immunoblots showing of BLM , FANCD2 PrimPol after BLM or FANCD2 downregulation in WT PRIMPOL KO for experiments in ( G ) . Ponceau - S is shown as loading control .

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

H. Immunoblots showing the levels of BLM, FANCD2 and PrimPol after BLM or FANCD2 downregulation in WT and PRIMPOL KO cells for experiments in (G). Ponceau-S is shown as loading control.


C . Representative confocal microscopy of EdU in WT PRIMPOL KO , 8 h after UVA ( control ) or TMP - UVA treatment ( 2 µM TMP , 2 h followed by 5 s irradiation ) . is counterstained with DAPI . When indicated , V5 - PrimPol was reintroduced into KO . Scale bar , 25 µm . Dot plots indicate distribution of EdU , mean values are indicated by horizontal red lines . Data from combination of three replicates ( n ≥ 200 per condition ) . Statistical was conducted with Kruskal - Wallis Dunns post - . p - values of comparisons are indicated .

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

C. Representative confocal microscopy images of EdU staining in WT and PRIMPOL KO cells, 8 h after UVA (control) or TMP-UVA treatment (2 µM TMP, 2 h followed by 5 s irradiation). Nuclear DNA is counterstained with DAPI. When indicated, V5-PrimPol was reintroduced into KO cells. Scale bar, 25 µm. Dot plots indicate the distribution of nuclear EdU intensity, and the mean values are indicated by horizontal red lines. Data from the combination of three replicates (n ≥200 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunns post-test. p-values of individual comparisons are indicated.


A . Confocal microscopy of FANCD2 immunofluorescence in control ( UVA ) or TMP - UVA - treated ( 2 µM TMP , 2 h followed by 5 s irradiation ) WT PRIMPOL KO . is counterstained with DAPI . Scale bar , 25 µm .

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A. Confocal microscopy images of FANCD2 immunofluorescence staining in control (UVA) or TMP-UVA-treated (2 µM TMP, 2 h followed by 5 s irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.


D . Histograms show number of chromosomal aberrations per metaphase ( average ) in each condition . Data taken from three replicates are combined ( 50 metaphases per replicate ) . Statistical was conducted with one - way ANOVA followed by Bonferroni post - . p - values of comparisons are indicated .

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D. Histograms show the number of chromosomal aberrations per metaphase (average and SEM) in each condition. Data taken from three replicates are combined (50 metaphases per replicate). Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.


A . Kaplan - Meier survival curves of WT PRIMPOL KO mice after administration of a single dose of MMC ( 7 . 5 , 10 or 15 mg / kg ) . Dashed vertical lines represent median survival values for WT ( blue ) KO ( orange ) mice . Statistical was conducted with Log - rank ( Mantel - Cox ) ; p - value for highest dose of MMC ( 15 mg / kg ) is indicated .

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A. Kaplan-Meier survival curves of WT and PRIMPOL KO mice after the administration of a single dose of MMC (7.5, 10 or 15 mg/kg). Dashed vertical lines represent median survival values for WT (blue) and KO (orange) mice. Statistical analysis was conducted with Log-rank (Mantel-Cox) test; p-value for the highest dose of MMC (15 mg/kg) is indicated.


B . Haematoxylin - eosin ( H & E ) Ki - 67 immunohistochemistry ( ) stainings of bone marrow sections derived from WT PRIMPOL KO mice following administration of 10 mg / kg MMC . Control mice ( PBS ) were sacrificed when MMC - injected mice reached humane endpoint . Scale bar , 2 . 5 mm . In each image , a relevant is shown at higher magnification ( 10x ) .

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B. Haematoxylin-eosin (H&E) and Ki-67 immunohistochemistry (IHC) stainings of bone marrow sections derived from WT and PRIMPOL KO mice following the administration of 10 mg/kg MMC. Control mice (PBS) were sacrificed when MMC-injected mice reached the humane endpoint. Scale bar, 2.5 mm. In each image, a relevant area is shown at higher magnification (10x).


E . IF detection of FANCD2 foci in WT PRIMPOL KO 48 h after TMP - UVA ( 2 µM TMP , 2 h followed by 5 s irradiation ) with or without MCM3 downregulation . is counterstained with DAPI . Scale bar , 25 µm .

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E. IF detection of FANCD2 foci in WT and PRIMPOL KO cells 48 h after TMP-UVA (2 µM TMP, 2 h followed by 5 s irradiation) with or without MCM3 downregulation. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm.


G . survival assays of WT PRIMPOL KO with or without MCM3 downregulation in presence of increasing amounts of MMC or TMP ( followed by 90 sec UVA irradiation ) for 72 h . same control curves without MCM3 downregulation are also shown as part of Figure EV6D . Each point in curve represents of surviving ( average SD of three assays for MMC two assays for TMP ) . Statistical was conducted with two - way ANOVA followed by Bonferroni post - . p - values of comparisons between KO KO - siMCM3 are indicated .

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G. Cell survival assays of WT and PRIMPOL KO cells with or without MCM3 downregulation in the presence of increasing amounts of MMC or TMP (followed by 90 sec UVA irradiation) for 72 h. The same control curves without MCM3 downregulation are also shown as part of Figure EV6D. Each point in the curve represents the percentage of surviving cells (average and SD of three assays for MMC and two assays for TMP). Statistical analysis was conducted with two-way ANOVA followed by Bonferroni post-test. p-values of comparisons between KO and KO-siMCM3 are indicated.


A ) Representative zooms of WT MEFs transfected with GFP - tagged Miro1 Miro2 ( GFPMiro1 GFPMiro2 ) . Control is GFP fused to first 70 of Tom70 . Tom20 catalase stain mitochondria peroxisomes , respectively . Merge is of Miro ( green ) catalase ( magenta ) with colocalisation shown as . arrowheads highlight an where an isolated peroxisome is situated . B ) of extent of GFP signal on peroxisomes . Data information : B ) One - way ANOVA with Newman - Keuls post hoc was used for comparisons ( n = 18 per condition over 3 independent experiments ) . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 in comparison to Control , respectively , # # # # # denote p < 0 . 01 p < 0 . 001 in comparison to GFPMiro1 , respectively . Data are represented as mean ± . Scale bar is 5 μm .

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

A) Representative zooms of WT MEFs transfected with GFP-tagged Miro1 and Miro2 (GFPMiro1 and GFPMiro2). Control is GFP fused to the first 70 amino acids of Tom70. Tom20 and catalase stain mitochondria and peroxisomes, respectively. Merge is of Miro (green) and catalase (magenta) with colocalisation shown as white. White arrowheads highlight an area where an isolated peroxisome is situated. B) Quantification of the extent of GFP signal on peroxisomes. Data information: B) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.


C ) Pulldown of overexpressed Pex19 in Cos7 with a Pex19 antibody shows interaction with full - Miro1 , but not Miro1 lacking transmembrane domain .

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C) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full-length Miro1, but not Miro1 lacking the transmembrane domain.


E ) Representative of Miro1 truncation constructs expressed in DKO MEFs . Mitochondria peroxisomes are stained with Tom20 catalase , respectively . arrowheads highlight an where an isolated peroxisome is situated .

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E) Representative images of Miro1 truncation constructs expressed in DKO MEFs. Mitochondria and peroxisomes are stained with Tom20 and catalase, respectively. White arrowheads highlight an area where an isolated peroxisome is situated.F


. F ) of extent of peroxisomal localisation of Miro1 truncation constructs . Data information : F ) One - way ANOVA with Newman - Keuls post hoc was used for comparisons ( n = 18 per condition over 3 independent experiments ) . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 in comparison to Control , respectively , # # # # # denote p < 0 . 01 p < 0 . 001 in comparison to GFPMiro1 , respectively . Data are represented as mean ± . Scale bar is 5 μm .

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.F) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs. Data information: F) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.


G ) Co - immunoprecipation of Miro1 truncation constructs mycPex19 following transfection into Cos7 . GFP - tagged Miro1 truncation constructs were pulled down with GFP - Trap agarose beads Pex19 was probed with myc antibody .

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G) Co-immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells. GFP-tagged Miro1 truncation constructs were pulled down with GFP-Trap agarose beads and Pex19 was probed with myc antibody.


A ) Six consecutive frames of live trafficking of peroxisomes ( pxDsRed signal at 1 . 5 seconds per frame ) . Yellow arrows show trajectory of a fast - moving peroxisome in both WT DKO MEFs . Scale bar is 5 μm .

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A) Six consecutive frames of live trafficking of peroxisomes (pxDsRed signal at 1.5 seconds per frame). Yellow arrows show the trajectory of a fast-moving peroxisome in both WT and DKO MEFs. Scale bar is 5 μm.


B ) Blind scoring of number of long - range peroxisomal trafficking events in WT , Miro1KO Miro2KO DKO MEFs ( n = 42 over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For B ) a Kruskall - Wallis with a Dunn ' s correction posthoc was used to for significance . For B ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± .

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B) Blind scoring of the number of long-range peroxisomal trafficking events in WT, Miro1KO and Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For B) a Kruskall-Wallis with a Dunn's correction posthoc test was used to test for significance. For B) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


C ) D ) of track velocity , respectively ( n = 24 over three independent experiments ) . Data information : for C ) , D ) is a one - way ANOVA with Newman - Keuls post - hoc . * * denotes p < 0 . 01 . For C ) , D ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± .

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C) and D) Quantification of individual track length and velocity, respectively (n=24 cells over three independent experiments). Data information: Analysis for C), D) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For C), D) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


E ) Representative blots of Miro1 , Miro2 actin from whole lysates of wild - type Miro1 - floxed ERT Cre - recombinase MEFs treated with without 4 - OH tamoxifen for 48 hours . Lysates were taken one day after end of treatment .

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E) Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild-type and Miro1-floxed ERT Cre-recombinase MEFs treated with and without 4-OH tamoxifen for 48 hours. Lysates were taken one day after the end of treatment.


F ) of long - range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two - minute movie ( n = 18 per condition over three independent experiments ) . Data information : Statistical significance in F ) was calculated by two - way ANOVA . For F ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± .

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F) Quantification of long-range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two-minute movie (n=18 cells per condition over three independent experiments). Data information: Statistical significance in F) was calculated by two-way ANOVA. For F) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


G ) Representative blot of Miro1 Miro2 following knockdown of either Miro1 or Pex14 in HeLa .

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G) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.


H ) of long - range peroxisomal trafficking events ( visualised with pxGFP ) in HeLa transfected with scrambled , Miro1 Pex14 siRNA , by blind scoring of a five - minute movie ( n = 18 over three independent experiments ) . Data information : For H ) is a one - way ANOVA with Newman - Keuls post - hoc . * * denotes p < 0 . 01 . For H ) , no statistical difference between conditions was observed , unless stated . Data are represented as mean ± .

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

H) Quantification of long-range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a five-minute movie (n=18 cells over three independent experiments). Data information: For H) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


A ) Representative of WT , Miro1KO , Miro2KO DKO MEFs seeded onto Y - shaped fibronectin micropatterns stained for mitochondria ( Tom20 in red ) peroxisomes ( Catalase in ) . Scale bar represents 10 μm . Data information : n = 60 over three independent experiments .

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A) Representative images of WT, Miro1KO, Miro2KO and DKO MEFs seeded onto Y-shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (Catalase in white). Scale bar represents 10 μm. Data information: n = 60 cells over three independent experiments.


B ) Normalised cumulative distribution of mitochondria in WT , Miro1KO , Miro2KO DKO MEFs . C ) at which 95 % of mitochondria are distributed ( Mito95 ) for four genotypes of MEF . Data information : n = 60 over three independent experiments . For C ) one - way ANOVA with Newman - Keuls post - hoc was used to for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± .

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B) Normalised cumulative distribution of mitochondria in WT, Miro1KO, Miro2KO and DKO MEFs. C) Distance at which 95% of mitochondria are distributed (Mito95) for all four genotypes of MEF. Data information: n = 60 cells over three independent experiments. For C) one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


D ) Normalised cumulative distribution curves comparing peroxisomal distribution from centre of to periphery of WT , Miro1KO , Miro2KO DKO MEFs . E ) Bar graph comparing average at which 95 % of peroxisomal signal is distributed ( Perox95 ) . Data information : n = 60 over three independent experiments . For E ) a one - way ANOVA with Newman - Keuls post - hoc was used to for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± .

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D) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1KO, Miro2KO and DKO MEFs. E) Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox95). Data information: n = 60 cells over three independent experiments. For E) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


A ) Snapshots tracks of pxDsRed signal in WT , Miro1KO , Miro2KO DKO MEFs following live imaging at 1 . 5 seconds per frame for two minutes . B ) of median net displacement of peroxisomes ( pxDsRed signal ) for WT , Miro1KO , Miro2KO DKO MEFs ( n = 42 over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For multiple comparisons in B ) a one - way ANOVA with Newman - Keuls post - hoc was used to for statistical significance .

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A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance.


C ) Still every 30 seconds of ER peroxisomes ( ER - DsRed pseudo - coloured to green pxGFP pseudo - coloured to magenta , respectively ) in WT DKO MEFs taken from two - minute movies by live - spinning - disk microscopy . Arrows track peroxisomes associated with an ER tubule . Scale bar is 5 μm .

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C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.


D ) of relative ER displacement over a two - minute movie using ER - DsRed signal in WT DKO MEFs ( n = 20 per condition over three independent experiments ) . Data information : Student ' s t - was used to calculate statistical significance . * * * * represent p < 0 . 05 p < 0 . 001 , respectively . Data are represented as mean ± .

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D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


E ) Median net displacement of peroxisomes in WT DKO MEFs . n = 20 per condition over three independent experiments .

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E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments.


F ) Median net displacement of peroxisomes in DKO MEFs with without myc - tagged variant 4 of Miro1 ( mycv4 ) ( n = 12 per condition over three independent experiments ) . Data information : Student ' s t - was used to calculate statistical significance . * * * * represent p < 0 . 05 p < 0 . 001 , respectively . Data are represented as mean ± .

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F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


A ) Representative of catalase ( peroxisomes ) in WT , Miro1KO , Miro2KO DKO MEFs .

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A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs.


B ) Bar graph comparing average peroxisomal of WT Miro1KO , Miro2KO DKO MEFs ( n = 60 per condition over three independent experiments ) . Data information : Calculation of statistical significance in B ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in B) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


C ) Comparison of total peroxisomal number between WT , Miro1KO , Miro2KO DKO MEFs . n = 60 per condition over three independent experiments . Data information : Calculation of statistical significance in C ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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

C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments. Data information: Calculation of statistical significance in C) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


D ) Representative of Control ( GFP - tagged 1 - 70 of Tom70 ) , GFPMiro1 GFPMiro2 in WT MEFs stained with catalase ( peroxisomes ) .

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

D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).


E ) Representative of Pex14 ( peroxisomes ) in DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) .

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

E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4).


F ) Bar graph comparing average peroxisomal of Control , GFPMiro1 GFPMiro2 overexpressing WT MEFs ( n = 60 per condition over three independent experiments ) . Data information : Calculation of statistical significance in F ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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

F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


G ) Comparison of average of peroxisomes between DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) ( n = 30 per condition over three independent experiments ) . Data information : Statistical used in G ) was a two - Student ' s t . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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

G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments). Data information: Statistical test used in G) was a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


A ) Representative of proximity - ligation ( PLA ) of Fis1 Drp1 in WT DKO MEFs . Scale bar is 10 μm . Red dots indicate interaction of Fis1 Drp1 . Blue is DAPI . B ) of dots from PLA of Fis1 Drp1 in WT DKO MEFs . Dotted indicates sum of Fis1 Drp1 single antibody controls ( n = 3 experiments ) . Data information : For B ) statistical significance was quantified by a two - Student ' s t . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± .

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

A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments). Data information: For B) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


C ) Representative of a Fis1 - Drp1 PLA in DKO MEFs expressing either GFP , GFPv1 ( GFP - tagged variant 1 of Miro1 ) or GFPv4 ( GFP - tagged variant 4 of Miro1 ) n = 30 per condition over three independent experiments ) . Scale bar is 10 μm . Red dots indicate interaction of Fis1 Drp1 . Blue is DAPI . D ) of PLA dots per between GFP , GFPv1 or GFPv4 transfected DKO MEFs ( n = 30 over three independent experiments ) . Data information : In D ) a one - way ANOVA with a Newman - Keuls post - hoc was used to calculate statistical significance . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± .

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

C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


E ) Western blot of Drp1 , Fis1 actin from WT DKO whole lysates . F ) of normalised band of Drp1 Fis1 in WT DKO MEFs ( n = 3 ) . Data information F ) , statistical significance was quantified by a two - Student ' s t .

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

E) Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates. F) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n=3). Data information F), statistical significance was quantified by a two-tailed Student's t test.


G ) Zooms of endogenous Drp1 , mitochondria ( Mitotracker ) peroxisomes ( PMP70 ) in WT DKO MEFs . Scale bar is 5 μm . H ) Integrated density of Drp1 signal on Mitotracker - positive PMP70 - negative structures in WT DKO MEFs . I ) Integrated density of Drp1 signal on PMP70 - positive Mitotracker - negative structures in WT DKO MEFs . Data information G - I n = 42 per condition over three independent experiments . For , H ) statistical significance was quantified by a two - Student ' s t . * , * * * * * denote p < 0 . 05 , p < 0 . 01 p < 0 . 001 , respectively . Data are represented as mean ± .

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

G) Zooms of endogenous Drp1, mitochondria (Mitotracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm. H) Integrated density of Drp1 signal on Mitotracker-positive and PMP70-negative structures in WT and DKO MEFs. I) Integrated density of Drp1 signal on PMP70-positive and Mitotracker-negative structures in WT and DKO MEFs. Data information G-I n=42 cells per condition over three independent experiments. For , H) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


( B - C ) Representative confocal their of Gb3 accumulation within lysosome , detected by - conjugated Shiga toxin , from WT , CLN3 - KO CLN7 - KO ARPE - 19 treated with PDMP or silenced for Gb3 synthase ( siGb3S ) ( * * * vs WT , ° ° ° vs DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(B-C) Representative confocal images and their quantification of Gb3 accumulation within the lysosome, detected by fluorescent-conjugated Shiga toxin, from WT, CLN3-KO and CLN7-KO ARPE-19 cells treated with PDMP or silenced for Gb3 synthase (siGb3S) (*** vs WT, °°° vs DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( A - B ) Representative confocal of Gb3 accumulation , revealed by STX , in brain sections from CLN7Δex2 mice at 3 7 . 5 months of compared with CLN7 WT mice . Scale bars : 80 µm .

O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-TISSUE O O B-GENEPROD B-ORGANISM O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O

(A-B) Representative confocal images of Gb3 accumulation, revealed by STX staining, in brain sections from CLN7Δex2 mice at 3 and 7.5 months of age compared with CLN7 WT mice. Scale bars: 80 µm.


( E ) Lipidomic of neurons isolated from CLN7 WT CLN7Δex2 mouse forebrain . Data are presented as mean ± SD , * * : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas )

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(E) Lipidomic analysis of neurons isolated from CLN7 WT and CLN7Δex2 mouse forebrain. Data are presented as mean ± SD, **: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (n= 3 biological replicas)


( A ) Representative confocal of SCMAS within lysosome of ARPE - 19 WT CLN3 KO upon depletion of Gb3S ( siGb3S ) , LacCer ( siLCS ) , GM3s ( siGM3S ) . ( * * * vs WT , ° ° ° vs CLN3 KO siSCR ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(A) Representative confocal images and quantification of SCMAS staining within the lysosome of ARPE-19 WT and CLN3 KO upon depletion of Gb3S (siGb3S), LacCer (siLCS), and GM3s (siGM3S). (*** vs WT, °°° vs CLN3 KO siSCR). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( D ) Representative confocal of STX within lysosome of ARPE - 19 WT CLN7 KO in DMSO or treated 48h with Tamoxifen . ( * * * vs WT , ° ° ° vs CLN3 KO DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(D) Representative confocal images and quantification of STX staining within the lysosome of ARPE-19 WT and CLN7 KO in DMSO or treated 48h with Tamoxifen. (*** vs WT, °°° vs CLN3 KO DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( E ) Representative confocal of STX within lysosome of NPCs WT derived from CLN7Pa474 IPSCs in DMSO or treated with Tamoxifen for 48h . ( * * * vs WT , ° ° ° vs CLN7Pa474 DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(E) Representative confocal images and quantification of STX staining within the lysosome of NPCs WT and derived from CLN7Pa474 patient IPSCs in DMSO or treated with Tamoxifen for 48h. (*** vs WT, °°° vs CLN7Pa474 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( A ) Representative confocal of STX within lysosome in U2 - OS HeLa after acute silencing of CLN3 ( siCLN3 ) in DMSO or treated 48h with Tamoxifen . ( * * * vs siSCR DMSO , ° ° ° vs siCLN3 DMSO ) . Data are presented as mean ± SD , ° ° ° / * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(A) Representative confocal images and Quantification of STX within the lysosome in U2-OS and HeLa cells after acute silencing of CLN3 (siCLN3) in DMSO or treated 48h with Tamoxifen. (*** vs siSCR DMSO, °°° vs siCLN3 DMSO). Data are presented as mean ± SD, °°°/***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( B ) Representative confocal image of Gb3 in ARPE - 19 CLN3 KO silenced with siRNA against scramble TFEB ( siTFEB ) for 72h treated for last 48h with DMSO or Tamoxifen . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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(B) Representative confocal image and quantification of Gb3 in ARPE-19 CLN3 KO cells silenced with siRNA against scramble sequence and TFEB (siTFEB) for 72h and treated for the last 48h with DMSO or Tamoxifen. Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( C ) Representative confocal image of TFEB in U2 - OS silenced with siRNA against scramble CLN3 ( siCLN3 ) for 72h treated for last 48h with DMSO or Tamoxifen ( 5 µM 10 µM ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(C) Representative confocal image and quantification of TFEB in U2-OS cells silenced with siRNA against scramble sequence and CLN3 (siCLN3) for 72h and treated for the last 48h with DMSO or Tamoxifen (5 µM and 10 µM). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


( C ) Representative confocal image of TFEB localization in HeLa TFEB - GFP transfected with RagC for 48h treated for last 3h with DMSO , Torin1 , Tamoxifen or Ospemifene . Ratios of to TFEB localization in RagC non - expressing ( RagC - ) RagC - expressing ( RagC + ) are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

(C) Representative confocal image and quantification of TFEB localization in HeLa TFEB-GFP transfected with RagC for 48h and treated for the last 3h with DMSO, Torin1, Tamoxifen or Ospemifene. Ratios of nuclear to cytosolic TFEB localization in RagC non-expressing (RagC-) and RagC-expressing cells (RagC+) are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


D ) Representative confocal of STX in ARPE - 19 CLN3 KO transfected with empty vector or HA - RagC for 48h treated for 48h with DMSO or Tamoxifen . STX average spot presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( n = 3 biological replicas in duplicate ) . Scale bars : 20 µm .

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

D) Representative confocal images and quantification of STX in ARPE-19 CLN3 KO cells transfected with empty vector or HA-RagC for 48h and treated for 48h with DMSO or Tamoxifen. STX average spot area presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (n=3 biological replicas in duplicate). Scale bars: 20 µm.


Representative confocal of STX in Cortex , Hippocampus , Cerebellum brain section derived from 7 . 5 months - old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen ( Tamox ) . of confocal , plot shows of STX average spot normalized for number of Hoechst positive . ( * * * vs WT , ° ° ° / ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * / oo : P ≤ 0 . 001 , * * * / ooo : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 60 µm .

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

Representative confocal images of STX in the Cortex, Hippocampus, and Cerebellum brain section derived from 7.5 months-old mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen (Tamox). Quantification of confocal images, the plot shows the quantification of the STX average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°°/°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, **/oo: P ≤ 0.001, ***/ooo: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 60 µm.


Representative confocal of SCMAS in Cortex , Hippocampus , Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen . of confocal , plot shows of SCMAS average spot normalized for number of Hoechst positive . ( * * * vs WT , ° ° ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological ) . Scale bars : 60 µm .

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Representative confocal images of SCMAS in the Cortex, Hippocampus, and Cerebellum brain section derived from mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen. Quantification of confocal images, the plot shows the quantification of the SCMAS average spot area normalized for the number of Hoechst positive cells. (*** vs WT, °°° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological). Scale bars: 60 µm.


( A - B ) Representative confocal of IBA - 1 in Cortex , Hippocampus , Cerebellum brain section derived from WT or CLN7Δex2 mice injected with vehicle or Tamoxifen . ( * * * / * * / * vs WT , ° ° ° / ° ° / ° vs CLN7Δex2 Vehicle ) . Data are presented as mean ± SD , * / o : P ≤ 0 . 01 , * * / oo : P ≤ 0 . 001 , * * * : P ≤ 0 . 0001 , as determined by ANOVA ( N ≥ 3 biological replicas ) . Scale bars : 50 µm .

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(A-B) Representative confocal images and quantification of IBA-1 in the Cortex, Hippocampus, and Cerebellum brain section derived from WT or CLN7Δex2 mice injected with the vehicle or Tamoxifen. (***/**/* vs WT, °°°/°°/° vs CLN7Δex2 Vehicle). Data are presented as mean ± SD, */o: P ≤ 0.01, **/oo: P ≤ 0.001, ***: P ≤ 0.0001, as determined by ANOVA (N≥3 biological replicas). Scale bars: 50 µm.


( D ) Representative of hindlimb clasping in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen .

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(D) Representative Images of hindlimb clasping test in mouse WT or CLN7Δex2 injected with vehicle or Tamoxifen.


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) control mice were infected for 2 months with H . pylori . A Representative H & E from antral ( upper row ) . dotted marks base , which expands upon infection in Lgr5 - / - control mice . Immunofluorescence labeling for GSII Ki67 ( middle row ) GIF Muc5ac ( lower row ) of antral epithelium from Lgr5 - / - control mice uninfected after 2 months of infection . Scale bars : 50 µm .

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Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. A Representative H&E staining from antral glands (upper row). The dotted line marks the gland base cell area, which expands upon infection in Lgr5-/- and control mice. Immunofluorescence labeling for GSII and Ki67 (middle row) and GIF and Muc5ac (lower row) of antral epithelium from Lgr5-/- and control mice uninfected and after 2 months of infection. Scale bars: 50 µm.


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) control mice were infected for 2 months with H . pylori . B of antral height in Lgr5 - / - compared to control mice . Uninfected control ( n = 5 ) versus uninfected Lgr5 - / - ( n = 4 ) versus infected control ( n = 4 ) versus infected Lgr5 - / - ( n = 4 ) . Data information : B , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison for B ,

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Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. B Quantification of antral gland height in Lgr5-/- compared to control mice. Uninfected control (n = 5) versus uninfected Lgr5-/- (n = 4) versus infected control (n = 4) versus infected Lgr5-/- (n = 4). Data information: B, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for B,


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) control mice were infected for 2 months with H . pylori . C Quantifications of GSII + ( n = 4 - 5 biological replicates per group , n = 10 per mouse ) , Ki67 as of total DAPI - stained ( n = 3 - 4 biological replicates per group , n = 20 per mouse ) , GIF + ( n = 4 - 5 biological replicates per group , n = 10 per mouse ) , Muc5ac + ( n = 4 - 5 biological replicates per group , n = 10 per mouse ) . Data information : For ( one - way ANOVA + Tukey ´ s multiple comparison for C ,

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Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Ki67 as percentage of total DAPI-stained cells (n = 3-4 biological replicates per group, n = 20 glands per mouse), GIF+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse). Data information: For (one-way ANOVA + Tukey´s multiple comparison test for C,


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) control mice were infected for 2 months with H . pylori . D H . pylori CFUs in Lgr5 - / - ( n = 5 ) control mice ( n = 5 ) . Data information : For D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant t - for

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Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. D H. pylori CFUs in Lgr5-/- (n = 5) and control mice (n = 5). Data information: For D, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant t-test for D


Lgr5fl / fl / Axin2CreErt2 ( Lgr5 - / - ) control mice were infected for 2 months with H . pylori . E qPCR expression of Lgr4 , Lgr5 , Axin2 , base markers ( Gif , Pgc , Tff2 , Muc6 ) , pit marker Muc5ac , Itln1 in uninfected Lgr5 - / - ( n = 6 - 10 ) control mice ( n = 4 - 10 ) . F qPCR expression of Lgr4 , Lgr5 , base markers ( Gif , Pgc , Muc6 ) , Itln1 in Lgr5 - / - ( n = 5 ) control mice ( n = 5 ) after 2 months of infection . Data information : For E F , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; ns = not significant t - for E F ) .

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Lgr5fl/fl/Axin2CreErt2 (Lgr5-/-) and control mice were infected for 2 months with H. pylori. E qPCR expression levels of Lgr4, Lgr5, Axin2, gland base cell markers (Gif, Pgc, Tff2, Muc6), pit cell marker Muc5ac, and Itln1 in uninfected Lgr5-/- (n = 6-10) and control mice (n = 4-10). F qPCR expression levels of Lgr4, Lgr5, gland base cell markers (Gif, Pgc, Muc6), and Itln1 in Lgr5-/- (n = 5) and control mice (n = 5) after 2 months of infection. Data information: For E and F, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; ns = not significant t-test for E and F).


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) control mice were infected for 2 months with H . pylori . A Representative H & E from antral ( upper row ) . dotted marks base , which does not expand in infected Lgr4 - / - but does expand in corresponding control mice . Immunofluorescence labeling for GSII Ki67 ( middle row ) GIF Muc5ac ( bottom row ) of antral epithelium from Lgr4 - / - control mice uninfected after 2 months of infection . Scale bars : 50 µm .

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Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. A Representative H&E staining from antral glands (upper row). The dotted line marks the gland base cell area, which does not expand in infected Lgr4-/- but does expand in the corresponding control mice. Immunofluorescence labeling for GSII and Ki67 (middle row) and GIF and Muc5ac (bottom row) of antral epithelium from Lgr4-/- and control mice uninfected and after 2 months of infection. Scale bars: 50 µm.


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) control mice were infected for 2 months with H . pylori . B of antral height in Lgr4 - / - compared to control mice . Uninfected control ( n = 2 ) versus uninfected Lgr4 - / - ( n = 4 ) versus infected control ( n = 4 ) versus infected Lgr4 - / - ( n = 4 ) . Data information : For B , , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison for

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Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. B Quantification of antral gland height in Lgr4-/- compared to control mice. Uninfected control (n = 2) versus uninfected Lgr4-/- (n = 4) versus infected control (n = 4) versus infected Lgr4-/- (n = 4). Data information: For B, , data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for B


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) control mice were infected for 2 months with H . pylori . C Quantifications of GSII + ( n = 2 - 4 biological replicates per group , n = 10 per mouse ) , Ki67 as of total DAPI - stained ( n = 2 - 4 biological replicates per group , n = 20 per mouse ) , GIF + ( n = 2 - 4 biological replicates per group , n = 10 per mouse ) , Muc5ac + ( n = 4 - 5 biological replicates per group , n = 10 per mouse ) . Data information : For C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant ( one - way ANOVA + Tukey ´ s multiple comparison for C ,

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Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. C Quantifications of GSII+ area (n = 2-4 biological replicates per group, n = 10 glands per mouse), Ki67 as percentage of total DAPI-stained cells (n = 2-4 biological replicates per group, n = 20 glands per mouse), GIF+ area (n = 2-4 biological replicates per group, n = 10 glands per mouse), Muc5ac+ area (n = 4-5 biological replicates per group, n = 10 glands per mouse). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant (one-way ANOVA + Tukey´s multiple comparison test for C,


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) control mice were infected for 2 months with H . pylori . D H . pylori CFUs show higher colonization in Lgr4 - / - ( n = 5 ) compared to control mice ( n = 4 ) . Data information : For D , , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant t - for

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Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. D H. pylori CFUs show higher colonization in Lgr4-/- (n = 5) compared to control mice (n = 4). Data information: For D, , data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant t-test for D


Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) control mice were infected for 2 months with H . pylori . E qPCR expression of Lgr4 , Lgr5 , Axin2 , base markers ( Gif , Pgc , Tff2 , Muc6 ) , pit marker Muc5ac , Itln1 in uninfected Lgr4 - / - ( n = 4 - 10 ) control mice ( n = 4 - 6 ) . F qPCR expression of Lgr4 , Lgr5 , base markers ( Gif , Pgc , Muc6 ) , Itln1 in Lgr4 - / - ( n = 5 ) control mice ( n = 5 ) after 2 months of infection . Data information : For E F , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ; ns = not significant t - for E F ) .

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Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) and control mice were infected for 2 months with H. pylori. E qPCR expression levels of Lgr4, Lgr5, Axin2, gland base cell markers (Gif, Pgc, Tff2, Muc6), pit cell marker Muc5ac, and Itln1 in uninfected Lgr4-/- (n = 4-10) and control mice (n = 4-6). F qPCR expression levels of Lgr4, Lgr5, gland base cell markers (Gif, Pgc, Muc6), and Itln1 in Lgr4-/- (n = 5) and control mice (n = 5) after 2 months of infection. Data information: For E and F, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001; ns = not significant t-test for E and F).


A , B GSEA comparing microarray data from 2 - month infected Lgr4fl / fl / Axin2CreErt2 ( Lgr4 - / - ) mice with publicly available data demonstrating downregulation of antral signature ( A ) beta - catenin targets ( B ) .

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A, B GSEA comparing microarray data from 2-month infected Lgr4fl/fl/Axin2CreErt2 (Lgr4-/-) mice with publicly available data demonstrating downregulation of antral stem cell signature (A) and beta-catenin targets (B).


C qPCR expression for beta catenin targets Axin2 Sox9 in uninfected infected Lgr4 - / - control mice ( n = 2 - 9 ) . Data information : For C , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison ) .

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C qPCR expression levels for beta catenin targets Axin2 and Sox9 in uninfected and infected Lgr4-/- and control mice (n = 2-9). Data information: For C, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).


D GSEA comparing microarray data from 2 - month infected Lgr4 - / - mice with publicly available data demonstrating downregulation of TNF - α signaling via NF - κB .

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D GSEA comparing microarray data from 2-month infected Lgr4-/- mice with publicly available data demonstrating downregulation of the TNF-α signaling via NF-κB.


E Immunofluorescence labeling for p65 , Ki67 GSII in mouse antrum tissue from uninfected infected Lgr4 - / - control mice . Insets show magnified . Scale bars : 50 µm .

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E Immunofluorescence labeling for p65, Ki67 and GSII in the mouse antrum tissue from uninfected and infected Lgr4-/- and control mice. Insets show magnified images. Scale bars: 50 µm.


F of p65 + as of total DAPI - stained of uninfected infected Lgr4 - / - control mice ( n = 4 biological replicates per group , n = 20 per mouse ) . Data information : For data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison ) .

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F Quantification of p65+ area as percentage of total DAPI-stained cells of uninfected and infected Lgr4-/- and control mice (n = 4 biological replicates per group, n = 20 glands per mouse). Data information: For data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).


G Immunofluorescence co - localization of p65 acetyl K310 ( activated form of NF - κB ) in mouse antrum tissue of infected Lgr4 - / - in control mice . Insets show magnified . Scale bars : 50µm . Data information : For G represent findings that were reproduced at least twice in laboratory in independent biological replicates .

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G Immunofluorescence co-localization of p65 acetyl K310 (activated form of NF-κB) in the mouse antrum tissue of infected Lgr4-/- and in control mice. Insets show magnified images. Scale bars: 50µm. Data information: For G images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.


H qPCR expression of Cxcl1 , Cxcl2 , Cxcl10 , Ccl2 in uninfected infected Lgr4 - / - mice ( n = 4 - 5 ) control mice ( n = 2 - 5 ) . Data information : For H , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( one - way ANOVA + Tukey ´ s multiple comparison ) .

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H qPCR expression levels of Cxcl1, Cxcl2, Cxcl10, Ccl2 in uninfected and infected Lgr4-/- mice (n = 4-5) and control mice (n = 2-5). Data information: For H, data are mean + SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-way ANOVA + Tukey´s multiple comparison test).


I Immunofluorescence labeling for Cxcl1 in mouse antrum tissue in infected Lgr4 - / - in control mice . Scale bars : 50 µm . Data information : For I represent findings that were reproduced at least twice in laboratory in independent biological replicates .

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I Immunofluorescence labeling for Cxcl1 in the mouse antrum tissue in infected Lgr4-/- and in control mice. Scale bars: 50 µm. Data information: For I images represent findings that were reproduced at least twice in the laboratory in independent biological replicates.


A Immunofluorescence labeling for p65 E - cadherin in mouse antrum tissue of 2 infected B6 - Myh11CreErt2 / Rspondin3fl / fl ( Rspo3 - / - ) control mice . Arrows in infected control mice indicate p65 that is not observed in Rspo3 - / - mice . Scale bars : 50 µm .

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A Immunofluorescence labeling for p65 and E-cadherin in the mouse antrum tissue of 2 weeks infected B6-Myh11CreErt2/Rspondin3fl/fl (Rspo3-/-) and control mice. Arrows in infected control mice indicate nuclear p65 that is not observed in Rspo3-/- mice. Scale bars: 50 µm.


B of p65 + as of total DAPI - stained of infected Rspo3 - / - control mice ( n = 3 biological replicates per group , n = 20 per mouse ) . Data information : For B , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - for

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B Quantification of p65+ area as percentage of total DAPI-stained cells of infected Rspo3-/- and control mice (n = 3 biological replicates per group, n = 20 glands per mouse). Data information: For B, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for B


C qPCR expression level of Cxcl1 Cxcl2 in 2 - week infected Rspo3 - / - mice ( n = 3 ) versus infected control mice ( n = 4 ) . D qPCR expression level of Cxcl1 Cxcl2 in 2 - month infected Rspo3 - / - mice ( n = 5 ) versus infected control mice ( n = 6 ) . Data information : For C , D , data are mean + SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; ns = not significant ( t - for C

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C qPCR expression level of Cxcl1 and Cxcl2 in 2-week infected Rspo3-/- mice (n = 3) versus infected control mice (n = 4). D qPCR expression level of Cxcl1 and Cxcl2 in 2-month infected Rspo3-/- mice (n = 5) versus infected control mice (n = 6). Data information: For C, D, data are mean + SD. * = p < 0.05; ** = p < 0.01; ns = not significant (t-test for C and D