( B ) Screen shots from the UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti - EZH2 antibody in LNCaP and C4 - 2 cells .

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(B) Screen shots from the UCSC genome browser showing signal profiles of p53 mRNA immunoprecipitated by anti-EZH2 antibody in LNCaP and C4-2 cells.


( C ) C4 - 2 cells were subjected to UV - RIP assay . RT - qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti - EZH2 antibody . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * P < 0 . 01 .

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(C) C4-2 cells were subjected to UV-RIP assay. RT-qPCR measurement of p53 mRNA immunoprecipitated by IgG or anti-EZH2 antibody. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test. ** P<0.01.


( E ) Top , RT - qPCR analysis of p53 mRNA in C4 - 2 cell lysate pulled down by GST or GST - EZH2 recombinant proteins EZ1 - EZ4 . Bottom , western blotting analysis of GST or GST - EZH2 proteins used for GST pull - down assay . Asterisks indicate the protein bands at expected molecular weight . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * P < 0 . 01 .

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(E) Top, RT-qPCR analysis of p53 mRNA in C4-2 cell lysate pulled down by GST or GST-EZH2 recombinant proteins EZ1-EZ4. Bottom, western blotting analysis of GST or GST-EZH2 proteins used for GST pull-down assay. Asterisks indicate the protein bands at expected molecular weight. Data shown as means ± SD (n=3). Statistical significance was determined by two-tailed Student's t-test.** P<0.01.


( F ) C4 - 2 cells were transfected with Myc - tagged EZH2 - WT or EZH2 - ∆ 336 - 554 for 24 h and cells were harvested for RIP with IgG or anti - Myc tag antibody . Transfected proteins and pull - down p53 mRNAs were analyzed by western blot and RT - qPCR , respectively . Data shown are means ± SD ( n = 3 ) . * P < 0 . 01 . ERK2 , a loading control .

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(F) C4-2 cells were transfected with Myc-tagged EZH2-WT or EZH2-∆336-554 for 24 h and cells were harvested for RIP with IgG or anti-Myc tag antibody. Transfected proteins and pull-down p53 mRNAs were analyzed by western blot and RT-qPCR, respectively. Data shown are means ± SD (n=3). * P<0.01. ERK2, a loading control.


( G ) GST pull - down assay using in vitro transcribed different fragments of p53 mRNA and indicated GST proteins followed by RT - qPCR analysis of pull - down p53 mRNA . FL , full length ; ORF , open reading frame ; UTR , untranslated region .

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(G) GST pull-down assay using in vitro transcribed different fragments of p53 mRNA and indicated GST proteins followed by RT-qPCR analysis of pull-down p53 mRNA. FL, full length; ORF, open reading frame; UTR, untranslated region.


( H , I ) RNA EMSA evaluation of EZh2 binding of p53 mRNA . GST - EZH2 recombinant proteins ( EZ1 to EZ4 ) were incubated with biotin - labeled in vitro transcribed p53 5 ' UTR ( biotin - labeled probe ) in the presence or absence of 100 - fold unlabeled p53 5 ' UTR ( unlabeled probe ) , followed by PAGE and immune blotting with HPR - conjugated streptavidin .

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(H, I) RNA EMSA evaluation of EZh2 binding of p53 mRNA. GST-EZH2 recombinant proteins (EZ1 to EZ4) were incubated with biotin-labeled in vitro transcribed p53 5'UTR (biotin-labeled probe) in the presence or absence of 100-fold unlabeled p53 5'UTR (unlabeled probe), followed by PAGE and immune blotting with HPR-conjugated streptavidin.


( A ) Biotin pull down assay by incubating biotin - labelled different fragments of p53 mRNA and HOTAIR ( positive control ) with C4 - 2 cell lysate followed by western blot with EZH2 antibody .

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(A) Biotin pull down assay by incubating biotin-labelled different fragments of p53 mRNA and HOTAIR (positive control) with C4-2 cell lysate followed by western blot with EZH2 antibody.


( B ) Biotin pull down assay as in ( A ) using unmutated and various internally deleted mutants of 5 ' UTR of p53 mRNA . Top , diagram of different p53 5 ' UTR deletion mutants .

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(B) Biotin pull down assay as in (A) using unmutated and various internally deleted mutants of 5'UTR of p53 mRNA. Top, diagram of different p53 5'UTR deletion mutants.


( C ) Upper , the linear map of the pRF bicistronic report plasmid . The SV40 promoter ( purple box ) was used to drive firefly luciferase ( Fluc ) and Renilla luciferase ( Rluc ) gene transcription . Different p53 5 ' UTR fragments were inserted between the Fluc and Rluc genes . Lower , at 24 h after transfection , cells were lysed and luciferase activities were measured using a dual - luciferase kit and the ratio of Fluc / Rluc was calculated . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - tailed Student ' s t - test . * P < 0 . 01 .

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(C) Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5'UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual-luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01.


( D ) EZH2 knockdown C4 - 2 cells were transfected with the bicistronic reporter vector in combination with empty vector , Myc - tagged EZH2 WT or deletion mutants followed by western blot analysis with indicated antibodies ( bottom ) and luciferase assay ( top ) . Data shown as means ± SD ( n = 3 ) . * P < 0 . 01 .

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(D) EZH2 knockdown C4-2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc-tagged EZH2 WT or deletion mutants followed by western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means±SD (n=3). * P<0.01.


( E ) C4 - 2 and U2OS cell lines were transfected with non - specific control ( siC ) or two independent EZH2 - specific siRNAs and harvested for western blot analysis with indicated antibodies . ERK2 and β - TUBULIN , loading controls .

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(E) C4-2 and U2OS cell lines were transfected with non-specific control (siC) or two independent EZH2-specific siRNAs and harvested for western blot analysis with indicated antibodies. ERK2 and β-TUBULIN, loading controls.


( F ) U2OS cells were transfected with control ( siC ) or EZH2 - specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins .

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(F) U2OS cells were transfected with control (siC) or EZH2-specific siRNAs and treated with 200 nM of CPT followed by western blot analysis for indicated proteins.


( G ) C4 - 2 cells were transfected with control ( siC ) or EZH2 - specific siRNAs and plasmids for empty vector , EZH2 WT or deletion mutants followed by western blot analysis for indicated proteins .

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(G) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs and plasmids for empty vector, EZH2 WT or deletion mutants followed by western blot analysis for indicated proteins.


( A ) List of top hits of EZH2 - bound proteins identified by TAP - MS . The number of peptides and coverage of each protein are indicated .

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(A) List of top hits of EZH2-bound proteins identified by TAP-MS. The number of peptides and coverage of each protein are indicated.


( B ) Reciprocal co - IP of endogenous EZH2 and eIF4G2 or PABP1 in C4 - 2 cells .

(B) Reciprocal co-IP of endogenous EZH2 and eIF4G2 or PABP1 in C4-2 cells.


( C ) Co - IP of endogenous EZH2 with eIF4G2 or PABP1 from C4 - 2 cell lysate pre - treated with RNaseA in 37oC for 30 min . The effectiveness of RNaseA treatment was monitored by RT - PCR analysis of the presence of GAPDH mRNA .

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(C) Co-IP of endogenous EZH2 with eIF4G2 or PABP1 from C4-2 cell lysate pre-treated with RNaseA in 37oC for 30 min. The effectiveness of RNaseA treatment was monitored by RT-PCR analysis of the presence of GAPDH mRNA.


( D ) C4 - 2 cells were transfected control ( siC ) or EZH2 - specific siRNA and lysed for polysome fractionation . RNA was extracted from even - number fractions followed by RT - qPCR analysis for p53 and GAPDH mRNA . The β - ACTIN mRNA was used as an internal control . Data shown as means ± SD ( n = 3 ) . Statistical significance was determined by two - tailed Student ' s t - test . * P < 0 . 01 ; NS , no significance .

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(D) C4-2 cells were transfected control (siC) or EZH2-specific siRNA and lysed for polysome fractionation. RNA was extracted from even-number fractions followed by RT-qPCR analysis for p53 and GAPDH mRNA. The β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). Statistical significance was determined by two-tailed Student's t-test. * P<0.01; NS, no significance.


( E ) C4 - 2 cells were transfected with control ( siC ) or EZH2 - specific siRNAs in combination with empty vector , EZH2 WT or different deletion mutants followed by polysome fractionation and RT - qPCR . β - ACTIN mRNA was used as an internal control . Data shown as means ± SD ( n = 3 ) . * P < 0 . 01 .

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(E) C4-2 cells were transfected with control (siC) or EZH2-specific siRNAs in combination with empty vector, EZH2 WT or different deletion mutants followed by polysome fractionation and RT-qPCR. β-ACTIN mRNA was used as an internal control. Data shown as means±SD (n=3). * P<0.01.


( A ) IHC analysis of Pten , Ezh2 and p53 proteins in prostatic tissues of 4 - month - old mice with different genotypes : Ptenpc - / - ( n = 11 ) ; Ezh2pc - / - ( n = 8 ) ; Ptenpc - / - ; Ezh2pc - / - ( n = 10 ) and ' wild - type ' littermate controls ( n = 10 ) . Scale bars ,

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(A) IHC analysis of Pten, Ezh2 and p53 proteins in prostatic tissues of 4-month-old mice with different genotypes: Ptenpc-/- (n=11); Ezh2pc-/- (n=8); Ptenpc-/-;Ezh2pc-/- (n=10) and 'wild-type' littermate controls (n=10). Scale bars, 50


( B ) Western blot analysis of expression of indicated proteins in four different groups of mice as described ( n = 2 mice / group ) .

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(B) Western blot analysis of expression of indicated proteins in four different groups of mice as described (n=2 mice/group).


( C ) Analysis of correlation of Ezh2 and p53 protein levels determined by IHC in Pten knockout ( Ptenpc - / - ) mice ( n = 11 ) .

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(C) Analysis of correlation of Ezh2 and p53 protein levels determined by IHC in Pten knockout (Ptenpc-/-) mice (n=11).


( D ) RT - qPCR analysis of p53 mRNA expression in four different genotypes of mice as described Statistical significance was determined by two - tailed Student ' s t - test . * P < 0 . 01 .

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(D) RT-qPCR analysis of p53 mRNA expression in four different genotypes of mice as described Statistical significance was determined by two-tailed Student's t-test. * P<0.01.


( E ) RT - qPCR analysis of mRNA expression of p53 downstream target gene Bax in four different genotypes of mice as indicated . Statistical significance was determined by two - tailed Student ' s t - test . * P < 0 . 01 .

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(E) RT-qPCR analysis of mRNA expression of p53 downstream target gene Bax in four different genotypes of mice as indicated. Statistical significance was determined by two-tailed Student's t-test. * P< 0.01.


( F ) H & E analysis of ventral prostate ( VP ) of 4 - month - old mice with indicated genotypes . The inset shows a high magnification image of the representative ( framed ) area in each panel . Scale bars , 50 µm . Scale bars in insets , 10 µm .

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(F) H&E analysis of ventral prostate (VP) of 4-month-old mice with indicated genotypes. The inset shows a high magnification image of the representative (framed) area in each panel. Scale bars, 50 µm. Scale bars in insets, 10 µm.


( G ) Quantification of nonmalignant , low - grade PIN ( LGPIN ) and high - grade PIN ( HGPIN ) or cancerous acini in the prostates ( including anterior prostate ( AP ) , ventral prostate ( VP ) and dorsolateral prostate ( DLP ) ) of mice with the indicated genotypes and number as in ( A ) .

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(G) Quantification of nonmalignant, low-grade PIN (LGPIN) and high-grade PIN (HGPIN) or cancerous acini in the prostates (including anterior prostate (AP), ventral prostate (VP) and dorsolateral prostate (DLP)) of mice with the indicated genotypes and number as in (A).


( A ) Correlation analysis of EZH2 and p53 mRNA expression measured by RT - qPCR in primary and metastatic prostate cancer specimens of a cohort of patients from University of Washington .

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

(A) Correlation analysis of EZH2 and p53 mRNA expression measured by RT-qPCR in primary and metastatic prostate cancer specimens of a cohort of patients from University of Washington.


( B ) IHC analysis of EZH2 and p53 protein expression in human prostate cancer tissues of a cohort of patients from Mayo Clinic . Case 1 and 2 show low and high expression in primary tissues , respectively ; case 3 shows high expression in lymph node metastasis . Scale bar , 50 µm . Scale bars in insets , 10 µm .

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(B) IHC analysis of EZH2 and p53 protein expression in human prostate cancer tissues of a cohort of patients from Mayo Clinic. Case 1 and 2 show low and high expression in primary tissues, respectively; case 3 shows high expression in lymph node metastasis. Scale bar, 50 µm. Scale bars in insets, 10 µm.


( C ) Correlation analysis of EZH2 and p53 protein expression in primary and metastatic prostate cancer specimens of the Mayo Clinic cohort .

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(C) Correlation analysis of EZH2 and p53 protein expression in primary and metastatic prostate cancer specimens of the Mayo Clinic cohort.


( D ) IHC analysis of EZH2 and p53 protein expression in commercially purchased TMA with patient specimens of brain , colorectal , pancreatic cancer and sarcoma . Scale bars in low magnification images , 200 µm ; Scale bars in high magnification images , 50 µm . Arrows , indicated the zoom out - box . ( E ) IHC data as shown in ( D ) were quantified and used for correlation analysis of EZH2 and p53 protein expression in patient specimens of brain , colorectal , pancreatic cancer and sarcoma . Pearson correlation r and P values in each cancer type are indicated .

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(D) IHC analysis of EZH2 and p53 protein expression in commercially purchased TMA with patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Scale bars in low magnification images, 200 µm; Scale bars in high magnification images, 50 µm. Arrows, indicated the zoom out-box. (E) IHC data as shown in (D) were quantified and used for correlation analysis of EZH2 and p53 protein expression in patient specimens of brain, colorectal, pancreatic cancer and sarcoma. Pearson correlation r and P values in each cancer type are indicated.


( A , B ) VCaP cells stably expressing control ( sh - control ) or p53 - specific shRNA were infected with lentivirus for empty vector ( EV ) or deletion mutants of EZH2 . Cell growth in 2D ( A ) and 3D ( B ) conditions were determined by MTS assay and measurement of clone size , respectively . Statistical significance was determined by two - tailed Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(A, B) VCaP cells stably expressing control (sh-control) or p53-specific shRNA were infected with lentivirus for empty vector (EV) or deletion mutants of EZH2. Cell growth in 2D (A) and 3D (B) conditions were determined by MTS assay and measurement of clone size, respectively. Statistical significance was determined by two-tailed Student's t-test. * P < 0.05, *** P < 0.001.


( C , D ) VCaP cells ( 1x107 ) infected with lentivirus were injected subcutaneously into NSG mice ( n = 8 / group ) . Tumors were measured by caliper twice a week . Tumors at the end point of measurement were isolated and photographed ( C ) , and data are shown as means ± SD ( D ) . Statistical significance was determined by two - tailed Student ' s t - test for tumors at day 56 . * * * P < 0 . 001 .

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(C, D) VCaP cells (1x107) infected with lentivirus were injected subcutaneously into NSG mice (n = 8/group). Tumors were measured by caliper twice a week. Tumors at the end point of measurement were isolated and photographed (C), and data are shown as means ± SD (D). Statistical significance was determined by two-tailed Student's t-test for tumors at day 56. *** P < 0.001.


( E - G ) Luciferase - expressing MIA PaCa - 2 cells ( 2 × 106 ) infected with lentivirus were injected via tail vein into NSG mice ( n = 6 / group ) . At 12 weeks after injection , mice were subjected to bioluminescent imaging , and images were recorded ( E ) and bioluminescent signals were quantified ( F ) . Bioluminescent flux ( photons s - 1 sr - 1 cm - 2 ) was determined for lesions in lung , the ends of the box are the upper and lower quartiles and the box spans the interquartile range ; the median is marked by a vertical line inside the box ; the whiskers are the two lines outside the box that extend to the highest and lowest observations . Lungs were isolated from mice , stained with Bouin ' s solution , and photographed ( G ) . The white spots on lungs ( stained in yellow ) are metastatic tumors . * P < 0 . 05 ; NS , no significance .

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

(E-G) Luciferase-expressing MIA PaCa-2 cells (2×106) infected with lentivirus were injected via tail vein into NSG mice (n = 6/group). At 12 weeks after injection, mice were subjected to bioluminescent imaging, and images were recorded (E) and bioluminescent signals were quantified (F). Bioluminescent flux (photons s-1 sr-1 cm-2) was determined for lesions in lung, the ends of the box are the upper and lower quartiles and the box spans the interquartile range; the median is marked by a vertical line inside the box; the whiskers are the two lines outside the box that extend to the highest and lowest observations. Lungs were isolated from mice, stained with Bouin's solution, and photographed (G). The white spots on lungs (stained in yellow) are metastatic tumors. * P < 0.05; NS, no significance.


( B ) Kaplan - Meier plots showing the association of EZH2 overexpression with biochemical recurrence of prostate cancer in patients from the TCGA cohort . Tumors were separated into two groups according to EZH2 expression levels ( high or low ) and three groups based on the mutation status of p53 ( WT , loss or mutated ) .

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(B) Kaplan-Meier plots showing the association of EZH2 overexpression with biochemical recurrence of prostate cancer in patients from the TCGA cohort. Tumors were separated into two groups according to EZH2 expression levels (high or low) and three groups based on the mutation status of p53 (WT, loss or mutated).


VCaP and C4 - 2 cells were treated with GSK - 126 , DZNep ( 5 µM for VCaP and 10 µM for C4 - 2 ) , and EZH2 ASO , followed by western blots with indicated antibodies ( C ) Data shown as means ± SD ( n = 6 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * * P < 0 . 001 .

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

VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by western blots with indicated antibodies (C) Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


VCaP and C4 - 2 cells were treated with GSK - 126 , DZNep ( 5 µM for VCaP and 10 µM for C4 - 2 ) , and EZH2 ASO , followed by MTT assay ( D ) . Data shown as means ± SD ( n = 6 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * * P < 0 . 001 .

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

VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by MTT assay (D). Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


( E , F ) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts . Mice ( n = 10 ) bearing the Ptenpc - / - ; Trp53pc - / - ( E ) and Ptenpc - / - ; Trp53pc - / R172H allografts ( F ) were treated with control ASOs ( 50 mg kg - 1 ) or Ezh2 ASOs at different doses ( 25 mg kg - 1 and 50 mg kg - 1 ) . Tumor growth was measured twice a week for 3 weeks and the data are shown in the bottom panels . Each allograft was isolated by the end of ASO treatment and photographed ( top panels ) . Data shown as means ± SD ( n = 10 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * * P < 0 . 001 .

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

(E, F) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts. Mice (n=10) bearing the Ptenpc-/-;Trp53pc-/- (E) and Ptenpc-/-;Trp53pc-/R172H allografts (F) were treated with control ASOs (50 mg kg-1) or Ezh2 ASOs at different doses (25 mg kg-1 and 50 mg kg-1). Tumor growth was measured twice a week for 3 weeks and the data are shown in the bottom panels. Each allograft was isolated by the end of ASO treatment and photographed (top panels). Data shown as means±SD (n=10). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


C - E Mapping of the binding region between myoVa and caldendrin using co - immunoprecipitation of cald - tagRFP and GFP - myoVa fragments expressed in HEK293T cells . Arrows in D indicate the size of the respective myosin fragment . F Summary of the results from C - E . Caldendrin binds specifically to a fragment containing IQ - motif 1 ( amino acids 742 - 791 ) , but not to any other region of myoVa , in a Ca2 + - dependent manner .

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

C-E Mapping of the binding region between myoVa and caldendrin using co-immunoprecipitation of cald-tagRFP and GFP-myoVa fragments expressed in HEK293T cells. Arrows in D indicate the size of the respective myosin fragment. F Summary of the results from C - E. Caldendrin binds specifically to a fragment containing IQ-motif 1 (amino acids 742-791), but not to any other region of myoVa, in a Ca2+-dependent manner.


B Western blot analysis of competition co - immunoprecipitation experiment in HEK293T cells . In the presence of Ca2 + , caldendrin binds strongly to the IQ1 fragment ( upper arrow ) , while tagRFP by itself does not bind ( lower arrow ) . Detection using a calmodulin antibody indicated that Ca2 + - dependent binding of caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control ( tagRFP only ) . MW = molecular weight marker . C Quantification of the normalized relative intensity of cald - tagRFP ( left ) and CaM bands ( right ) in western blot analysis of three independent experiments as shown in B . Shown is mean ± SEM . Upper : Ca2 + greatly increases cald - tagRFP binding to GFP - myoVa - IQ1 . Lower : When Ca2 + is available , the simultaneous presence of cald - tagRFP leads to a reduced binding of CaM compared to the control ( tagRFP ) .

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

B Western blot analysis of competition co-immunoprecipitation experiment in HEK293T cells. In the presence of Ca2+, caldendrin binds strongly to the IQ1 fragment (upper arrow), while tagRFP by itself does not bind (lower arrow). Detection using a calmodulin antibody indicated that Ca2+-dependent binding of caldendrin reduced the amount of CaM bound to the IQ1 motif relative to the control (tagRFP only). MW = molecular weight marker. C Quantification of the normalized relative intensity of cald-tagRFP (left) and CaM bands (right) in western blot analysis of three independent experiments as shown in B. Shown is mean ± SEM. Upper: Ca2+ greatly increases cald-tagRFP binding to GFP-myoVa-IQ1. Lower: When Ca2+ is available, the simultaneous presence of cald-tagRFP leads to a reduced binding of CaM compared to the control (tagRFP). D


F Native MS analysis of ion saturation for CaM . Ca2 + carried along from purification is detected on CaM in native MS . Without further supplementation with ions , intact CaM shows primarily its high affinity calcium binding sites occupied ( left ) , while in the same analysis myoVa peptide selectively and preferentially binds only to the fully Ca2 + - saturated CaM species ( right ) . Additional unassigned peaks represent unspecific adducts of Na + and Mg2 + ions .

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

F Native MS analysis of ion saturation for CaM. Ca2+ carried along from purification is detected on CaM in native MS. Without further supplementation with ions, intact CaM shows primarily its high affinity calcium binding sites occupied (left), while in the same analysis myoVa peptide selectively and preferentially binds only to the fully Ca2+-saturated CaM species (right). Additional unassigned peaks represent unspecific adducts of Na+ and Mg2+ ions.


G Native MS analysis of ion saturation for caldendrin . Supplemented by an excess of 200 µM Ca2 + and 100 µM Mg2 + , caldendrin displays myoVa peptide binding to both fully Ca2 + - saturated and 2 Ca2 + - bound species ( right ) , which are also both detectable for the intact caldendrin protein in the same analysis ( left ) .

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

G Native MS analysis of ion saturation for caldendrin. Supplemented by an excess of 200 µM Ca2+ and 100 µM Mg2+, caldendrin displays myoVa peptide binding to both fully Ca2+-saturated and 2 Ca2+-bound species (right), which are also both detectable for the intact caldendrin protein in the same analysis (left).


C Visualization of F - actin gliding caused by myoVa activity . The first ( red ) and last ( green ) frame of a 30 second time - lapse image stack were color - coded and overlaid to visualize movement . Yellow = actin filament did not move during 30 seconds of imaging . In the presence of only CaM ( left ) , the majority of actin filaments are gliding . In the presence of both CaM and cald ( right ) , a large number of filaments are immobile . Scale bar = 10 µm .

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

C Visualization of F-actin gliding caused by myoVa activity. The first (red) and last (green) frame of a 30 second time-lapse image stack were color-coded and overlaid to visualize movement. Yellow = actin filament did not move during 30 seconds of imaging. In the presence of only CaM (left), the majority of actin filaments are gliding. In the presence of both CaM and cald (right), a large number of filaments are immobile. Scale bar = 10 µm.


B Co - sedimentation assay of GFP - tagged myosin fragment lacking the globular tail domain ( GFP - myoVa - motor + CC ) with F - actin shows that the presence of Ca2 + - caldendrin does not prevent myoV from binding to actin filaments . GFP - myoVa - motor + CC , as visualized by an anti - GFP antibody via western blot , co - sediments with F - actin ( comassie blue stained SDS - PAGE ) , both in presence and absence of Ca2 + , regardless of the presence of caldendrin .

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

B Co-sedimentation assay of GFP-tagged myosin fragment lacking the globular tail domain (GFP-myoVa-motor+CC) with F-actin shows that the presence of Ca2+-caldendrin does not prevent myoV from binding to actin filaments. GFP-myoVa-motor+CC, as visualized by an anti-GFP antibody via western blot, co-sediments with F-actin (comassie blue stained SDS-PAGE), both in presence and absence of Ca2+, regardless of the presence of caldendrin.


D Left : Representative image of COS7 cell expressing the inducible dimerization system described in C . Red : Peroxisomes ( PEX - RFP - FKBP ) , Green : KIF17 - GFP - PEX13 and myoV - GFP - FRB . Scale bar = 10 µm . Middle and right : Individual tracks of kinesin - driven peroxisome movements reconstructed over 20 seconds ( yellow ) before ( middle ) and after ( right ) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes . Blue outlines represent manually drawn cellular border and nucleus ( excluded from analysis ) .

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

D Left: Representative image of COS7 cell expressing the inducible dimerization system described in C. Red: Peroxisomes (PEX-RFP-FKBP), Green: KIF17-GFP-PEX13 and myoV-GFP-FRB. Scale bar = 10 µm. Middle and right: Individual tracks of kinesin-driven peroxisome movements reconstructed over 20 seconds (yellow) before (middle) and after (right) addition of rapalog indicate that the recruitment of myoV slows down the motility of peroxisomes. Blue outlines represent manually drawn cellular border and nucleus (excluded from analysis).


E Analysis of the mean squared displacement ( MSD ) of peroxisomes over time ( left ) suggests a linear increase for all conditions . Slope analysis for MSD curves for individual cells ( right ) indicates that addition of rapalog induces efficient stalling ( 2 - way ANOVA , * * * p < 0 . 0001 as compared before and after rapalog ) . However , no difference between control and caldendrin ( cald ) transfected cells could be observed ( 2 - way ANOVA , Mean ± SEM ; n control = 14 cells , n cald = 23 cells ; 3 experiments ) .

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

E Analysis of the mean squared displacement (MSD) of peroxisomes over time (left) suggests a linear increase for all conditions. Slope analysis for MSD curves for individual cells (right) indicates that addition of rapalog induces efficient stalling (2-way ANOVA, *** p<0.0001 as compared before and after rapalog). However, no difference between control and caldendrin (cald) transfected cells could be observed (2-way ANOVA, Mean ± SEM; n control=14 cells, n cald=23 cells; 3 experiments).


A Example confocal images from 10 min time - lapse imaging of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat primary hippocampal neurons ( DIV17 ) expressing an ER - marker ( ER - tDimer ) . Yellow arrows show dendritic protrusions with a stably inserted ER ( > 10 min ) . Blue arrows indicate protrusions with a transient ER entry . Scale bar = 5 µm . B Quantification of dynamic SER presence in protrusions of control ( GFP ) or caldendrin ( cald - GFP ) transfected neurons . In protrusions of cald - GFP neurons , the percentage of stably localized ER ( > 10 min ) is increased , while transient SER visits are decreased compared to the control . ( mean ± SEM , 2 - way ANOVA , * p = 0 . 0386 for interaction , n GFP = 9 , n cald - GFP = 10 ; 2 experiments )

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

A Example confocal images from 10 min time-lapse imaging of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons (DIV17) expressing an ER-marker (ER-tDimer). Yellow arrows show dendritic protrusions with a stably inserted ER (>10 min). Blue arrows indicate protrusions with a transient ER entry. Scale bar = 5 µm. B Quantification of dynamic SER presence in protrusions of control (GFP) or caldendrin (cald-GFP) transfected neurons. In protrusions of cald-GFP neurons, the percentage of stably localized ER (> 10 min) is increased, while transient SER visits are decreased compared to the control. (mean ± SEM, 2-way ANOVA, * p=0.0386 for interaction, n GFP = 9, n cald-GFP = 10; 2 experiments)


C Representative confocal images of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat primary hippocampal neurons expressing an ER - marker ( ER - tDimer ) . To pre - load overexpressed caldendrin with Ca2 + , neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1 - antibody to visualize synapse - containing spines . Arrows indicate spines that contain ER . Scale bar = 5 µm . D - F Quantification of protrusion - and spine density and the number of SER - positive spines as shown in C . ( mean ± SEM , n GFP ctrl = 24 cells , n Cald - GFP = 25 cells ; 3 experiments ) . ( D ) The density of dendritic protrusions ( spines and filopodia ) is increased in cald - GFP overexpressing neurons compared to the control . ( * p = 0 . 0119 , unpaired t - test ) . ( E ) The density of homer1 - positive dendritic spines is increased in cald - GFP overexpressing neurons compared to the control . ( * * p = 0 . 0049 , unpaired t - test ) . ( F ) The percentage of ER - containing dendritic spines is increased in cald - GFP overexpressing neurons compared to the control . ( * p = 0 . 0179 , unpaired t - test )

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

C Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons expressing an ER-marker (ER-tDimer). To pre-load overexpressed caldendrin with Ca2+, neurons were stimulated with 50 µM bicuculline for 5 min 8 hours prior to fixation and stained with a homer1-antibody to visualize synapse-containing spines. Arrows indicate spines that contain ER. Scale bar = 5 µm. D - F Quantification of protrusion- and spine density and the number of SER-positive spines as shown in C. (mean ± SEM, n GFP ctrl = 24 cells, n Cald-GFP = 25 cells; 3 experiments). (D) The density of dendritic protrusions (spines and filopodia) is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0119, unpaired t-test). (E) The density of homer1-positive dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (** p=0.0049, unpaired t-test). (F) The percentage of ER-containing dendritic spines is increased in cald-GFP overexpressing neurons compared to the control. (* p=0.0179, unpaired t-test) G


G Representative confocal images of control ( GFP ) or caldendrin ( cald - GFP ) transfected rat primary hippocampal neurons pre - treated with bicuculline as in C and stained against homer1 and the spine apparatus - marker synaptopodin . Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft , blue arrows indicate spine - localized synaptopodin . Scale bar = 5 µm . H - J Quantification of spine density , synaptopodin clusters and synaptopodin - positive spines as shown in G . ( mean ± SEM , n GFP ctrl = 21 cells , n cald - GFP = 30 cells ; 2 experiments ) . ( H ) Spine density is increased in cald - GFP overexpressing neurons compared to the control ( in line with 5e ) . ( * * p = 0 . 0024 , unpaired t - test ) . ( I ) The total number of synaptopodin clusters is not affected by caldendrin overexpression . ( n . s . , unpaired t - test ) . ( J ) The percentage of synapotpodin - positive spines is increased in cald - GFP overexpressing neurons compared to the control . ( * * * * p < 0 . 0001 , unpaired t - test ) .

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

G Representative confocal images of control (GFP) or caldendrin (cald-GFP) transfected rat primary hippocampal neurons pre-treated with bicuculline as in C and stained against homer1 and the spine apparatus-marker synaptopodin. Yellow arrows indicate synaptopodin clusters localized inside the dendritic shaft, blue arrows indicate spine-localized synaptopodin. Scale bar = 5 µm. H - J Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in G. (mean ± SEM, n GFP ctrl = 21 cells, n cald-GFP = 30 cells; 2 experiments). (H) Spine density is increased in cald-GFP overexpressing neurons compared to the control (in line with 5e). (** p = 0.0024, unpaired t-test). (I) The total number of synaptopodin clusters is not affected by caldendrin overexpression. (n.s., unpaired t-test). (J) The percentage of synapotpodin-positive spines is increased in cald-GFP overexpressing neurons compared to the control. (**** p < 0.0001, unpaired t-test).


A 10 min time - lapse imaging of DIV12 wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO + cald - GFP ) or caldendrin knock - out treated with jasplakinolide ( cald KO + JPK ) mouse primary hippocampal neurons , additionally expressing a cell fill ( maxGFP ) and an ER - marker ( ER - tDimer ) . Yellow arrows indicate protrusions with a stably anchored ER ( > 10 min ) . Blue arrows indicate protrusions that show transient presence of ER . Scale bar = 5 µm .

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A 10 min time-lapse imaging of DIV12 wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO + cald-GFP) or caldendrin knock-out treated with jasplakinolide (cald KO + JPK) mouse primary hippocampal neurons, additionally expressing a cell fill (maxGFP) and an ER-marker (ER-tDimer). Yellow arrows indicate protrusions with a stably anchored ER (>10 min). Blue arrows indicate protrusions that show transient presence of ER. Scale bar = 5 µm.


A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin . The groups compared are wild - type ( cald WT ) , caldendrin knock - out ( cald KO ) , caldendrin knock - out expressing caldendrin - GFP ( cald KO cald ) , and caldendrin knock - out treated with jasplakinolide ( cald KO JPK ) . Scale bar = 5 µm .

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A Representative confocal images of mouse primary hippocampal neurons stained against homer1 and synaptopodin. The groups compared are wild-type (cald WT), caldendrin knock-out (cald KO), caldendrin knock-out expressing caldendrin-GFP (cald KO cald), and caldendrin knock-out treated with jasplakinolide (cald KO JPK). Scale bar = 5 µm.


B - C Quantification of spine density , synaptopodin clusters and synaptopodin - positive spines as shown in A ( mean ± SEM . Kruskal - Wallis test with Dunn ' s multiple comparisons post - test . * * * p < 0 . 0001 . cald wt n = 34 cells , 2 experiments ; cald KO n = 33 cells , 3 experiments ; cald KO cald n = 10 cells , 1 experiment ; cald KO JPK n = 14 cells , 1 experiment ) . ( B ) The total number of synaptopodin clusters is unaffected by the loss of caldendrin . ( C ) The number of synaptopodin - positive spines is reduced in cald KO compared to the WT neurons . This phenotype could be rescued by over - expression of caldendrin ( cald KO + cald ) , but not by treatment with JPK ( cald KO + JPK ) .

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B - C Quantification of spine density, synaptopodin clusters and synaptopodin-positive spines as shown in A (mean ± SEM. Kruskal-Wallis test with Dunn's multiple comparisons post-test. *** p < 0.0001. cald wt n = 34 cells, 2 experiments; cald KO n = 33 cells, 3 experiments; cald KO cald n = 10 cells, 1 experiment; cald KO JPK n = 14 cells, 1 experiment). (B) The total number of synaptopodin clusters is unaffected by the loss of caldendrin. (C) The number of synaptopodin-positive spines is reduced in cald KO compared to the WT neurons. This phenotype could be rescued by over-expression of caldendrin (cald KO + cald), but not by treatment with JPK (cald KO + JPK).


Media from soluble TREM2 - Fc transfected HEK293T cells incubated with Aβ42 fibrils . Top left panel shows original media samples . Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet . The bottom two panels represent the same samples as the top , but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils . Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet . The bottom two panels represent the same samples as the top , but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1 . All experiments were replicated 3 independent

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Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10 Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM2 - Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Left panels show original media samples . Right panels show the purified soluble TREM2 - Fc ( bottom ) and the presence of APOE3 with the purified soluble TREM2 - Fc ( top ) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1 . All experiments were replicated 3 independent

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Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top) Western blo had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times


Media from soluble TREM - Fc family member transfected HEK293T cells incubated with Aβ42 fibrils . Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet . Bottom panel shows original media and Aβ42 pellet probed with

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Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10


Media from soluble TREM - Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti - Human IgG Fc . Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3 . Bottom panel shows original media and purified soluble TREM - Fc family members probed with anti -

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Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5


Schematic representation of Aβ42 oligomers ( right panel ) and APOE4 ( left panel ) binding with soluble TREM2 - Fc at various concentrations . Curves were fit by setting buffer control ( no ligand ) to y =

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Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0


BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2 - Fc pre incubated with Aβ42 oligomers . Schematic representations of two sequential associations of ligand on soluble TREM2 - Fc are represented by I and II . Soluble TREM2 - Fc loaded on a sensor is first incubated with APOE3 ( association I ) followed by a second incubation with Aβ42 oligomers ( association II ) or the reverse - incubated with Aβ42 oligomers ( association I ) followed by a second incubation with APOE3 ( association II ) . All experiments were replicated 3 independent times

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BioLayer Interferometry experiments were performed to evaluate the binding capacity of APOE3 on soluble TREM2-Fc pre incubated with Aβ42 oligomers. Schematic representations of two sequential associations of ligand on soluble TREM2-Fc are represented by I and II. Soluble TREM2-Fc loaded on a sensor is first incubated with APOE3 (association I) followed by a second incubation with Aβ42 oligomers (association II) or the reverse- incubated with Aβ42 oligomers (association I) followed by a second incubation with APOE3 (association II). All experiments were replicated 3 independent times


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2 - Fc shows a dose dependent signal . Results were expressed as the OD450 averaged ± standard error ( n = 3 ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

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ELISA plates were coated with purified soluble TREM2-Fc, and then incubated with various concentrations of Aβ42 monomer Binding of Aβ42 monomers with soluble TREM2-Fc shows a dose dependent signal. Results were expressed as the OD450 averaged ± standard error (n =3; * p<0.05, ** p<0.01, *** p<0.001, **** p < 0.0001, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


ELISA plates were coated with purified soluble TREM2 - Fc , and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM - Fc family members . Results were expressed as the OD450 averaged ± standard error ( n = 4 , * p < 0 . 05 , * * * * p < 0 . 0001 , ANOVA , Bonferroni multiple comparisons , see Appendix table S2 for p value )

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ELISA plates were coated with purified soluble TREM2-Fc, and then incubated wit 1 µM Aβ42 oligomers Binding of 1 µM Aβ42 oligomers with soluble TREM-Fc family members. Results were expressed as the OD450 averaged ± standard error (n = 4, * p < 0.05, **** p<0.0001, ANOVA, Bonferroni multiple comparisons, see Appendix table S2 for p value)


2B4 NFAT - GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours . GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage ± GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with mock , * * * * p < 0 . 0001 compared with mock , # p < 0 . 05 compared with TREM2 , # # # # p < 0 . 0001 compared with TREM2 , Two - way ANOVA , Bonferroni multiple comparisons , see Appendix table S3 for p value )

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2B4 NFAT-GFP reporter cells transduced with TREM2 or AD variant R47H were incubated with various concentrations of Aβ42 oligomers for 12 hours. GFP expression was detected at 1 µM Aβ42 oligomers with TREM2 transduced cells. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage ± GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with mock, **** p< 0.0001 compared with mock, # p < 0.05 compared with TREM2, #### p < 0.0001 compared with TREM2, Two-way ANOVA, Bonferroni multiple comparisons, see Appendix table S3 for p value)


B - 2B4 NFAT - GFP reporter cells transduced with TREM2 or variants ( R47H , R62H and T96K ) were incubated with 10 µM Aβ42 oligomers for 12 hours . No GFP expression was detected with Aβ42 oligomers from control transduced cells . Results were expressed as the percentage GFP cells averaged of ± standard error ( n = 3 , * p < 0 . 05 compared with TREM2 , * * p < 0 . 05 compared with TREM2 , * * * * p < 0 . 0001 compared with TREM2 , # # p < 0 . 01 compared with TREM2 T96K , # # # # p < 0 . 0001 compared with TREM2 T96K , ANOVA , Dunnett ' s multiple comparisons test , see Appendix table S3 for p value

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B-2B4 NFAT-GFP reporter cells transduced with TREM2 or variants (R47H, R62H and T96K) were incubated with 10 µM Aβ42 oligomers for 12 hours. No GFP expression was detected with Aβ42 oligomers from control transduced cells. Results were expressed as the percentage GFP cells averaged of ± standard error (n = 3, * p < 0.05 compared with TREM2, ** p < 0.05 compared with TREM2, **** p< 0.0001 compared with TREM2, ## p < 0.01 compared with TREM2 T96K, #### p < 0.0001 compared with TREM2 T96K, ANOVA, Dunnett's multiple comparisons test, see Appendix table S3 for p value


HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA . Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant . Results were averaged ± standard error ( n = 9 , # p < 0 . 05 compared with TREM2 , # # p < 0 . 01 compared with TREM2 , * * * p < 0 . 001 compared with mock , * * * * p < 0 . 0001 compared with mock , ANOVA , Tukey ' s Multiple Comparison Test , see Appendix table S3 for p value )

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HEK293T cells transfected with TREM2 or AD variant incubated with 500 nM Aβ42 monomers were lysed for Aβ42 quantifications by ELISA. Expression of TREM2 increases Aβ42 level detected compared to mock control as well the AD variant. Results were averaged ± standard error (n = 9, # p < 0.05 compared with TREM2, ## p< 0.01 compared with TREM2, *** p< 0.001 compared with mock, **** p <0.0001 compared with mock, ANOVA, Tukey's Multiple Comparison Test, see Appendix table S3 for p value)


HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours . Conditioned media and cell lysate were analyzed by Western blot for TREM2 ( RIPA lysate ) and soluble TREM2 ( media ) detection . Experiment was replicated 3 independent

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HEK293T cells transfected with TREM2 were incubated with Aβ42 oligomers for 16 hours. Conditioned media and cell lysate were analyzed by Western blot for TREM2 (RIPA lysate) and soluble TREM2 (media) detection. Experiment was replicated 3 independent times


HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification . Bottom panel in D shows the presence of TREM2 in the purified product . Endogenous APP and β - tubulin were used as controls for the purification ( middle and upper panels ) . Experiment was replicated 3 independent

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HEK293T cells transfected with TREM2 were subjected to cell membrane biotinylation for purification. Bottom panel in D shows the presence of TREM2 in the purified product. Endogenous APP and β-tubulin were used as controls for the purification (middle and upper panels). Experiment was replicated 3 independent time


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen , scale bar 100

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(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control of tamoxifen, scale bar 100 μm


( B Quantification of GLUT1 ( hypoxia marker Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e .

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(B Quantification of GLUT1 (hypoxia marker Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m


( C ) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

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(C) Relative values of protein levels for Glut1 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


, D ) Quantification o CD31 ( endothelial cell marker ) . Control ( n = 5 ) , 2mg ( n = 5 ) , and 5mg ( n = 4 ) . In all cases , bars represent mean ± s . e . m . (

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,D) Quantification o CD31 (endothelial cell marker). Control (n=5), 2mg (n=5), and 5mg (n=4). In all cases, bars represent mean ± s.e.m. (C


( E , F ) Expression levels of DEGs relevant to response to hypoxia ( left ) and blood vessel morphogenesis ( right ) . The values were normalized by tubulin family

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(E,F) Expression levels of DEGs relevant to response to hypoxia (left) and blood vessel morphogenesis (right). The values were normalized by tubulin family genes


( G ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen , scale bar 100

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(G) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control and 5mg of tamoxifen, scale bar 100 μm


( H ) Quantification of HIF - 1A in PDAC tissues Control ( n = 5 ) , and 5mg ( n = 4 ) . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum

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(H) Quantification of HIF-1A in PDAC tissues Control (n=5), and 5mg (n=4). In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value


( I ) qPCR levels of HIF - 1A in PSCs , normalized to RPLP0 and relative to

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

(I) qPCR levels of HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Western blot bands for protein expression in PSCs ( p - Tmod is post - translational modification ) . The plot shows the quantification of the sum of band intensities corresponding to isoform 1 , isoform 2 , isoform 3 , and post - transcriptionally modified HIF - 1A ( n = 8 control and n = 8 tam )

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

(J) Western blot bands for protein expression in PSCs (p-Tmod is post-translational modification). The plot shows the quantification of the sum of band intensities corresponding to isoform 1, isoform 2, isoform 3, and post-transcriptionally modified HIF-1A (n=8 control and n=8 tam)


( A ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 and relative to

(A) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot levels of LOX - L2 in PSCs ( n = 3 experimental replicates , * * P < 0 . 01 )

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

(B) Western blot levels of LOX-L2 in PSCs (n=3 experimental replicates, **P<0.01)


( C ) Expression of LOX family genes obtained from RNA - seq data in control and tamoxifen treated PSCs ( n = 3 experimental replicates ) . Expression value was normalized by tubulin family

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

(C) Expression of LOX family genes obtained from RNA-seq data in control and tamoxifen treated PSCs (n = 3 experimental replicates). Expression value was normalized by tubulin family genes


( D ) qPCR levels of LOX family in PSCs , normalized to RPLP0 and relative to

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

(D) qPCR levels of LOX family in PSCs, normalized to RPLP0 and relative to control


( E ) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O

(E) Relative values of protein levels for LOX members in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates


( F ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 50 μm . ( G ) Quantification of LOX - L2 for images in F . n ≥ 4 mice per condition , and n > 10 sections per

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

(F) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 50 μm. (G) Quantification of LOX-L2 for images in F. n ≥ 4 mice per condition, and n> 10 sections per animal


( H ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to 1

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

(H) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to 1 kPa


( I ) qPCR levels of LOX - L2 and HIF - 1A in PSCs , normalized to RPLP0 and relative to

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

(I) qPCR levels of LOX-L2 and HIF-1A in PSCs, normalized to RPLP0 and relative to control


( J ) Quantification of average forces applied by PSCs on pillars . BBI = blebbistatin . In the box and whisker plot , the central box represents values from the lower and upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum . * * * P < 0 . 001 Three experimental

O O O O O O O O O B-CELL_TYPE 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 O O O

(J) Quantification of average forces applied by PSCs on pillars. BBI = blebbistatin. In the box and whisker plot, the central box represents values from the lower and upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum. ***P<0.001 Three experimental repeats


( K ) qPCR levels of LOX - L2 in PSCs , normalized to RPLP0 ( 60S acidic ribosomal protein P0 ) and relative to

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

(K) qPCR levels of LOX-L2 in PSCs, normalized to RPLP0 (60S acidic ribosomal protein P0) and relative to control


( A ) Images of matrigel collagen gels previously remodelled by PSCs , second harmonic generation signal for fibrillar collagen ( green ) and F - actin ( red ) , scale bar 100 μm . ( B ) Fiber thickness colour - code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness , scale bar 100 μm . ( C ) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm . Insets show FFTs of fibrillar collagen - I images , representing alignment with respect to the elliptical distribution of the FFT central maxima . Circular behaviour ( values approaching 1 ) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction . Scale bar 20 μm ( D - F ) Quantification of fiber thickness , length , and alignment for images in A - C . ( G ) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B , H . n ≥ 6 matrices per condition . In the scattered plots each point represents a section . In the box - and - whisker plot , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum value . Histogram bars represent mean ± s . e . m . * * * P < 0 . 001 ( t - test for D , E , F and ; Anova and Tukey " s post hoc test for G ) . Three experimental replicates for all

O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR 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 O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Images of matrigel collagen gels previously remodelled by PSCs, second harmonic generation signal for fibrillar collagen (green) and F-actin (red), scale bar 100 μm. (B) Fiber thickness colour-code map in a represented through the BoneJ plugin where larger spheres fit along fibers represent greater thickness, scale bar 100 μm. (C) SHG fibrillar collagen images used for calculation of alignment through the FFT algorithm. Insets show FFTs of fibrillar collagen-I images, representing alignment with respect to the elliptical distribution of the FFT central maxima. Circular behaviour (values approaching 1) represent no aligned orientation and lower values represent fiber orientation as alignment is displayed as a power distribution orthogonal to the orientation direction. Scale bar 20 μm (D-F) Quantification of fiber thickness, length, and alignment for images in A-C. (G) Quantification of collagen fiber thickness using the BoneJ algorithm for images in B,H. n≥6 matrices per condition. In the scattered plots each point represents a section. In the box-and-whisker plot, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value. Histogram bars represent mean±s.e.m. ***P<0.001 (t-test for D, E, F and ; Anova and Tukey"s post hoc test for G). Three experimental replicates for all p


( A ) qPCR levels of collagen in PSCs , normalized to RPLP0 and relative to

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

(A) qPCR levels of collagen in PSCs, normalized to RPLP0 and relative to control


( B ) Western blot analysis of collagen normalised to total protein and relative to

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

(B) Western blot analysis of collagen normalised to total protein and relative to control


( C ) Quantification of time - lapse collagen synthesis and deposition by

(C) Quantification of time-lapse collagen synthesis and deposition by PSCs


( D ) Representative immunofluorescent images used for the quantification in C , scale bar 20 μm , collagen - I was assessed with a specific primary antibody

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

(D) Representative immunofluorescent images used for the quantification in C, scale bar 20 μm, collagen-I was assessed with a specific primary antibody staining


( E ) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O

(E) Relative values of protein levels for collagen in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


( F ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to

(F) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to contro


( G ) MMP - 2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography ; above signal intensity of the representative bands used for the quantification presented in the plot

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

(G) MMP-2 activity on control and tamoxifen treated PSCs assayed by gelatin zymography; above signal intensity of the representative bands used for the quantification presented in the plot below


quantification of MMP - 2 levels in PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of tamoxifen , scale bar 100 μm ( n = 4 animals per condition and n ≥ 5 sections per animal )

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quantification of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxifen, scale bar 100 μm (n=4 animals per condition and n≥ 5 sections per animal)


Immunohistochemistry image of MMP - 2 levels in PDAC tissues from KPC mice treated with vehicle ( control ) , and 2mg and 5mg of

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

Immunohistochemistry image of MMP-2 levels in PDAC tissues from KPC mice treated with vehicle (control), and 2mg and 5mg of tamoxife


( J ) Relative values of protein levels for MMP - 2 in PDAC tumors assessed by proteomic analysis ( 6 mice per condition and samples were analysed in duplicates )

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

(J) Relative values of protein levels for MMP-2 in PDAC tumors assessed by proteomic analysis (6 mice per condition and samples were analysed in duplicates)


( K ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to 1

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

(K) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to 1 kPa


( L ) qPCR levels of MMP - 2 in PSCs , normalized to RPLP0 and relative to control . Histogram bars represent mean ± s . e . m .

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

(L) qPCR levels of MMP-2 in PSCs, normalized to RPLP0 and relative to control. Histogram bars represent mean±s.e.m.


( A ) qPCR levels of fibronectin ( FN ) , fibronectin extracellular domain A ( FN - EDA ) , and fibronectin extracellular domain B ( FN - EDB ) in PSCs , normalised to RPLP0 and relative to

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

(A) qPCR levels of fibronectin (FN), fibronectin extracellular domain A (FN-EDA), and fibronectin extracellular domain B (FN-EDB) in PSCs, normalised to RPLP0 and relative to control


( B ) Western blot analysis of FN normalised to total protein and relative to

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

(B) Western blot analysis of FN normalised to total protein and relative to control


( C ) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by

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

(C) Quantification of fibronectin intensity density o FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs


( D ) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by

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

(D) FN fluorescence and SHG collagen images for matrigel collagen gels remodelled by PSCs


( E ) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control , and 2mg , and 5mg of tamoxifen . ( n = 4 animals per condition , and n ≥ 5 sections per animal ) . ( F ) FN fiber thickness colour - code map in E represented through the BoneJ

O O O B-EXP_ASSAY O O B-DISEASE B-TISSUE O O B-ORGANISM 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 B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O

(E) Immunohistochemistry images of PDAC tissues from KPC mice treated with vehicle control, and 2mg, and 5mg of tamoxifen. (n=4 animals per condition, and n ≥ 5 sections per animal). (F) FN fiber thickness colour-code map in E represented through the BoneJ plugin


( G ) Relative values of protein levels for FN in PDAC tumors assessed by proteomic

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

(G) Relative values of protein levels for FN in PDAC tumors assessed by proteomic analysis


( H - J ) Quantification of fibronectin immunohistochemistry staining , fiber thickness and alignment scored of images presented in e . In all cases , histogram bars represent mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test for A , B , C ; Anova and Tukey " s post hoc test for H , I , J ) . Scale bar is

O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY 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 O O O 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-J) Quantification of fibronectin immunohistochemistry staining, fiber thickness and alignment scored of images presented in e. In all cases, histogram bars represent mean ± s.e.m.; *P<0.05,**P<0.01,***P<0.001 (t-test for A, B, C; Anova and Tukey"s post hoc test for H, I, J). Scale bar is 1


( A ) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control , and 2mg of tamoxifen , scale bar is 100 μm . Upper panels : Ki67 staining is used as a surrogate of proliferation . White arrows indicate Ki67 positive nuclei in epithelial cells . Lower panels : Cc3 staining shows the cells undergoing caspase - mediated apoptosis . Tamoxifen panels show higher levels of yellow staining , which indicates higher percentage of apoptotic epithelial cells . ( B ) Quantification of staining in panel A ( n = 4 animals per condition , and n ≥ 5 sections per animal , two experimental repetitions ) . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t -

O O O B-EXP_ASSAY O O B-DISEASE B-TISSUE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE I-CELL_TYPE O O 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 O O O O O O O O O O O O

(A) Immunofluorescence images of PDAC tissues from KPC mice treated with vehicle control, and 2mg of tamoxifen, scale bar is 100 μm. Upper panels: Ki67 staining is used as a surrogate of proliferation. White arrows indicate Ki67 positive nuclei in epithelial cells. Lower panels: Cc3 staining shows the cells undergoing caspase-mediated apoptosis. Tamoxifen panels show higher levels of yellow staining, which indicates higher percentage of apoptotic epithelial cells. (B) Quantification of staining in panel A (n=4 animals per condition, and n≥ 5 sections per animal, two experimental repetitions). Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test


( A , E , F ) Immunofluorescence images of control and tamoxifen treated suit - 2 cells , scale bar is 20 μm . Panel A represents HIF - 1A staining in hypoxia and non - hypoxia conditions , panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase - mediated apoptosis , respectively . Panel E : Red - F - actin , green - Ki67 , blue - nuclei . Tamoxifen negatively regulates HIF - 1A in hypoxia and non - hypoxia conditions . ( B , C , D ) Quantification of immunofluorescence staining in panels A , E , F . For quantification , eight fields of view ( n > 50 cells ) per condition . Histogram bars represent mean ± s . e . m . ; * * * P < 0 . 001 , t - test . All panels include data collected during 3 independent

O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E, F) Immunofluorescence images of control and tamoxifen treated suit-2 cells, scale bar is 20 μm. Panel A represents HIF-1A staining in hypoxia and non-hypoxia conditions, panels E and F show Ki67 and Cc3 staining as markers of proliferation and caspase-mediated apoptosis, respectively. Panel E: Red - F-actin, green - Ki67, blue - nuclei. Tamoxifen negatively regulates HIF-1A in hypoxia and non-hypoxia conditions. (B,C,D) Quantification of immunofluorescence staining in panels A, E, F. For quantification, eight fields of view (n>50 cells) per condition. Histogram bars represent mean ± s.e.m.; ***P<0.001, t-test. All panels include data collected during 3 independent experiments


A Montage of class averages and 3D volumes of humanTFG ( amino acids 1 ‐ 193 ; EMDataBank accession code EMD ‐ 6076 ) . Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference ‐ free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left . Scale bars , 50 Å .

O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Montage of class averages and 3D volumes of humanTFG (amino acids 1‐193; EMDataBank accession code EMD‐6076). Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference‐free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left. Scale bars, 50 Å.


B Montage of class averages and 3D volumes of C . elegans TFG ( amino acids 1 ‐ 195 ; EMDataBank accession code EMD ‐ 6075 ) , generated as described in ( A ) . Scale bars , 50 Å .

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B Montage of class averages and 3D volumes of C. elegans TFG (amino acids 1‐195; EMDataBank accession code EMD‐6075), generated as described in (A). Scale bars, 50 Å.


C Superposition of 3D volumes of full ‐ length human TFG and truncated TFG ( amino acids 1 ‐ 193 ) . The truncated form is depicted in yellow , while the full ‐ length form is depicted as gray mesh . The top view ( top ) exhibits limited differences in the structures , while the side view ( bottom ) shows extra density in the full ‐ length TFG isoform . Scale bar , 25 Å .

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

C Superposition of 3D volumes of full‐length human TFG and truncated TFG (amino acids 1‐193). The truncated form is depicted in yellow, while the full‐length form is depicted as gray mesh. The top view (top) exhibits limited differences in the structures, while the side view (bottom) shows extra density in the full‐length TFG isoform. Scale bar, 25 Å.


A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations . Error bars represent mean ± SEM ; n = 10 replicates .

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A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations. Error bars represent mean ± SEM; n = 10 replicates.


B Confocal microscopy images of recombinant , BODIPY ‐ labeled TFG in the presence of varying potassium acetate concentrations . Scale bar , 2 μm .

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

B Confocal microscopy images of recombinant, BODIPY‐labeled TFG in the presence of varying potassium acetate concentrations. Scale bar, 2 μm.


C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain ‐ EM . Large arrowheads highlight TFG polymers ( only found in the presence of elevated potassium acetate ) . Small arrowheads highlight individual 11 nm cup ‐ like TFG octamers . Scale bar , 30 nm .

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

C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain‐EM. Large arrowheads highlight TFG polymers (only found in the presence of elevated potassium acetate). Small arrowheads highlight individual 11 nm cup‐like TFG octamers. Scale bar, 30 nm.


D Circular dichroism spectroscopy was used to characterize the carboxyl ‐ terminus of C . elegans TFG ( amino acids 195 ‐ 486 ) . Samples were analyzed at different concentrations , and the data were normalized relative to one another . CD spectra were collected at 25 ° C in 25 mM sodium phosphate ( pH 7 . 2 ) using a 1 mm path length quartz cell . The spectra are characteristic of an intrinsically disordered protein .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

D Circular dichroism spectroscopy was used to characterize the carboxyl‐terminus of C. elegans TFG (amino acids 195‐486). Samples were analyzed at different concentrations, and the data were normalized relative to one another. CD spectra were collected at 25°C in 25 mM sodium phosphate (pH 7.2) using a 1 mm path length quartz cell. The spectra are characteristic of an intrinsically disordered protein.


A The distributions of endogenous TFG and ERGIC ‐ 53 were examined in hTERT ‐ RPE ‐ 1 cells stably expressing low levels of mApple ‐ Sec16B using SR ‐ SIM ( more than 15 cells analyzed on 3 separate occasions ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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

A The distributions of endogenous TFG and ERGIC‐53 were examined in hTERT‐RPE‐1 cells stably expressing low levels of mApple‐Sec16B using SR‐SIM (more than 15 cells analyzed on 3 separate occasions). Scale bar, 5 μm. Inset scale bar, 1 μm.


B Cells stably expressing low levels of GFP ‐ Sec16B and mRuby ‐ Sec23A ( Sec23A ) were fixed and imaged using SR ‐ SIM ( n = 12 ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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B Cells stably expressing low levels of GFP‐Sec16B and mRuby‐Sec23A (Sec23A) were fixed and imaged using SR‐SIM (n = 12). Scale bar, 5 μm. Inset scale bar, 1 μm.


C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby ‐ Sec23A ( Sec23A ) using SR ‐ SIM ( n = 10 ) . Scale bar , 5 μm . Inset scale bar , 1 μm .

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C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby‐Sec23A (Sec23A) using SR‐SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm.


D The distributions of endogenous TFG and Sec31A were examined using g ‐ STED ( n = 8 ) . Scale bar , 1 μm .

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D The distributions of endogenous TFG and Sec31A were examined using g‐STED (n = 8). Scale bar, 1 μm.