( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , Ndc80 - 9D or Ndc80 - 9A . Z - projection and 3D - reconstructed images show MTOCs ( mNeonGreen - Cep192 , green ) and chromosomes ( H2B - mCherry , magenta ) . Time in h : mm after NEBD . Scale bar , 10 μm .

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(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, Ndc80-9D or Ndc80-9A. Z-projection and 3D-reconstructed images show MTOCs (mNeonGreen-Cep192, green) and chromosomes (H2B-mCherry, magenta). Time in h:mm after NEBD. Scale bar, 10 μm.


( B ) Ndc80 - 9D is defective for MTOC sorting . The density map of MTOCs along the spindle axis in Zp3 - Cre Ndc80f / f oocytes expressing Ndc80 - WT , Ndc80 - 9D or Ndc80 - 9A . The color represents the percentage of MTOC volume coded from dark ( 0 % ) to white ( 50 % or more of the total volume ) .

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(B) Ndc80-9D is defective for MTOC sorting. The density map of MTOCs along the spindle axis in Zp3-Cre Ndc80f/f oocytes expressing Ndc80-WT, Ndc80-9D or Ndc80-9A. The color represents the percentage of MTOC volume coded from dark (0%) to white (50% or more of the total volume).


( C ) MTOCs do not accumulate at the spindle poles in Ndc80 - 9D - expressing oocytes . Ratio of MTOCs ( green ) or chromosomes ( magenta ) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD ( n = 19 , 19 and 22 oocytes , respectively , from at least 3 independent experiments ) .

(C) MTOCs do not accumulate at the spindle poles in Ndc80-9D-expressing oocytes. Ratio of MTOCs (green) or chromosomes (magenta) in the polar region versus those in the middle region of the spindle at 6 hours after NEBD (n=19, 19 and 22 oocytes, respectively, from at least 3 independent experiments).


( D ) Ndc80 - 9A decreases central MTOCs . The number of central MTOCs ( MTOCs that were positioned in the middle region of the spindle , was determined at each time point ( n = 19 , 21 oocytes , respectively , from at least 3 independent experiments ) .

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(D) Ndc80-9A decreases central MTOCs. The number of central MTOCs (MTOCs that were positioned in the middle region of the spindle , was determined at each time point (n=19, 21 oocytes, respectively, from at least 3 independent experiments).


( A ) Live imaging of Zp3 - Cre Ndc80f / f oocytes injected with Ndc80 - WT , - 9D or - 9A . Z - projection and 3D - reconstructed images show microtubules ( EGFP - Map4 , green ) and chromosomes ( H2B - mCherry , magenta ) . Scale bar , 10 μm .

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(A) Live imaging of Zp3-Cre Ndc80f/f oocytes injected with Ndc80-WT, -9D or -9A. Z-projection and 3D-reconstructed images show microtubules (EGFP-Map4, green) and chromosomes (H2B-mCherry, magenta). Scale bar, 10 μm.


( B ) Ndc80 - 9D is defective for limiting spindle elongation . The aspect ratio ( length / width ) of 3D - reconstructed spindles was measured over time ( n = 18 , 18 and 18 oocytes , respectively , from 3 independent experiments ) .

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(B) Ndc80-9D is defective for limiting spindle elongation. The aspect ratio (length/width) of 3D-reconstructed spindles was measured over time (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


( C ) Ndc80 - 9D causes excessive spindle elongation . Spindle lengths measured after 3D reconstruction are shown ( n = 18 , 18 and 18 oocytes , respectively , from 3 independent experiments ) .

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(C) Ndc80-9D causes excessive spindle elongation. Spindle lengths measured after 3D reconstruction are shown (n=18, 18 and 18 oocytes, respectively, from 3 independent experiments).


( A ) K - fibers are connected to MTOCs and extend to spindle poles . Oocytes 6 hours after NEBD were fixed following cold treatment . Microtubules ( α - tubulin , green ) , MTOCs ( pericentrin , magenta ) , kinetochores ( ACA , red ) and DNA ( Hoechst333342 ) are shown . Five oocytes from 2 independent experiments were analyzed . Scale bar , 10 μm .

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(A) K-fibers are connected to MTOCs and extend to spindle poles. Oocytes 6 hours after NEBD were fixed following cold treatment. Microtubules (α-tubulin, green), MTOCs (pericentrin, magenta), kinetochores (ACA, red) and DNA (Hoechst333342) are shown. Five oocytes from 2 independent experiments were analyzed. Scale bar, 10 μm.


( B ) A majority of MTOCs are attached to K - fibers . The volumes of individual MTOCs were measured after 3D reconstruction . The sum volume of MTOCs attached to K - fibers relative to the total volume of MTOCs was calculated for each oocyte ( n = 5 oocytes from 2 independent experiments ) .

(B) A majority of MTOCs are attached to K-fibers. The volumes of individual MTOCs were measured after 3D reconstruction. The sum volume of MTOCs attached to K-fibers relative to the total volume of MTOCs was calculated for each oocyte (n=5 oocytes from 2 independent experiments).


( C ) K - fibers extend to spindle poles . The length of K - fibers and distance between kinetochores and a spindle pole ( KT - to - Pole distance ) were measured . K - fiber length relative to the KT - to - Pole distance was calculated ( n = 90 K - fibers of 5 oocytes from 2 independent experiments ) .

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(C) K-fibers extend to spindle poles. The length of K-fibers and distance between kinetochores and a spindle pole (KT-to-Pole distance) were measured. K-fiber length relative to the KT-to-Pole distance was calculated (n=90 K-fibers of 5 oocytes from 2 independent experiments).


B Measurement of 2 - NBDG uptake in different cell lines by flow cytometry ( n = 4 biological replicates ) .

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B Measurement of 2-NBDG uptake in different cell lines by flow cytometry (n=4 biological replicates).


C Quantitative RT - PCR detection of glycolytic gene expression levels in HaCaT cells upon miR - 31 overexpression . β - actin was used as a house - keeping control gene ( n = 3 biological replicates )

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C Quantitative RT-PCR detection of glycolytic gene expression levels in HaCaT cells upon miR-31 overexpression. β-actin was used as a house-keeping control gene (n=3 biological replicates)


G and H Glycolysis stress test of HaCaT cells upon UK5099 ( G , n = 4 biological replicates ) or PC siRNA ( si - PC , H , n = 5 biological replicates ) in 1mM glucose condition . ECAR and OCR were normalized to the amount of protein .

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G and H Glycolysis stress test of HaCaT cells upon UK5099 (G, n=4 biological replicates) or PC siRNA (si-PC, H, n=5 biological replicates) in 1mM glucose condition. ECAR and OCR were normalized to the amount of protein.


B Western blot analysis of GLS , GS , GOT1 , GOT2 , AGC1 in HaCaT cells upon miR - 31 overexpression , Tubulin α was used as loading control .

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B Western blot analysis of GLS, GS, GOT1, GOT2, AGC1 in HaCaT cells upon miR-31 overexpression, Tubulin α was used as loading control.


C Violin plot of glutamine consumption , glutamate secretion , and cystine consumption of HaCaT cells upon different treatments ( n = 12 biological replicates ) .

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C Violin plot of glutamine consumption, glutamate secretion, and cystine consumption of HaCaT cells upon different treatments (n=12 biological replicates).


G Cellular and extracellular levels of metabolites were detected by targeted metabolomics using C13 glutamine tracing protocol in a time course ( n = 3 biological replicates ) .

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G Cellular and extracellular levels of metabolites were detected by targeted metabolomics using C13 glutamine tracing protocol in a time course (n=3 biological replicates).


B Cell viability of HaCaT cell was measured by MTS assay ( n = 4 biological replicates ) .

B Cell viability of HaCaT cell was measured by MTS assay (n=4 biological replicates).


C Quantitative RT - PCR detection of Has - miR - 31 - 5p expression levels in HaCaT cells exposed to a range of glucose ( n = 4 biological replicates ) . RNU6 - 1 was used as house control gene .

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C Quantitative RT-PCR detection of Has-miR-31-5p expression levels in HaCaT cells exposed to a range of glucose (n=4 biological replicates). RNU6-1 was used as house control gene.


D Western blot analysis of GS in HaCaT cells with miR - 31 overexpression under different concentrations of glucose or 2 - DG treatment , and Tubulin α was used as a loading control .

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D Western blot analysis of GS in HaCaT cells with miR-31 overexpression under different concentrations of glucose or 2-DG treatment, and Tubulin α was used as a loading control.


G and H FACS analysis of Th17 differentiation upon TFB - TBOA treatment ( G , n = 4 biological replicates ) or AOA and iGOT1 treatments ( H ) , n ≥ 3 biological replicates in each group .

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G and H FACS analysis of Th17 differentiation upon TFB-TBOA treatment (G, n=4 biological replicates) or AOA and iGOT1 treatments (H), n≥3 biological replicates in each group.


A Representative picture of in situ hybridization ( ISH ) staining of miR - 31 in skin biopsies of healthy individuals and psoriasis patients ( n = 5 individuals in each group ) . Quantification of results showing the average level of miR - 31 in skin epidermis ( right panel ) . B , basal layer ; S + G , spinous and granular layers . Scale bars : 100 μm .

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A Representative picture of in situ hybridization (ISH) staining of miR-31 in skin biopsies of healthy individuals and psoriasis patients (n=5 individuals in each group). Quantification of results showing the average level of miR-31 in skin epidermis (right panel). B, basal layer; S+G, spinous and granular layers. Scale bars: 100 μm.


B Representative pictures of Immunohistochemical staining of GLUT1 , GLS , and GS in skin biopsies of HC and Ps ( n = 5 individuals in each group ) . Scale bars : 200 μm .

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B Representative pictures of Immunohistochemical staining of GLUT1, GLS, and GS in skin biopsies of HC and Ps (n=5 individuals in each group). Scale bars: 200 μm.


Disease activity was evaluated by epidermal thickness ( E ) n = 5 mice in Vehicle and n = 6 mice in other groups .

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Disease activity was evaluated by epidermal thickness (E) n=5 mice in Vehicle and n=6 mice in other groups.


H FACS analysis of Th17 cells proportion in splenic lymphocytes of mice ( n = 5 mice in Vehicle and n = 6 mice in other groups ) .

H FACS analysis of Th17 cells proportion in splenic lymphocytes of mice (n=5 mice in Vehicle and n=6 mice in other groups).


I Enzyme - linked immunosorbent assay for the determination of IL - 17A levels in serum of mice ( n = 5 mice in Vehicle and n = 6 mice in other groups ) .

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I Enzyme-linked immunosorbent assay for the determination of IL-17A levels in serum of mice (n=5 mice in Vehicle and n=6 mice in other groups).


J Quantitative RT - PCR detection of mmu - miR - 31 - 5p expression levels in skin of mice ( n = 5 mice in each group ) .

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J Quantitative RT-PCR detection of mmu-miR-31-5p expression levels in skin of mice (n=5 mice in each group).


( A ) Immunoblotting for Dnmt1 , Dmnt3a and Dmnt3b in the spinal cords of AR - 97Q , AR - 24Q , and wild - type mice . ( B ) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up - regulated in AR - 97Q mice ( n = 3 ) . Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

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(A) Immunoblotting for Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of AR-97Q, AR-24Q, and wild-type mice. (B) Quantitative densitometry analysis indicated that Dnmt1 spinal cord level was up-regulated in AR-97Q mice (n = 3). Dnmt3a and Dnmt3b spinal cord level was not significantly different among wild-type, AR-24Q, and AR-97Q mice (n = 3).


( C ) Quantification of the Dnmts mRNA levels of the spinal cord in wild - type , AR - 24Q , and AR - 97Q mice using RT - qPCR ( n = 3 ) .

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(C) Quantification of the Dnmts mRNA levels of the spinal cord in wild-type, AR-24Q, and AR-97Q mice using RT-qPCR (n = 3).


( D and E ) Representative immunohistochemical images depicting the expression of Dnmt1 , Dmnt3a and Dmnt3b in the spinal cords of male AR - 97Q , AR - 24Q , and wild - type mice and quantification of the immunoreactivity for Dnmts ( n = 25 neurons from three mice of each group ) .

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(D and E) Representative immunohistochemical images depicting the expression of Dnmt1, Dmnt3a and Dmnt3b in the spinal cords of male AR-97Q, AR-24Q, and wild-type mice and quantification of the immunoreactivity for Dnmts (n = 25 neurons from three mice of each group).


( F ) Double - immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR - 97Q mouse .

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(F) Double-immunofluorescence staining for Dnmt1 and 1C2 in the spinal motor neurons of an AR-97Q mouse.


( G ) Immunoblotting for Dnmt1 of the skeletal muscle of wild - type and AR - 97Q mice . ( H ) Quantification of the signal intensity of Dnmt1 bands ( n = 3 ) .

(G) Immunoblotting for Dnmt1 of the skeletal muscle of wild-type and AR-97Q mice. (H) Quantification of the signal intensity of Dnmt1 bands (n = 3).


( A ) Immunoblotting of NSC34 cells expressing AR - 24Q or AR - 97Q ( NSC24Q and NSC97Q , respectively ) that were treated with DHT for Dnmt1 , Dnmt3a and Dnmt3b , and quantification of the signal intensities of bands corresponding to Dnmt1 , Dnmt3a and Dnmt3b ( n = 3 ) .

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(A) Immunoblotting of NSC34 cells expressing AR-24Q or AR-97Q (NSC24Q and NSC97Q, respectively) that were treated with DHT for Dnmt1, Dnmt3a and Dnmt3b, and quantification of the signal intensities of bands corresponding to Dnmt1, Dnmt3a and Dnmt3b (n = 3).


( B ) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT - qPCR ( n = 3 ) .

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(B) Relative mRNA levels of Dnmt1 in NSC24Q and NSC97Q cells measured with RT-qPCR (n = 3).


( C ) Protein levels of Dnmt1 , Dnmt3a and Dnmt3b in DHT - treated and - untreated NSC97Q cells by Western blotting analysis .

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(C) Protein levels of Dnmt1, Dnmt3a and Dnmt3b in DHT-treated and -untreated NSC97Q cells by Western blotting analysis.


( D ) RT - qPCR analysis of Dnmt1 in DHT - treated and - untreated NSC97Q cells ( n = 3 ) .

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(D) RT-qPCR analysis of Dnmt1 in DHT-treated and -untreated NSC97Q cells (n = 3).


( E , F ) Quantitative assessment of cell viability changes due to the siRNA - mediated knock down of Dnmt1 , Dnmt3a or Dnmt3b in NSC97Q cells with DHT ( E ) or without DHT ( F ) using the WST - 8 assay ( n = 3 ) .

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(E, F) Quantitative assessment of cell viability changes due to the siRNA-mediated knock down of Dnmt1, Dnmt3a or Dnmt3b in NSC97Q cells with DHT (E) or without DHT (F) using the WST-8 assay (n = 3).


( G ) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST - 8 assay ( n = 3 per group ) .

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(G) Quantitative assessment of the cell viability of NSC97Q cells treated with DHT and RG108 using the WST-8 assay (n = 3 per group).


( H ) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1 , Dnmt3a and Dnmt3b .

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(H) Immunoblotting of NSC97Q cells treated with DHT and RG108 for Dnmt1, Dnmt3a and Dnmt3b.


( I , J ) Cell viability analysis with siRNA - mediated knockdown of Dnmts in DHT - untreated NSC97Q ( I ) and DHT - treated NSC24Q ( J ) .

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(I, J) Cell viability analysis with siRNA-mediated knockdown of Dnmts in DHT-untreated NSC97Q (I) and DHT-treated NSC24Q (J).


( A - D ) Grip power ( A ) , body weight ( B ) , rotarod performance ( C ) ( Two - way ANOVA with Tukey ' s test ) and survival rate ( D ) ( Kaplan - Meier analysis and log - rank test ) of AR - 97Q mice treated with or without RG108 ( DMSO , n = 20 ; 0 . 5 mg / dL , n = 20 ; 2 . 0 mg / dL , n = 20 ) . All parameters were significantly improved in AR - 97Q mice treated with 2 . 0 mg / dL RG108 compared with those treated with DMSO : * P < 0 . 05 ( grip ) ; * P < 0 . 05 ( body weight ) ; * P < 0 . 05 ( rotarod ) ; and * P < 0 . 05 ( survival ) .

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(A-D) Grip power (A), body weight (B), rotarod performance (C) (Two-way ANOVA with Tukey's test) and survival rate (D) (Kaplan-Meier analysis and log-rank test) of AR-97Q mice treated with or without RG108 (DMSO, n =20; 0.5 mg/dL, n = 20; 2.0 mg/dL, n = 20). All parameters were significantly improved in AR-97Q mice treated with 2.0 mg/dL RG108 compared with those treated with DMSO: *P < 0.05 (grip); *P < 0.05 (body weight); *P < 0.05 (rotarod); and *P < 0.05 (survival).


( E ) Representative photograph of a 13 - week - old AR - 97Q mouse treated with DMSO ( left ) and an age - matched AR - 97Q mouse treated with 2 . 0 mg / dL RG108 ( right ) . AR - 97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO - treated AR - 97Q mouse of same age . The white compact masses ( arrow ) of mice head are the cement covering the surgical site .

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(E) Representative photograph of a 13-week-old AR-97Q mouse treated with DMSO (left) and an age-matched AR-97Q mouse treated with 2.0 mg/dL RG108 (right). AR-97Q mouse medicated with RG108 maintained muscle volume and body size compared with DMSO-treated AR-97Q mouse of same age. The white compact masses (arrow) of mice head are the cement covering the surgical site.


( F , G ) Immunoblots ( F ) and relative intensities ( G ) of the Dnmt1 - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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(F,G) Immunoblots (F) and relative intensities (G) of the Dnmt1-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3 per group).


( H , I ) Dnmt1 immunohistochemistry in spinal cord sections from AR - 97Q mice treated with or without RG108 ( n = 3 per group ) .

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(H,I) Dnmt1 immunohistochemistry in spinal cord sections from AR-97Q mice treated with or without RG108 (n = 3 per group).


( A ) Immunohistochemistry of the spinal cords of AR - 97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody . ( B ) Quantification of 1C2 - positive motor neurons in the spinal cords ( n = 5 per group ) .

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(A) Immunohistochemistry of the spinal cords of AR-97Q mice treated with DMSO or RG108 for polyglutamine using a 1C2 antibody. (B) Quantification of 1C2-positive motor neurons in the spinal cords (n = 5 per group).


( C ) Immunoblotting of the spinal cords of AR - 97Q mice treated with or without RG108 for AR . ( D ) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR - 97Q mice treated with DMSO or RG108 ( n = 3 ) .

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(C) Immunoblotting of the spinal cords of AR-97Q mice treated with or without RG108 for AR. (D) Densitometric analyses to quantify AR accumulation in the spinal motor neurons of AR-97Q mice treated with DMSO or RG108 (n = 3).


( E ) Relative mRNA expression levels of human AR in AR - 97Q mice spinal cord with or without RG108 treatment ( n = 3 ) .

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(E) Relative mRNA expression levels of human AR in AR-97Q mice spinal cord with or without RG108 treatment (n = 3).


Immunohistochemistry for ChAT in the spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

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Immunohistochemistry for ChAT in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).


quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR - 97Q mice ( n = 5 per group ) .

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quantitative analysis of the neuron sizes in the spinal cord anterior horns of AR-97Q mice (n = 5 per group).


( H , I ) Western blot analysis ( H ) and relative signal intensities ( I ) of the ChAT - immunoreactive bands of spinal cords from AR - 97Q mice treated with or without RG108 ( n = 3 ) .

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(H,I) Western blot analysis (H) and relative signal intensities (I) of the ChAT-immunoreactive bands of spinal cords from AR-97Q mice treated with or without RG108 (n = 3).


( A ) Immunoblotting of SH - SY5Y cells stably expressing AR - 24Q ( SH24Q ) or AR - 97Q ( SH97Q ) for Dnmt1 , Dnmt3a and Dnmt3b . ( B ) Densitometry quantification showing that Dnmt1 level was up - regulated in SH97Q cells compared with SH24Q cells ( n = 3 ) .

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(A) Immunoblotting of SH-SY5Y cells stably expressing AR-24Q (SH24Q) or AR-97Q (SH97Q) for Dnmt1, Dnmt3a and Dnmt3b. (B) Densitometry quantification showing that Dnmt1 level was up-regulated in SH97Q cells compared with SH24Q cells (n = 3).


( C , D ) Immunoblots for Dnmts of SH97Q with or without DHT treatment ( C ) and quantification data ( D ) ( n = 3 ) .

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(C, D) Immunoblots for Dnmts of SH97Q with or without DHT treatment (C) and quantification data (D) (n = 3).


( E ) Seven genes with DNA hyper - methylation in their CpG islands were identified by DNA methylation array analysis .

(E) Seven genes with DNA hyper-methylation in their CpG islands were identified by DNA methylation array analysis.


( F ) The relative mRNA levels of CDC25B , GFRA3 , NPY , HES5 , SCTR , LEF1 - AS1 and CABS1 , normalized to beta - 2 microglobulin , in DHT - treated SH24Q and SH97Q cells were measured using RT - qPCR ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The relative mRNA levels of CDC25B, GFRA3, NPY, HES5, SCTR, LEF1-AS1 and CABS1, normalized to beta-2 microglobulin, in DHT-treated SH24Q and SH97Q cells were measured using RT-qPCR (n = 3).


( G ) RT - qPCR analysis indicated the decrease of Hes5 mRNA level in DHT - treated SH97Q cells compared with DHT - untreated SH97Q cells ( n = 3 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

(G) RT-qPCR analysis indicated the decrease of Hes5 mRNA level in DHT-treated SH97Q cells compared with DHT-untreated SH97Q cells (n = 3).


( H ) Methylation - specific PCR of SH24Q and SH97Q cells that were treated with DHT .

(H) Methylation-specific PCR of SH24Q and SH97Q cells that were treated with DHT.


( I ) Hes5 immunoreactivity of spinal motor neurons in wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

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

(I) Hes5 immunoreactivity of spinal motor neurons in wild-type, AR-24Q, and AR-97Q mice (n = 3).


( J ) Hes5 mRNA levels in the spinal cords of wild - type , AR - 24Q , and AR - 97Q mice ( n = 3 ) .

O O O O O O 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) Hes5 mRNA levels in the spinal cords of wild-type, AR-24Q, and AR-97Q mice (n = 3).


DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

DNA methylation levels of the HES5 promoter region of SH97Q cells treated with DHT and RG108.


HES5 mRNA levels of SH97Q cells treated with DHT and RG108 .

HES5 mRNA levels of SH97Q cells treated with DHT and RG108.


DNA methylation levels of the Hes5 promoter region in DHT - treated NSC97Q cells treated with or without RG108 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

DNA methylation levels of the Hes5 promoter region in DHT-treated NSC97Q cells treated with or without RG108.


mRNA levels of Hes5 ( n = 3 ) in DHT - treated NSC97Q cells treated with or without RG108 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

mRNA levels of Hes5 (n = 3) in DHT-treated NSC97Q cells treated with or without RG108.


( E ) Hes5 mRNA levels in the spinal cords of AR - 97Q mice treated with or without RG108 ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

(E) Hes5 mRNA levels in the spinal cords of AR-97Q mice treated with or without RG108 (n = 3).


( F , G ) Hes5 immunoreactivity of spinal motor neurons in AR - 97Q mice treated with or without RG108 ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

(F, G) Hes5 immunoreactivity of spinal motor neurons in AR-97Q mice treated with or without RG108 (n = 3).


( A ) Viability of DHT - treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST - 8 assay ( n = 3 ) .

(A) Viability of DHT-treated NSC97Q cells treated with Hes5 and control siRNA measured by the WST-8 assay (n = 3).


( B ) Immunoblots of DHT - administrated NSC97Q cells treated with Hes5 siRNA ( n = 3 ) .

(B) Immunoblots of DHT-administrated NSC97Q cells treated with Hes5 siRNA (n = 3).


( C ) Quantitative cell viability analysis revealed that siRNA - mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT ( n = 3 ) , as determined by the WST - 8 assay .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantitative cell viability analysis revealed that siRNA-mediated Hes5 knock down negated the therapeutic effect of RG108 in NSC97Q cells that were treated with DHT (n = 3), as determined by the WST-8 assay.


Cell viability measured with the WST - 8 assay of DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 3 ) .

Cell viability measured with the WST-8 assay of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).


Cell viability measured with immunoblotting of DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 3 ) .

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

Cell viability measured with immunoblotting of DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 3).


( F ) Quantification of LDH release from DHT - treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector ( n = 4 ) .

O O O O 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) Quantification of LDH release from DHT-treated NSC97Q cells transfected with a mock plasmid or the Hes5 vector (n = 4).


( G ) Immunoblots of DHT - administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR . ( H ) AR aggregation as quantified by densitometry ( n = 3 ) .

O O O O O O O O O O O O O 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) Immunoblots of DHT-administrated NSC97Q cells transfected with a mock plasmid or the Hes5 vector for AR. (H) AR aggregation as quantified by densitometry (n = 3).


( A ) Immunoblots of NSC34 cells treated with Hes5 siRNA . ( B ) Quantification of protein levels of pSmad2 , Smad2 , phospho - IkBα and heat shock factor - 1 ( Hsf1 ) ( n = 3 ) .

O O O O O O O O O O O O O O O O O O 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) Immunoblots of NSC34 cells treated with Hes5 siRNA. (B) Quantification of protein levels of pSmad2, Smad2, phospho-IkBα and heat shock factor-1 (Hsf1) (n = 3).


( C ) siRNA - mediated knockdown of Hes5 down - regulated Hes5 protein level ( n = 3 ) .

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

(C) siRNA-mediated knockdown of Hes5 down-regulated Hes5 protein level (n = 3).


( D ) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment ( n = 3 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(D) Western blots of Smad2 and pSmad2 in NSC97Q cells with or without DHT treatment (n = 3).


( E ) Protein levels of Smad2 , pSmad2 , and Hes5 in NSC97Q cells transfected with Hes5 vector ( n = 3 ) .

O 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) Protein levels of Smad2, pSmad2, and Hes5 in NSC97Q cells transfected with Hes5 vector (n = 3).


( F ) Immunoblots of Smad2 , pSmad2 , and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q ( n = 3 ) .

(F) Immunoblots of Smad2, pSmad2, and Hes5 in primary cortical neurons treated with lentiviral vector containing AR24Q and AR97Q (n = 3).


( G ) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q ( n = 3 ) .

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

(G) Relative mRNA levels of Hes5 in primary cortical neurons expressing AR24Q and AR97Q (n = 3).


( H ) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q ( n = 6 ) .

(H) Relative mRNA levels of Hes5 in primary motor neurons expressing AR24Q and AR97Q (n = 6).


( I ) Western blots of Smad2 , pSmad2 , and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5 - containing lentiviral vector .

(I) Western blots of Smad2, pSmad2, and Hes5 in primary cortical neurons expressing AR97Q treated with mock plasmid or Hes5-containing lentiviral vector.


( B ) Structured illumination microscopy image of perimCherry / cytoGFP E . coli confirming localization of mCherry ( red ) in the periplasm and GFP ( green ) in the cytosol . Scale bar = 3 µm .

O O O O O O O O O O O O O O O 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) Structured illumination microscopy image of perimCherry/cytoGFP E. coli confirming localization of mCherry (red) in the periplasm and GFP (green) in the cytosol. Scale bar = 3 µm.


( C ) Outer membrane damage ( mCherry intensity ) and inner membrane damage ( % Sytox positive ) of perimCherry / cytoGFP E . coli bacteria exposed to ( different concentrations of ) human serum . Inner membrane damage correlates with killing ( samples where bacteria are killed are indicated with gray shadings and a cross , see CFU data in Fig EV1b ) . Data information : ( C - E ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of perimCherry/cytoGFP E. coli bacteria exposed to (different concentrations of) human serum. Inner membrane damage correlates with killing (samples where bacteria are killed are indicated with gray shadings and a cross, see CFU data in Fig EV1b). Data information: (C-E) Data represent mean ± SD of 3 independent experiments.


( D ) Serum - induced inner membrane damage ( % Sytox positive ) of different Gram - negative strains depends on MAC components C5 and C8 , but not on lysozyme ( 10 % serum ) . Dotted line represents the detection limit of the assay . Data information : ( D , E ) The cfu / ml ( Fig . 1E , 2C ) and sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " and " C5b6MAC " were all generated from the same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Serum-induced inner membrane damage (% Sytox positive) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


, E Serum - induced killing ( CFU / ml ) of different Gram - negative strains depends on MAC components C5 and C8 , but not on lysozyme ( 10 % serum ) . Dotted line represents the detection limit of the assay . Data information : ( D , E ) The cfu / ml ( Fig . 1E , 2C ) and sytox measurements ( Fig . 1D , 2E ) of " Buffer " , " Serum " , " ΔC5 " , " ΔC8 " " Δlysozym " and " C5b6MAC " were all generated from the same experiment . ( C - E ) Data represent mean ± SD of 3 independent experiments . ( D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Serum-induced killing (CFU/ml) of different Gram-negative strains depends on MAC components C5 and C8, but not on lysozyme (10% serum). Dotted line represents the detection limit of the assay. Data information: (D, E) The cfu/ml (Fig. 1E, 2C) and sytox measurements (Fig. 1D, 2E) of "Buffer", "Serum", "ΔC5", "ΔC8" "Δlysozym" and "C5b6MAC" were all generated from the same experiment. (C-E) Data represent mean ± SD of 3 independent experiments. (D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( B ) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7 . After washing , erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured . ( B - E ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysis of human erythrocytes after exposure to a concentration range of preassembled C5b6 in the presence of 100 nM C7. After washing, erythrocytes were exposed to 20 nM C8 and 100 nM C9 for 30 minutes after which the OD405 nm of the supernatant was measured. (B-E) Data represent mean ± SD of 3 independent experiments.


C ) Bacterial viability of three Gram - negative strains after exposure to buffer , 10 % human serum or C5b6MAC . Buffer and serum conditions are the same as Fig . 1E .

O O O O O O O O O O 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) Bacterial viability of three Gram-negative strains after exposure to buffer, 10% human serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1E.


( D ) Permeabilization of the outer , but not inner membrane of perimCherry / cytoGFP E . coli cells exposed to C5b6MAC ( different concentrations of C5b6 with fixed concentrations of C7 - C9 ) . ( B - E ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Permeabilization of the outer, but not inner membrane of perimCherry/cytoGFP E. coli cells exposed to C5b6MAC (different concentrations of C5b6 with fixed concentrations of C7-C9). (B-E) Data represent mean ± SD of 3 independent experiments.


E ) Inner membrane damage of three Gram - negative strains exposed to buffer , 10 % serum or C5b6MAC . Buffer and serum conditions are the same as Fig . 1D .

O O O O O O O O O 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) Inner membrane damage of three Gram-negative strains exposed to buffer, 10% serum or C5b6MAC. Buffer and serum conditions are the same as Fig. 1D.


( B ) Bacterial viability of convertase - labeled bacterial strains exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of convertase-labeled bacterial strains exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( C ) Bacterial viability of convertase - labeled E . coli MG1655 exposed to a concentration range of C5 in the presence of 100 nM C6 , 100 nM C7 , 20 nM C8 and 100 nM C9 . ' Ctrl ' indicates bacteria that are pretreated with heat - inactivated ΔC5 serum . Dotted line represents the detection limit of the assay . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of convertase-labeled E. coli MG1655 exposed to a concentration range of C5 in the presence of 100 nM C6, 100 nM C7, 20 nM C8 and 100 nM C9. 'Ctrl' indicates bacteria that are pretreated with heat-inactivated ΔC5 serum. Dotted line represents the detection limit of the assay. Data information: (B-E) Data represent mean ± SD of 3 independent experiments.


( D ) Bacterial viability of convertase labeled E . coli MG1655 exposed to C5 - C9 or conditions lacking one MAC component . As an extra control , convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of convertase labeled E. coli MG1655 exposed to C5-C9 or conditions lacking one MAC component. As an extra control, convertase formation was blocked during ΔC5 serum incubation by adding 5 µM compstatin. Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( E ) Bacterial viability of E . coli MG1655 exposed to FB depleted serum in the presence of 20 µg / ml OmCI ( to deposit C4b and C3b without Bb ) . After washing , bacteria were exposed to C5 - C9 in the presence or absence of C1 and C2 ( to generate classical pathway C5 convertases , C4b2aC3b ) . Data information : ( B - E ) Data represent mean ± SD of 3 independent experiments . ( B , D , E ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 or * * * * p ≤ 0 . 0001 .

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(E) Bacterial viability of E. coli MG1655 exposed to FB depleted serum in the presence of 20 µg/ml OmCI (to deposit C4b and C3b without Bb). After washing, bacteria were exposed to C5-C9 in the presence or absence of C1 and C2 (to generate classical pathway C5 convertases, C4b2aC3b). Data information: (B-E) Data represent mean ± SD of 3 independent experiments. (B, D, E) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05, ** p≤0.01, *** p≤0.001 or **** p≤0.0001.


( A ) Outer membrane damage ( mCherry intensity ) and inner membrane damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli cells incubated with a concentration range of C5 and fixed concentrations of C6 - C9 . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Outer membrane damage (mCherry intensity) and inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli cells incubated with a concentration range of C5 and fixed concentrations of C6-C9. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.


( B ) Inner membrane damage of perimCherry / cytoGFP E . coli exposed to a concentration range of ΔC5 serum and , after washing , to C5 - C9 . As controls , bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O 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) Inner membrane damage of perimCherry/cytoGFP E. coli exposed to a concentration range of ΔC5 serum and, after washing, to C5-C9. As controls, bacteria were incubated with heat inactivated ΔC5 serum or 5 µM compstatin was added to the ΔC5 serum to block C3b deposition. Data information: (A-C) Data represent mean ± SD of 3 independent experiments.


( C ) Inner membrane damage of three different convertase - labeled bacteria exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Data information : ( A - C ) Data represent mean ± SD of 3 independent experiments . ( C ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * p ≤ 0 . 05 or * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Inner membrane damage of three different convertase-labeled bacteria exposed to buffer (Conv) or C5-C9 (Conv-MAC). Data information: (A-C) Data represent mean ± SD of 3 independent experiments. (C) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as * p≤0.05 or ** p≤0.01.


( D ) Confocal microscopy images of convertase - labeled perimCherry / cytoGFP E . coli exposed to buffer ( Conv ) or C5 - C9 ( Conv - MAC ) . Unlabeled bacteria exposed to 1 % serum served as control . Green = GFP , Red = To - pro - 3 DNA dye . Scale bars = 3 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O

(D) Confocal microscopy images of convertase-labeled perimCherry/cytoGFP E. coli exposed to buffer (Conv) or C5-C9 (Conv-MAC). Unlabeled bacteria exposed to 1% serum served as control. Green = GFP, Red = To-pro-3 DNA dye. Scale bars = 3 µm.


( B ) Bacterial viability of convertase - labeled E . coli MG1655 exposed to Buffer ( Conv ) , preassembled C5b6 ( Conv + C5b6MAC ) or a mixture of C5 and C6 ( Conv - MAC ) , in the presence of C7 , C8 and C9 . Dotted line represents the detection limit of the assay . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bacterial viability of convertase-labeled E. coli MG1655 exposed to Buffer (Conv), preassembled C5b6 (Conv + C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of C7, C8 and C9. Dotted line represents the detection limit of the assay. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


( C ) Inner membrane damage ( % Sytox positive ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6 , in the presence of 100 nM C7 . After washing , bacteria were exposed to 20 nM C8 and 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 .

(C) Inner membrane damage (% Sytox positive) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


( D ) outer membrane damage ( mCherry ) of convertase - labeled perimCherry / cytoGFP E . coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6 , in the presence of 100 nM C7 . After washing , bacteria were exposed to 20 nM C8 and 100 nM C9 . Data information : ( B - D ) Data represent mean ± SD of 3 independent experiments . ( B ) Statistical analysis was done using a ratio paired two - tailed t - test and displayed only when significant as * * p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) outer membrane damage (mCherry) of convertase-labeled perimCherry/cytoGFP E. coli exposed to a concentration range of preassembled C5b6 or a mixture of C5 and C6, in the presence of 100 nM C7. After washing, bacteria were exposed to 20 nM C8 and 100 nM C9. Data information: (B-D) Data represent mean ± SD of 3 independent experiments. (B) Statistical analysis was done using a ratio paired two-tailed t-test and displayed only when significant as ** p≤0.01.


A , B Step - wise assembly of MAC on convertase - labeled bacteria . Convertase - labeled bacteria were incubated with C5 / C6 or C5 / C6 / C7 for 15 minutes , and subsequently washed ( @ ) or treated with 10 μg / ml Eculizumab ( Ecu ) . Then , the remaining MAC components ( C7 - 9 for C5 / C6 or C8 - 9 for C5 / C6 / C7 respectively ) were added to the incubation mixture . In the control conditions ( Conv - MAC ) , the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor . ( A ) Outer membrane ( mCherry ) and ( B ) inner membrane damage ( % Sytox positive ) were determined . Data information : ( A - B ) Data represent mean ± SD of 3 independent experiments . Statistical analysis was done using a ratio paired two - tailed t - test in which the test conditions were compared to Conv - MAC and displayed only when significant as * p ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Step-wise assembly of MAC on convertase-labeled bacteria. Convertase-labeled bacteria were incubated with C5/C6 or C5/C6/C7 for 15 minutes, and subsequently washed (@) or treated with 10 μg/ml Eculizumab (Ecu). Then, the remaining MAC components (C7-9 for C5/C6 or C8-9 for C5/C6/C7 respectively) were added to the incubation mixture. In the control conditions (Conv-MAC), the remaining MAC components were added to the incubation mixture without washing or adding an inhibitor. (A) Outer membrane (mCherry) and (B) inner membrane damage (% Sytox positive) were determined. Data information: (A-B) Data represent mean ± SD of 3 independent experiments. Statistical analysis was done using a ratio paired two-tailed t-test in which the test conditions were compared to Conv-MAC and displayed only when significant as * p≤0.05.


( A ) Outer and inner membrane damage of convertase - labeled bacteria exposed to different combinations of MAC components . ' @ ' indicates a washing step . Data information : Data represent mean ± SD of 3 independent experiments . ( A ) Statistical analysis was done using a one - way ANOVA and displayed only when significant as * * p ≤ 0 . 01 or * * * * p ≤ 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Outer and inner membrane damage of convertase-labeled bacteria exposed to different combinations of MAC components. '@' indicates a washing step. Data information: Data represent mean ± SD of 3 independent experiments. (A) Statistical analysis was done using a one-way ANOVA and displayed only when significant as ** p≤0.01 or **** p≤0.0001.


( B ) Outer and inner membrane damage of convertase - labeled bacteria exposed to C5 - C8 and after washing , to a concentration range of C9 . Data information : Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O 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) Outer and inner membrane damage of convertase-labeled bacteria exposed to C5-C8 and after washing, to a concentration range of C9. Data information: Data represent mean ± SD of 3 independent experiments.


( A ) Convertase - labeled bacteria were exposed to a concentration range of either preassembled C5b6 ( C5b6MAC ) or a mixture of C5 and C6 ( Conv - MAC ) , in the presence of 100 nM C7 . After washing , 20 nM C8 and 100 nM C9 - Cy3 was added . Controls at 0 nM C5b6 or C5 - C6 confirm that the detected C9 - Cy3 deposition is specifically related to MAC formation ( solid lines ) . Proper insertion of pores was assessed by a previously described shaving method with trypsin ( Moskovich & Fishelson , 2007 ) . Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg / ml trypsin for 15 min at 37 ° C ( dotted lines ) . Data information : ( A ) Data represent mean ± SD of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Convertase-labeled bacteria were exposed to a concentration range of either preassembled C5b6 (C5b6MAC) or a mixture of C5 and C6 (Conv-MAC), in the presence of 100 nM C7. After washing, 20 nM C8 and 100 nM C9-Cy3 was added. Controls at 0 nM C5b6 or C5-C6 confirm that the detected C9-Cy3 deposition is specifically related to MAC formation (solid lines). Proper insertion of pores was assessed by a previously described shaving method with trypsin (Moskovich & Fishelson, 2007). Bacteria were first incubated with MAC components for 30 minutes and subsequently treated with 20 µg/ml trypsin for 15 min at 37°C (dotted lines). Data information: (A) Data represent mean ± SD of 3 independent experiments.


( B ) Convertase labeled perimCherry / cytoGFP bacteria ( Green ) exposed to C5b6MAC or Conv - MAC . Conditions were similar to those in ( A ) , however C9 - Cy5 was used to detect MAC pores ( Red ) . 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7 , 20 nM C8 and 100 nM C9 - Cy5 . Conv + C5b6MAC and conv - MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9 - Cy5 to properly visualize pore distribution . Scale bars = 3 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Convertase labeled perimCherry/cytoGFP bacteria (Green) exposed to C5b6MAC or Conv-MAC. Conditions were similar to those in (A), however C9-Cy5 was used to detect MAC pores (Red). 100 nM of C5 and C6 or C5b6 were used in combination with 100 nM C7, 20 nM C8 and 100 nM C9-Cy5. Conv + C5b6MAC and conv-MAC images were taken in separate experiments in which laser settings were adjusted to the staining intensity of C9-Cy5 to properly visualize pore distribution. Scale bars = 3 µm.


( C Atomic force microscopy analysis of E . coli BL21 and MG1655 immobilized using the Poly - L - Lysine protocol . ( C ) Entire bacteria and high - resolution comparisons of untreated and convertase - labeled E . coli BL21 exposed to C5 - C9 ( Conv - MAC ) for 10 minutes . Scale bars : 800 nm ( left ) and 30 nm ( right ) . Height Scales : 1 . 01 µm ( left ) , 8 nm ( top right ) , 22 nm ( bottom right ) . Width of magnification boxes ( left ) : 42 nm , height scales : 8 nm ( top left ) and 13 nm ( bottom left ) . Arrows highlight E . coli porin structures , an asterisk highlights the C5b - 7 stalk . Height profiles ( bottom ) are shown for the white dashed lines in the images .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Atomic force microscopy analysis of E. coli BL21 and MG1655 immobilized using the Poly-L-Lysine protocol. (C) Entire bacteria and high-resolution comparisons of untreated and convertase-labeled E. coli BL21 exposed to C5-C9 (Conv-MAC) for 10 minutes. Scale bars: 800 nm (left) and 30 nm (right). Height Scales: 1.01 µm (left), 8 nm (top right), 22 nm (bottom right). Width of magnification boxes (left): 42 nm, height scales: 8 nm (top left) and 13 nm (bottom left). Arrows highlight E. coli porin structures, an asterisk highlights the C5b-7 stalk. Height profiles (bottom) are shown for the white dashed lines in the images.


( D ) Atomic force microscopy ( height and phase images ) of convertase - labeled E . coli MG1655 exposed to a mixture of C5 and C6 ( Conv - MAC ) or preassembled C5b6 ( Conv + C5b6MAC ) , in the presence of C7 , C8 , C9 , FB and FD . Images were generated in the same experiment . Scale bars : 50 nm . Height scales : 15 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atomic force microscopy (height and phase images) of convertase-labeled E. coli MG1655 exposed to a mixture of C5 and C6 (Conv-MAC) or preassembled C5b6 (Conv + C5b6MAC), in the presence of C7, C8, C9, FB and FD. Images were generated in the same experiment. Scale bars: 50 nm. Height scales: 15 nm.


Immunoblots and qRT - PCRs showing that expression levels of miR - 203 , miR - 200 , and E - cadherin are increased after ZEB1 knockdown in Panc1 , MDA - MB - 231 . Vice versa , the drug - resistant clones of BxPC3 , H358 , and DU - 145 show increased expression of ZEB1 and decreased expression of the miRNAs and E - cadherin . n = 3 , mean ± SEM , except for H358 ( data from microarray ) . Unpaired Student ' s t - test .

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

Immunoblots and qRT-PCRs showing that expression levels of miR-203, miR-200, and E-cadherin are increased after ZEB1 knockdown in Panc1, MDA-MB-231. Vice versa, the drug-resistant clones of BxPC3, H358, and DU-145 show increased expression of ZEB1 and decreased expression of the miRNAs and E-cadherin. n = 3, mean ± SEM, except for H358 (data from microarray). Unpaired Student's t-test.


Lentiviral overexpression of miR - 200c and miR - 203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay . For the changes in EC50 values , see Table1 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Lentiviral overexpression of miR-200c and miR-203 in Panc1 and hPaca1 induces sensitivity to gemcitabine treatment as measured by MTT assay. For the changes in EC50 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Overexpression of miR - 203 decreases expression of the anti - apoptotic factor survivin and sensitizes to gemcitabine - triggered apoptosis as evaluated by cleaved caspase - 3 in Western blot and immunofluorescence . Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine , respectively , for 48 h . Scale bar 20 μm .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O O O O O O O

Overexpression of miR-203 decreases expression of the anti-apoptotic factor survivin and sensitizes to gemcitabine-triggered apoptosis as evaluated by cleaved caspase-3 in Western blot and immunofluorescence. Panc1 and hPaca1 were treated with 50 and 5 nM gemcitabine, respectively, for 48 h. Scale bar 20 μm.


MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR - 203 or all miR - 200 members . For the changes in EC80 values , see Table1 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MTT assay showing increase in gemcitabine resistance after inhibition of endogenous miRNAs in hPaca2 by specific antagomirs against miR-203 or all miR-200 members. For the changes in EC80 values, see Table1. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Overexpression of miR - 203 shows reduced numbers of the CD24 / CD44 double - positive cancer stem cell population as determined by FACS analysis . The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR - 203 overexpression in hPaca1 cells .

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

Overexpression of miR-203 shows reduced numbers of the CD24/CD44 double-positive cancer stem cell population as determined by FACS analysis. The arrow indicates the reduction in the CD24 high subpopulation and reduction in CD133 by miR-203 overexpression in hPaca1 cells.


Cancer stem cell sphere assay showing reduced sphere - forming capacity of Panc1 and hPaca1 in miR - 203 overexpression cells . Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres . n = 3 , mean ± SEM , Mann - Whitney U - test .

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

Cancer stem cell sphere assay showing reduced sphere-forming capacity of Panc1 and hPaca1 in miR-203 overexpression cells. Colonies with a diameter greater than 75 μM for Panc1 and greater 30 μM for hPaca1 cells were counted as spheres. n = 3, mean ± SEM, Mann-Whitney U-test.


B , C Histone marks were analyzed using ChIP coupled to qRT - PCR for Panc1 control versus shZEB , MDA - MB - 231 control versus shZEB ( B ) , and BxPC3 control versus gemcitabine resistant ( gr ) ( C ) . In MDA - MB - 231 and Panc1 , the active histone marks H3K4me3 , H3ac , H4ac , and H3K9ac were enriched . Vice versa , in the drug - resistant clones of BxPC3 , the active marks were reduced in the CpG islands . The repressive histone mark H3K27me3 was not detectable in the miR - 200 loci , but in the loci of miR - 203 and E - cadherin in Panc1 and MDA - MB - 231 . DNA methylation status was determined by bisulfite sequencing . Depletion of ZEB1 in MDA - MB - 231 resulted in almost complete demethylation , whereas the selection of drug - resistant , ZEB1 - expressing clones in BxPC3 induced complete methylation . n = 2 ( Panc1 ) or 3 ( MDA - MB - 231 and BxPC3 ) , mean ± SEM ; unpaired Student ' s t - test .

O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Histone marks were analyzed using ChIP coupled to qRT-PCR for Panc1 control versus shZEB, MDA-MB-231 control versus shZEB (B), and BxPC3 control versus gemcitabine resistant (gr) (C). In MDA-MB-231 and Panc1, the active histone marks H3K4me3, H3ac, H4ac, and H3K9ac were enriched. Vice versa, in the drug-resistant clones of BxPC3, the active marks were reduced in the CpG islands. The repressive histone mark H3K27me3 was not detectable in the miR-200 loci, but in the loci of miR-203 and E-cadherin in Panc1 and MDA-MB-231. DNA methylation status was determined by bisulfite sequencing. Depletion of ZEB1 in MDA-MB-231 resulted in almost complete demethylation, whereas the selection of drug-resistant, ZEB1-expressing clones in BxPC3 induced complete methylation. n = 2 (Panc1) or 3 (MDA-MB-231 and BxPC3), mean ± SEM; unpaired Student's t-test.


Heat map showing the relative expression levels after drug treatment for 48 h in Panc1 . Values measured by qRT - PCR were depicted with the software GENE - E . Only mocetinostat upregulated the miRNAs and downregulated ZEB1 .

O O O O O O O O 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-CONTROLLED_VAR O O O O O O O

Heat map showing the relative expression levels after drug treatment for 48 h in Panc1. Values measured by qRT-PCR were depicted with the software GENE-E. Only mocetinostat upregulated the miRNAs and downregulated ZEB1.


Relative expression of indicated genes in Panc1 measured by qRT - PCR after treatment with different HDAC inhibitors . Note the downregulation of ZEB1 and upregulation of miR - 203 , miR - 200 , and E - cadherin by mocetinostat . n = 3 , mean ± SEM ; unpaired Student ' s t - test . For significance , see Supplementary Table S1 .

O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Relative expression of indicated genes in Panc1 measured by qRT-PCR after treatment with different HDAC inhibitors. Note the downregulation of ZEB1 and upregulation of miR-203, miR-200, and E-cadherin by mocetinostat. n = 3, mean ± SEM; unpaired Student's t-test. For significance, see Supplementary Table S1.


Immunoblot and immunofluorescence showing that mocetinostat treatment ( 1 μM , 48 h ) reduced ZEB1 expression and induced E - cadherin in Panc1 . Expression of histone deacetylases was not altered by mocetinostat , but histone acetylation was induced . Scale bar 10 μm .

O O O O O B-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O O O

Immunoblot and immunofluorescence showing that mocetinostat treatment (1 μM, 48 h) reduced ZEB1 expression and induced E-cadherin in Panc1. Expression of histone deacetylases was not altered by mocetinostat, but histone acetylation was induced. Scale bar 10 μm.


Chromatin immunoprecipitation analysis validated mocetinostat - induced ( 1 μM , 48 h ) enrichment of the active histone marks H3ac , H4ac , H3K9ac , and H3K4me3 at ZEB1 target gene loci in Panc1 . n = 3 , mean ± SEM ; unpaired Student ' s t - test .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromatin immunoprecipitation analysis validated mocetinostat-induced (1 μM, 48 h) enrichment of the active histone marks H3ac, H4ac, H3K9ac, and H3K4me3 at ZEB1 target gene loci in Panc1. n = 3, mean ± SEM; unpaired Student's t-test.


Mocetinostat treatment reduced expression of the anti - apoptotic miR - 203 target survivin and sphere - forming capacity in Panc1 when pre - treated with mocetinostat for 48 h . n = 3 , mean ± SEM ; Mann - Whitney U - test .

B-CONTROLLED_VAR O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Mocetinostat treatment reduced expression of the anti-apoptotic miR-203 target survivin and sphere-forming capacity in Panc1 when pre-treated with mocetinostat for 48 h. n = 3, mean ± SEM; Mann-Whitney U-test.


Immunoblot and immunofluorescence for cleaved caspase - 3 showing strong increase in apoptosis in gemcitabine - resistant Panc1 after combined treatment with mocetinostat ( 1 μM ) and gemcitabine ( 50 nM ) . Scale bar 20 μm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O

Immunoblot and immunofluorescence for cleaved caspase-3 showing strong increase in apoptosis in gemcitabine-resistant Panc1 after combined treatment with mocetinostat (1 μM) and gemcitabine (50 nM). Scale bar 20 μm.


MTT assay for Panc1 treated with the indicated concentrations of mocetinostat ( left ) or SAHA ( middle ) and gemcitabine ( 72 h ) . Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability . In contrast , a combination with SAHA had no effect . For calculation of the CI and synergy between the drugs , see Table2 . Comparison of mocetinostat and SAHA alone ( right ) . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * * * P < 0 . 001 and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MTT assay for Panc1 treated with the indicated concentrations of mocetinostat (left) or SAHA (middle) and gemcitabine (72 h). Combined treatment of mocetinostat and gemcitabine significantly reduced cell viability. In contrast, a combination with SAHA had no effect. For calculation of the CI and synergy between the drugs, see Table2. Comparison of mocetinostat and SAHA alone (right). n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are ***P < 0.001 and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient - derived pancreatic cancer cells . Note that hPaca1 behaves similar to Panc1 , but mocetinostat had no significant effect in hPaca2 . Scale bar 20 μm . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 , * * P = 0 . 001 - 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; for exact P - values , see Supplementary Table S4 ) .

O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Effects of mocetinostat on cleaved caspase expression and susceptibility to gemcitabine in the patient-derived pancreatic cancer cells. Note that hPaca1 behaves similar to Panc1, but mocetinostat had no significant effect in hPaca2. Scale bar 20 μm. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05, **P = 0.001-0.01, ***P < 0.001, and ****P < 0.0001; for exact P-values, see Supplementary Table S4).


MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU - 145 and the docetaxel - resistant subclone DU - 145 DR ( left ) . Mocetinostat treatment of DU - 145 DR downregulates ZEB1 , upregulates E - cadherin , miR - 200 , and miR - 203 expression . For relative miRNA expression , the expression levels in original DU - 145 were set to 1 ( middle panels , the immunoblot panel derives from the same experiment shown in Fig1A ) . Mocetinostat sensitizes DU - 145 DR to docetaxel ( right ) . For calculation of the CI and synergy between the drugs , see Table2 . n = 3 , mean ± SEM , Dunnett ' s multiple comparisons test ( P - values in the graphs are * P = 0 . 01 - 0 . 05 ; for exact P - values , see Supplementary Table S4 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MTT assays comparing the effects of docetaxel and mocetinostat in the prostate cancer cell line DU-145 and the docetaxel-resistant subclone DU-145 DR (left). Mocetinostat treatment of DU-145 DR downregulates ZEB1, upregulates E-cadherin, miR-200, and miR-203 expression. For relative miRNA expression, the expression levels in original DU-145 were set to 1 (middle panels, the immunoblot panel derives from the same experiment shown in Fig1A). Mocetinostat sensitizes DU-145 DR to docetaxel (right). For calculation of the CI and synergy between the drugs, see Table2. n = 3, mean ± SEM, Dunnett's multiple comparisons test (P-values in the graphs are *P = 0.01-0.05; for exact P-values, see Supplementary Table S4).


Relative tumor volume ( RTV ) of Panc1 - derived tumors in NMRI nu / nu mice . Eleven days after implantation , mice were randomized according to tumor volume . Treatment with mocetinostat ( 60 or 90 mg / kg / day ) and gemcitabine ( 25 mg / kg / day ) was implemented ( day 0 ) as depicted in the scheme . Shown are the group medians of the RTVs over time ( left ) and the individual RTVs on day 32 ( right ) . n = 5 for each treatment group ; nonparametric Mann - Whitney U - test .

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Relative tumor volume (RTV) of Panc1-derived tumors in NMRI nu/nu mice. Eleven days after implantation, mice were randomized according to tumor volume. Treatment with mocetinostat (60 or 90 mg/kg/day) and gemcitabine (25 mg/kg/day) was implemented (day 0) as depicted in the scheme. Shown are the group medians of the RTVs over time (left) and the individual RTVs on day 32 (right). n = 5 for each treatment group; nonparametric Mann-Whitney U-test.


Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E - cadherin in tumor tissues of mice treated with mocetinostat . Scale bar 40 μm , inserts for higher magnifications 10 μm . Squares indicate magnified regions .

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Representative immunohistochemical stainings of serial sections showing reduced ZEB1 and increased E-cadherin in tumor tissues of mice treated with mocetinostat. Scale bar 40 μm, inserts for higher magnifications 10 μm. Squares indicate magnified regions.


Representative pictures of in situ hybridization for miR - 203 and control probe showing gain of miR - 203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat - treated xenograft tumors . Scale bar 40 μm , inserts for higher magnifications 5 μm . Squares indicate magnified regions .

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Representative pictures of in situ hybridization for miR-203 and control probe showing gain of miR-203 and associated loss of ZEB1 detected by immunohistochemistry in serial sections of mocetinostat-treated xenograft tumors. Scale bar 40 μm, inserts for higher magnifications 5 μm. Squares indicate magnified regions.


Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice . Panc1 cells were pre - treated with mocetinostat ( 1 μM ) and / or gemcitabine ( 50 nM ) for 48 h , followed by a 7 - day recovery period before being injected subcutaneously ( left ) . Equal numbers of viable cells were injected in 75 μl volume . At day 9 after injection , tumor growth was detectable in all groups ( lower right ) . To better visualize and compare tumor growth , the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted ( upper right ) . The individual absolute tumor volumes on day 37 ( lower left ) and the group medians of the absolute tumor volumes over time ( lower right ) are shown . For cells pre - treated with the combination of mocetinostat and gemcitabine , tumor growth was arrested . n = 4 for each treatment group ; nonparametric Mann - Whitney U - test .

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Schematic outline and results for xenografts of ex vivo treated Panc1 in Foxn1 nude mice. Panc1 cells were pre-treated with mocetinostat (1 μM) and/or gemcitabine (50 nM) for 48 h, followed by a 7-day recovery period before being injected subcutaneously (left). Equal numbers of viable cells were injected in 75 μl volume. At day 9 after injection, tumor growth was detectable in all groups (lower right). To better visualize and compare tumor growth, the tumor volume at day 9 was set to 1 and the increasing slope of the tumor volume to day 37 is depicted (upper right). The individual absolute tumor volumes on day 37 (lower left) and the group medians of the absolute tumor volumes over time (lower right) are shown. For cells pre-treated with the combination of mocetinostat and gemcitabine, tumor growth was arrested. n = 4 for each treatment group; nonparametric Mann-Whitney U-test.


A , B Relative expression levels of miR - 203 and miR - 200c in pancreatic adenocarcinomas . ( A ) In normal versus tumor tissue of the same case ( n = 6 cases ) , ( B ) in the non - recurrence ( n = 10 cases ) versus the recurrence group ( n = 11 cases ) . The mean value of the lower group in each figure was set to 1 . Nonparametric Mann - Whitney U - test .

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A, B Relative expression levels of miR-203 and miR-200c in pancreatic adenocarcinomas. (A) In normal versus tumor tissue of the same case (n = 6 cases), (B) in the non-recurrence (n = 10 cases) versus the recurrence group (n = 11 cases). The mean value of the lower group in each figure was set to 1. Nonparametric Mann-Whitney U-test.


Normalized RPF count profile averaged for all E . coli transcripts . Profiles generated for cells grown in the absence and presence of IPTG ( 1 mM ) . Start and stop codons are shaded .

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Normalized RPF count profile averaged for all E. coli transcripts. Profiles generated for cells grown in the absence and presence of IPTG (1 mM). Start and stop codons are shaded.


Bar chart of all measured RBS initiation rates ranked by their strength . Strong RBSs with initiation rates > 1 ribosome / s are highlighted in red . ( D ) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength . Stop codons with translation termination efficiency > 0 . 99 are highlighted in red . ( E ) Distribution of initiation rates for cells grown in the absence and presence of IPTG ( 1 mM ) . ( F ) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG ( 1 mM ) .

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Bar chart of all measured RBS initiation rates ranked by their strength. Strong RBSs with initiation rates >1 ribosome/s are highlighted in red. (D) Bar chart of all measured translation termination efficiencies at stop codons ranked by their strength. Stop codons with translation termination efficiency >0.99 are highlighted in red. (E) Distribution of initiation rates for cells grown in the absence and presence of IPTG (1 mM). (F) Distribution of translation termination efficiencies for cells grown in the absence and presence of IPTG (1 mM).


Comparison of protein synthesis rate of endogenous E . coli genes measured using Ribo - seq from this study ( in molecules / s units ) and from that by Li et al . ( Li et al , 2014 ) ( in molecules / generation units ) . Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate ( giving RNAP / ribosome ) capturing whether transcription ( light yellow ) or translation ( dark blue ) is more dominant .

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Comparison of protein synthesis rate of endogenous E. coli genes measured using Ribo-seq from this study (in molecules/s units) and from that by Li et al. (Li et al, 2014) (in molecules/generation units). Each point corresponds to a single gene and color denotes the ratio of transcription initiation rate to translation initiation rate (giving RNAP/ribosome) capturing whether transcription (light yellow) or translation (dark blue) is more dominant.


Transcription ( bottom ) and translation ( top ) profiles for yggN , rpoH and greA , computed from the RNA - seq and Ribo - seq data without induction . Positions of the genetic parts and gene are shown below the profiles . ( C ) Promoter strengths in RNAP / s units and RBS initiation rates in ribosome / s units .

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Transcription (bottom) and translation (top) profiles for yggN, rpoH and greA, computed from the RNA-seq and Ribo-seq data without induction. Positions of the genetic parts and gene are shown below the profiles. (C) Promoter strengths in RNAP/s units and RBS initiation rates in ribosome/s units.


Transcription ( bottom ) and translation ( top ) profiles for lacZ . Profiles are shown for cells in the absence and presence of IPTG ( 1 mM ) . Position of genetic parts and gene is shown below the profiles . RBS is omitted from the genetic design due to its size . ( E ) Measured promoter strength in RNAP / s units , RBS initiation rate in ribosomes / s units , and the transcriptional terminator and translation termination efficiency for lacZ . Data shown for cells in the absence and presence of IPTG ( 1 mM ) .

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Transcription (bottom) and translation (top) profiles for lacZ. Profiles are shown for cells in the absence and presence of IPTG (1 mM). Position of genetic parts and gene is shown below the profiles. RBS is omitted from the genetic design due to its size. (E) Measured promoter strength in RNAP/s units, RBS initiation rate in ribosomes/s units, and the transcriptional terminator and translation termination efficiency for lacZ. Data shown for cells in the absence and presence of IPTG (1 mM).


Translation profiles for the PK - LacZ construct in cells cultured in the absence ( bottom ) and presence ( top ) of IPTG ( 1 mM ) . The gene10 , middle , and lacZ regions are labelled above the profiles . Shaded region denotes the PK , and dashed lines denote the start codon and stop codons of gene10 and LacZ .

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Translation profiles for the PK-LacZ construct in cells cultured in the absence (bottom) and presence (top) of IPTG (1 mM). The gene10, middle, and lacZ regions are labelled above the profiles. Shaded region denotes the PK, and dashed lines denote the start codon and stop codons of gene10 and LacZ.


Fraction of the total RPFs and mRNA reads in each reading frame for the gene10 , PK or middle , and lacZ regions schematically shown below and are of the PK - LacZ construct . Data shown separately for cells cultured in the absence and presence of IPTG ( 1 mM ) .

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Fraction of the total RPFs and mRNA reads in each reading frame for the gene10, PK or middle, and lacZ regions schematically shown below and are of the PK-LacZ construct. Data shown separately for cells cultured in the absence and presence of IPTG (1 mM).


Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E . coli transcripts .

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Violin plots of the distributions of fractions of total RPFs and mRNA reads in each reading frame for all E. coli transcripts.


Change in expression of chromosomal genes in E . coli cells following induction of PK - lacZ expression ( 1 mM IPTG ) . Each point represents a transcript . Differentially expressed genes ( mRNA count : P < 0 . 001 and absolute log2 fold - change > 1 . 37 ; translation efficiency : P < 0 . 01 ) are highlighted in color and by an alternative point shape ( transcriptional regulation : purple cross ; translational regulation : orange open circle ) .

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Change in expression of chromosomal genes in E. coli cells following induction of PK-lacZ expression (1 mM IPTG). Each point represents a transcript. Differentially expressed genes (mRNA count: P < 0.001 and absolute log2 fold-change > 1.37; translation efficiency: P < 0.01) are highlighted in color and by an alternative point shape (transcriptional regulation: purple cross; translational regulation: orange open circle).


Translation initiation rates for all E . coli RBSs in cells harboring the LacZ and PK - LacZ constructs in the absence and presence of IPTG ( 1 mM ) . Solid line shows the same initiation rate for both conditions . Dotted lines denote linear regressions for the data with no offset .

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Translation initiation rates for all E. coli RBSs in cells harboring the LacZ and PK-LacZ constructs in the absence and presence of IPTG (1 mM). Solid line shows the same initiation rate for both conditions. Dotted lines denote linear regressions for the data with no offset.


Fractions of mRNA reads and RPFs mapping to each synthetic expression construct ( LacZ and PK - LacZ ) and E . coli transcripts , which are divided into three major categories : ribosomal , metabolic , and other functions . Data shown for cells cultured in the absence and presence of IPTG ( 1 mM ) .

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Fractions of mRNA reads and RPFs mapping to each synthetic expression construct (LacZ and PK-LacZ) and E. coli transcripts, which are divided into three major categories: ribosomal, metabolic, and other functions. Data shown for cells cultured in the absence and presence of IPTG (1 mM).


Growth phenotypes of the wild - type ( WT , Col - 0 ) , bri1 - null mutant , and two independent ubp12i / ubp13 double mutant lines grown on 1 / 2MS medium supplemented with 10 μM DEX ( DEX medium ) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution ( 0 . 1 % [ v / v ] EtOH ) for additionally 8 days . Scale bars : 10 mm .

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Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm.


Phenotype of the WT and ubp12i / ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days . Scale bars : 10 mm . Hypocotyl length relative to the mock control of seedlings ( B ) . Experiments were done in triplicate ( n > 15 seedlings for each line ) .

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Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.   Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).


Western blot ( WB ) analysis of BES1 dephosphorylation after BL treatment with the α - BES1 antibody . Total proteins were isolated from 6 - day - old seedlings grown on DEX medium in the light , either in the presence of 100 nM BL or mock . PEPC was used as a loading control . Percentage of dephosphorylated BES1 relative to the total BES1 from ( D ) ( n = 6 biological replicates ) .

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Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.   Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).


Western blot analysis of BRI1 protein amount in ubp12i / ubp13 plants with the α - BRI1 antibody . Total proteins were isolated from 18 - day - old plants grown on 1 / 2MS medium supplemented with 10 μM DEX ( + ) or not ( - ) . Western blot analysis of BRI1 protein level in concanamycin A ( ConcA ) - treated plants . Eighteen - day - old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation .

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Western blot analysis of BRI1 protein amount in ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from 18-day-old plants grown on 1/2MS medium supplemented with 10 μM DEX (+) or not (-).   Western blot analysis of BRI1 protein level in concanamycin A (ConcA)-treated plants. Eighteen-day-old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation.


Deubiquitinating activity of UBP13 against K48 - and K63 - linked ubiquitination . Diubiquitins linked through K48 or K63 ( K48 Di - Ub or K63 Di - Ub ) were incubated alone or with GST - UBP13 or GST - UBP13C207S for 18 h . Deubiquitination of the diubiquitins was analyzed by western blot with the α - ubiquitin ( α - Ub , P4D1 ) antibody . The presence of GST - UBP13 / UBP13C207S recombinant proteins was confirmed by the α - GST antibody .

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Deubiquitinating activity of UBP13 against K48- and K63-linked ubiquitination. Diubiquitins linked through K48 or K63 (K48 Di-Ub or K63 Di-Ub) were incubated alone or with GST-UBP13 or GST-UBP13C207S for 18 h. Deubiquitination of the diubiquitins was analyzed by western blot with the α-ubiquitin (α-Ub, P4D1) antibody. The presence of GST-UBP13/UBP13C207S recombinant proteins was confirmed by the α-GST antibody.


UBP13 deubiquitination of K63 - ubiquitinated BRI1 in vitro . Polyubiquitinated MBP - BRI1CD was generated by incubation with GST - PUB13 ( Ub reaction ) and then with GST - UBP13 or GST - UBP13C207S ( DUB reaction - MBP pull - down , input ) . After in vitro deubiquitination , MBP - BRI1CD was purified by an MBP pull - down to analyze the BRI1CD ubiquitination ( DUB reaction - MBP pull - down , SE ) . The ubiquitinated proteins were detected by western blot with an α - ubiquitin antibody ( FK2 ) . Ubiquitin chain specificity was detected with an α - K63Ub antibody ( Apu3 ) . The presence of recombinant proteins was confirmed by the α - GST and α - MBP antibodies .

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UBP13 deubiquitination of K63-ubiquitinated BRI1 in vitro. Polyubiquitinated MBP-BRI1CD was generated by incubation with GST-PUB13 (Ub reaction) and then with GST-UBP13 or GST-UBP13C207S (DUB reaction-MBP pull-down, input). After in vitro deubiquitination, MBP-BRI1CD was purified by an MBP pull-down to analyze the BRI1CD ubiquitination (DUB reaction-MBP pull-down, SE). The ubiquitinated proteins were detected by western blot with an α-ubiquitin antibody (FK2). Ubiquitin chain specificity was detected with an α-K63Ub antibody (Apu3). The presence of recombinant proteins was confirmed by the α-GST and α-MBP antibodies.


Growth phenotype comparison of BRI1 - mCit / bri1 / ubp12i / ubp13 and BRI125KR - mCit / bri1 / ubp12i / ubp13 . The indicated transgenic lines were grown on DEX medium for 18 days . Scale bars : 10 mm .

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Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm.


Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR - mCit / bri1 / ubp12i / ubp13 plants with the α - BRI1 antibody . Total proteins were isolated from the roots of the lines indicated ( A ) . PEPC was used as a loading control .

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Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR-mCit/bri1/ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from the roots of the lines indicated (A). PEPC was used as a loading control.


A - D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 ( A ) or BRI125KR ( C ) tagged with mCitrine ( mCit ) in either bri1 / UBP12 / UBP13 or bri1 / ubp12i / ubp13 background . Six - day - old seedlings were grown on DEX medium , and kept in the dark for 2 h in the presence of 50 μM CHX before imaging . Confocal images of epidermal cells from root meristem are shown . Scale bars : 10 μm . Measurements of the relative PM / intracellular BRI1 - mCit fluorescence intensity . For each line , at least 55 and 25 cells from over 25 and 15 seedlings were measured in ( B ) and ( D ) , respectively .

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A-D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 (A) or BRI125KR (C) tagged with mCitrine (mCit) in either bri1/UBP12/UBP13 or bri1/ubp12i/ubp13 background. Six-day-old seedlings were grown on DEX medium, and kept in the dark for 2 h in the presence of 50 μM CHX before imaging. Confocal images of epidermal cells from root meristem are shown. Scale bars: 10 μm. Measurements of the relative PM/intracellular BRI1-mCit fluorescence intensity. For each line, at least 55 and 25 cells from over 25 and 15 seedlings were measured in (B) and (D), respectively.