B ( Right panel ) Immunofluorescence for SOX9 ( green ) and HNF4α ( red ) in WT fibrotic mice . Arrowheads ( orange / yellow staining ) indicate SOX9 + / HNF4α + hepatocytes and star ( * ; red ) indicates SOX - / HNF4α + hepatocytes . ( Left panel ) Immunofluorescence for SOX9 ( green nucleus ) and CK19 ( red cytoplasm ) in WT fibrotic mice . Arrowheads ( green ) indicate SOX9 + hepatocytes and star ( * ) indicates SOX - hepatocytes identified by DAPI staining in Appendix Figure S1 .

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B (Right panel) Immunofluorescence for SOX9 (green) and HNF4α (red) in WT fibrotic mice. Arrowheads (orange/yellow staining) indicate SOX9+/HNF4α+ hepatocytes and star (*; red) indicates SOX-/ HNF4α+ hepatocytes. (Left panel) Immunofluorescence for SOX9 (green nucleus) and CK19 (red cytoplasm) in WT fibrotic mice. Arrowheads (green) indicate SOX9+ hepatocytes and star (*) indicates SOX- hepatocytes identified by DAPI staining in Appendix Figure S1.


C Co - localization by immunohistochemistry ( top panel ) and in situ hybridization ( lower panel ) for SOX9 ( red ) and αSMA ( brown ) in wild - type ( WT ) mice following CCl4 or BDL induced fibrosis .

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C Co-localization by immunohistochemistry (top panel) and in situ hybridization (lower panel) for SOX9 (red) and αSMA (brown) in wild-type (WT) mice following CCl4 or BDL induced fibrosis.


D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control .

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D Expression of SOX9 protein by immunoblotting of in vivo activated HSCs extracted from WT mice following CCl4 injections compared to olive oil control.


E Immunohistochemistry for SOX9 ( brown ) in control ( Sox9fl / fl ; RosaCre - ) or Sox9 - null ( Sox9fl / fl ; RosaCre + ) mice following fibrosis . Bile ducts ( bd ) and hepatocytes ( arrowheads ) indicated . SOX9 is absent , even in bile ducts in Sox9 - null liver . Size bar , 25 μm ( A , B and E ) , 10 μm ( C ) .

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E Immunohistochemistry for SOX9 (brown) in control (Sox9fl/fl;RosaCre-) or Sox9-null (Sox9fl/fl;RosaCre+) mice following fibrosis. Bile ducts (bd) and hepatocytes (arrowheads) indicated. SOX9 is absent, even in bile ducts in Sox9-null liver. Size bar, 25 μm (A, B and E), 10 μm (C).


A - J Representative images and quantification shown for olive oil treated ( n = 6 ) or chronic CCl4 ( n = 5 Sox9fl / fl ; RosaCreER - ; n = 8 Sox9fl / fl ; RosaCreER + ) induced fibrosis or following sham operation ( n = 5 ) or BDL ( n = 7 Sox9fl / fl ; RosaCreER - ; n = 5 Sox9fl / fl ; RosaCreER + ) ) induced fibrosis . A Picrosirius red ( PSR ) staining ( collagen deposition in red ) counterstained with fast green ( top row ) and immunohistochemistry for α - SMA ( brown staining bottom row ; activated HSC / myofibroblast marker ) in olive oil treated ( left ) or chronic CCl4 induced fibrosis ( right ) in control and Sox9 - null mice . Size bar = 200 μm .

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A-J Representative images and quantification shown for olive oil treated (n=6) or chronic CCl4 (n=5 Sox9fl/fl;RosaCreER-; n=8 Sox9fl/fl;RosaCreER+) induced fibrosis or following sham operation (n=5) or BDL (n=7 Sox9fl/fl;RosaCreER-; n=5 Sox9fl/fl;RosaCreER+)) induced fibrosis. A Picrosirius red (PSR) staining (collagen deposition in red) counterstained with fast green (top row) and immunohistochemistry for α-SMA (brown staining bottom row; activated HSC / myofibroblast marker) in olive oil treated (left) or chronic CCl4 induced fibrosis (right) in control and Sox9-null mice. Size bar = 200 μm.


B , C Quantification of surface area covered by the PSR staining or α - SMA in control ( Cnt ) and Sox9 - null ( Null ) in ( A ) .

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B, C Quantification of surface area covered by the PSR staining or α-SMA in control (Cnt) and Sox9-null (Null) in (A).


D PSR staining ( red ; top row ) and immunohistochemistry for α - SMA ( brown ; middle row ) and CK19 ( brown ; bottom row ) in control and Sox9 - null mice following sham operation ( left ) or BDL induced fibrosis ( right ) . Size bar = 500 μm .

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D PSR staining (red; top row) and immunohistochemistry for α-SMA (brown; middle row) and CK19 (brown; bottom row) in control and Sox9-null mice following sham operation (left) or BDL induced fibrosis (right). Size bar = 500 μm.


E , F Quantification of surface area covered by the PSR staining or α - SMA in ( D ) .

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E, F Quantification of surface area covered by the PSR staining or α-SMA in (D).


G , H Liver function is improved in Sox9 - null mice following CCl4 ( G ) or BDL ( H ) induced fibrosis compared to control mice . Reduction in serum alanine aminotransferase ( ALT ; G , H ) and bilirubin ( H ) down to levels shown in non - fibrotic mice ( olive oil treated groups ; Oil ) .

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G, H Liver function is improved in Sox9-null mice following CCl4 (G) or BDL (H) induced fibrosis compared to control mice. Reduction in serum alanine aminotransferase (ALT; G, H) and bilirubin (H) down to levels shown in non-fibrotic mice (olive oil treated groups; Oil).


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I Sox9 - loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4 . Olive oil treated mice had no bridging fibrosis , in line with a non - fibrotic liver histology in ( A ) .

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I Sox9-loss improved severity of fibrosis compared to control mice indicated by quantification of bridging fibrosis in PSR sections following CCl4. Olive oil treated mice had no bridging fibrosis, in line with a non-fibrotic liver histology in (A).


J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in ( D ) is reduced in Sox9 - null mice compared to control following BDL . All mice were treated with tamoxifen ( Tam ) which did not induce ectopic expression of SOX9 in non - fibrotic livers ( Figure 2A , E and Appendix Figure S3 ) . All experiments are n ≥ 5 as indicated . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . P values indicated .

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J Ductal hyperplasia as quantified by the surface area covered by CK19 positive ducts in (D) is reduced in Sox9-null mice compared to control following BDL. All mice were treated with tamoxifen (Tam) which did not induce ectopic expression of SOX9 in non-fibrotic livers (Figure 2A, E and Appendix Figure S3). All experiments are n≥5 as indicated. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated.


A - C Representative images shown for olive oil treated ( n = 5 ) or chronic CCl4 ( n = 5 ) induced fibrosis or following sham operation ( n = 6 Sox9fl / fl ; AlbCre - ; n = 5 Sox9fl / fl ; AlbCre + ) or BDL ( n = 5 Sox9fl / fl ; AlbCre - ; n = 8 Sox9fl / fl ; AlbCre + ) induced fibrosis . SOX9 immunohistochemistry ( brown ; A ) , in situ hybridization for Sox9 ( brown ) and α - Sma ( red ) and collagen deposition by PSR staining ( red ; B ) in control ( Sox9fl / fl ; AlbCre - ) or Sox9 - null ( Sox9fl / fl ; AlbCre + ) mice following fibrosis . Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl / fl ; AlbCre + mice ( A ) .

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A-C Representative images shown for olive oil treated (n=5) or chronic CCl4 (n=5) induced fibrosis or following sham operation (n=6 Sox9fl/fl;AlbCre-; n=5 Sox9fl/fl;AlbCre+) or BDL (n=5 Sox9fl/fl;AlbCre-; n=8 Sox9fl/fl;AlbCre+) induced fibrosis. SOX9 immunohistochemistry (brown; A), in situ hybridization for Sox9 (brown) and α-Sma (red) and collagen deposition by PSR staining (red; B) in control (Sox9fl/fl;AlbCre-) or Sox9-null (Sox9fl/fl;AlbCre+) mice following fibrosis. Higher magnified image of SOX9 localization in discrete cells within the scar is shown for CCl4 and BDL in the Sox9fl/fl;AlbCre+ mice (A).


D , E . Quantification of surface area covered by the PSR staining in ( C ) .

D, E. Quantification of surface area covered by the PSR staining in (C).


F Expression of SOX9 protein by immunohistochemistry in quiescent ( Q ) and activated ( A ) HSCs extracted from Sox9fl / fl ; AlbCre + mice .

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F Expression of SOX9 protein by immunohistochemistry in quiescent (Q) and activated (A) HSCs extracted from Sox9fl/fl;AlbCre+ mice.


G Individual fluorescent channels showing localization and expression of α - SMA ( left panel ) , SOX9 ( middle panel ) and composite image for α - SMA ( green ) and SOX9 ( red ) in right panel in activated Sox9fl / fl ; AlbCre + HSCs ( genotyping shown in Appendix Figure S9 ) . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . Size bars = 100 μm ( A ) , 10 μm ( B ) 200 μm ( C ) , 25 μm ( G ) .

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G Individual fluorescent channels showing localization and expression of α-SMA (left panel), SOX9 (middle panel) and composite image for α-SMA (green) and SOX9 (red) in right panel in activated Sox9fl/fl;AlbCre+ HSCs (genotyping shown in Appendix Figure S9). Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. Size bars = 100 μm (A), 10 μm (B) 200 μm (C), 25 μm (G).


A , B Flow cytometry of CD45 + CD11B + cells in control ( Sox9fl / fl ; RosaCre - ; A ) and Sox9 - null ( Sox9fl / fl ; RosaCre + ; B ) livers stained for Ly6C and MHCII ( far left ) to identify four populations of cells with differences in their expression of MerTK , CD64 , F4 / 80 and MHCII .

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A, B Flow cytometry of CD45+CD11B+ cells in control (Sox9fl/fl;RosaCre-; A) and Sox9-null (Sox9fl/fl;RosaCre+; B) livers stained for Ly6C and MHCII (far left) to identify four populations of cells with differences in their expression of MerTK, CD64, F4/80 and MHCII.


C Graphical representation of Ly6C and F4 / 80 staining in macrophages from population 3 and 4 .

C Graphical representation of Ly6C and F4/80 staining in macrophages from population 3 and 4.


D , E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9 - null livers . Data are representative of four independent experiments .

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D, E Graphical representation of cell numbers in the four Ly6C and MHCII cell populations in control and Sox9-null livers. Data are representative of four independent experiments.


F F4 / 80 immunohistochemistry ( brown staining ) in control and Sox9 - null mice livers following 4 - weeks CCl4 induced fibrosis . Size bar = 100μm . Two - tailed unpaired t - test was used for statistical analysis . Data in bar charts show means ± s . e . m . P values indicated . All experiments are n = 4 .

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F F4/80 immunohistochemistry (brown staining) in control and Sox9-null mice livers following 4-weeks CCl4 induced fibrosis. Size bar = 100μm. Two-tailed unpaired t-test was used for statistical analysis. Data in bar charts show means ± s.e.m. P values indicated. All experiments are n=4.


A , B Immunofluorescence of activated mouse HSCs cultured on plastic ( A ) or hydrogels ( A , B ) . Individual fluorescent channels showing localization and expression of SOX9 ( left ) , YAP ( middle ) and composite image for SOX9 ( red ) and YAP ( green ) in right panel . ( B ) Individual and composite images for αSMA ( green ) and TAZ ( red ) or TEAD ( red ) . Size bar , 25μm .

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A, B Immunofluorescence of activated mouse HSCs cultured on plastic (A) or hydrogels (A, B). Individual fluorescent channels showing localization and expression of SOX9 (left), YAP (middle) and composite image for SOX9 (red) and YAP (green) in right panel. (B) Individual and composite images for αSMA (green) and TAZ (red) or TEAD (red). Size bar, 25μm.


C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin ( VP ) in activated mouse HSCs expressed relative to DMSO control ( n = 3 ) .

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C Quantified decrease in SOX9 protein levels following inhibition of YAP using verteporfin (VP) in activated mouse HSCs expressed relative to DMSO control (n=3).


D Immunohistochemistry for SOX9 ( brown ) following CCl4 or BDL induced fibrosis in mice treated with DMSO ( CCl4 n = 4 , BDL n = 7 ) or verteporfin ( CCl4 n = 3 , BDL n = 5 ) .

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D Immunohistochemistry for SOX9 (brown) following CCl4 or BDL induced fibrosis in mice treated with DMSO (CCl4 n=4, BDL n=7) or verteporfin (CCl4 n=3, BDL n=5).


E , F quantification of SOX9 cell numbers in the HSC / Hep population . Size bar , 50 μm .

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E, F quantification of SOX9 cell numbers in the HSC/Hep population. Size bar, 50 μm.


( C ) Survival curves after the injection of EL4 lymphoma cells ( n = 6 / group ) .

(C) Survival curves after the injection of EL4 lymphoma cells (n = 6/group).


( H ) Ifng protein and Ifng mRNA levels ( n = 4 / group ) were determined by ELISA and quantitative PCR .

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(H) Ifng protein and Ifng mRNA levels (n = 4/group) were determined by ELISA and quantitative PCR.


( A ) m6A dot blot assay using total RNA from NK92 - siCTRL or NK92 - siFTO cells .

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(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.


( D ) Western blotting for the detection of GZMB in NK92 - siCTRL and NK92 - siFTO cells after treatment with IL - 2 .

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(D) Western blotting for the detection of GZMB in NK92-siCTRL and NK92-siFTO cells after treatment with IL-2.


( E ) Levels of IFNγ ( n = 5 biological replicates ) and TNFα ( n = 3 biological replicates ) in the culture media 24 hours after IL - 2 treatment ( left ) or co - culture media with K562 cells for 4 hours ( right ) were determined by ELISA .

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(E) Levels of IFNγ (n = 5 biological replicates) and TNFα (n = 3 biological replicates) in the culture media 24 hours after IL-2 treatment (left) or co-culture media with K562 cells for 4 hours (right) were determined by ELISA.


( B ) Western blotting analysis . NK92 - siCTRL or NK92 - siFTO cells were incubated with 20 ng / ml IL - 2 for 24 hours , washed , and cultured without IL - 2 ( IL - 2 depletion ) for the indicated times ( n = 3 ) . ( C ) A western blotting analysis of NK92 + MOCK or NK92 + FTO cells incubated with 20 ng / ml IL - 2 for the indicated times was performed ( n = 3 ) .

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(B) Western blotting analysis. NK92-siCTRL or NK92-siFTO cells were incubated with 20 ng/ml IL-2 for 24 hours, washed, and cultured without IL-2 (IL-2 depletion) for the indicated times (n = 3). (C) A western blotting analysis of NK92+MOCK or NK92+FTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed (n = 3). D


( C ) m6A methylated RNA immunoprecipitation & quantitative PCR ( MeRIP - qPCR ) analysis using a m6A antibody in NK92 - siFTO ( upper ) or NK92 + FTO ( lower ) cells compared to NK92 - siCTRL or NK92 + MOCK cells .

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(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.


( D ) SOCS3 3 ' - UTR reporter assay using the AANAT reporter system . Plasmid information for the AANAT reporter system with or without the SOCS3 3 ' - UTR sequence ( upper ) . Levels of Aanat protein ( left ) and Aanat mRNA ( right ) expression in HEK - 293T cells co - transfected with plasmid - expressing AANAT reporter containing SOCS3 3 ' - UTR and siCTRL or siFTO .

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(D) SOCS3 3'-UTR reporter assay using the AANAT reporter system. Plasmid information for the AANAT reporter system with or without the SOCS3 3'-UTR sequence (upper). Levels of Aanat protein (left) and Aanat mRNA (right) expression in HEK-293T cells co-transfected with plasmid-expressing AANAT reporter containing SOCS3 3'-UTR and siCTRL or siFTO.


( B ) Relative mRNA expression levels of JAK / STAT target genes , IFNγ ( n = 10 biological replicates ) , GZMB ( n = 6 biological replicates ) and PRF1 ( n = 8 biological replicates ) were determined by qPCR in CD56 + - siCTRL or CD56 + - siFTO cells .

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(B) Relative mRNA expression levels of JAK/STAT target genes, IFNγ (n = 10 biological replicates), GZMB (n = 6 biological replicates) and PRF1 (n = 8 biological replicates) were determined by qPCR in CD56+-siCTRL or CD56+-siFTO cells.


( C ) Western blotting of CD56 + - siCTRL or CD56 + - siFTO cells incubated with 20 ng / ml IL - 15 and 20 ng / ml IL - 21 for the indicated times was performed . Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated ( p - ) and total proteins ( n = 2 ~ 3 ) . Data are representative of three times independent experiments with similar results . The protein levels were expressed as the relative band density of the corresponding protein ( lower ) .

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(C) Western blotting of CD56+-siCTRL or CD56+-siFTO cells incubated with 20 ng/ml IL-15 and 20 ng/ml IL-21 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p-) and total proteins (n = 2~3). Data are representative of three times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (lower).


( E ) The images of the ventral bioluminescence ( BLI ) following the injection of NK92 - TRC or NK92 - shFTO cells .

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(E) The images of the ventral bioluminescence (BLI) following the injection of NK92-TRC or NK92-shFTO cells.


( G ) The survival curves after the injection of K562 - Luc cells .

(G) The survival curves after the injection of K562-Luc cells.


Kaplan - Meier analysis of overall survival in breast cancer patients with recurrent lethal disease , classified according to p - JNK expression . p - JNK was determined by immunostaining of breast cancer samples in a tissue microarray ; p - JNK low , n = 31 ; p - JNK high , n = 10 . P value and hazard ratio ( HR ) were calculated by log - rank test .

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Kaplan-Meier analysis of overall survival in breast cancer patients with recurrent lethal disease, classified according to p-JNK expression. p-JNK was determined by immunostaining of breast cancer samples in a tissue microarray; p-JNK low, n = 31; p-JNK high, n = 10. P value and hazard ratio (HR) were calculated by log-rank test.


Immunostaining of p - c - Jun ( arrows ) in patient - matched breast tumor and lung metastasis . Scale bar , 50 μm .

Immunostaining of p-c-Jun (arrows) in patient-matched breast tumor and lung metastasis. Scale bar, 50 μm.


Quantification of p - c - Jun positive ( p - c - Jun + ) cells per tumor area in matched primary tumors and lung metastases . Boxes depict median from 12 patients with upper and lower quartiles .

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Quantification of p-c-Jun positive (p-c-Jun+) cells per tumor area in matched primary tumors and lung metastases. Boxes depict median from 12 patients with upper and lower quartiles.


Quantification of p - c - Jun positive ( p - c - Jun + ) cancer cells within mammary tumors and matched metastases in mice injected with MDA231 - LM2 cells .

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Quantification of p-c-Jun positive (p-c-Jun+) cancer cells within mammary tumors and matched metastases in mice injected with MDA231-LM2 cells.


Immunofluorescence analysis of p - c - Jun in micrometastatic ( day 7 ) and macrometastatic ( day 21 ) nodules in a xenograft mouse model injected intravenously with MDA231 - LM2 cells . DAPI was used to stain cell nuclei , and cancer cells express GFP as a marker . Scale bar , 50 μm . Quantification of p - c - Jun + cancer

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Immunofluorescence analysis of p-c-Jun in micrometastatic (day 7) and macrometastatic (day 21) nodules in a xenograft mouse model injected intravenously with MDA231-LM2 cells. DAPI was used to stain cell nuclei, and cancer cells express GFP as a marker. Scale bar, 50 μm. Quantification of p-c-Jun+ cancer cells


Mammary tumor growth curves from mice described in panel G . Each value represents the mean ± SEM . For MDA231 - LM2 , n = 12 tumors in 6 mice for each experimental group . For SUM159 - LM1 , n = 16 tumors in 8 mice for each experimental group .

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Mammary tumor growth curves from mice described in panel G. Each value represents the mean ± SEM. For MDA231-LM2, n = 12 tumors in 6 mice for each experimental group. For SUM159-LM1, n = 16 tumors in 8 mice for each experimental group.


Lung metastatic burden in MDA231 - LM2 or SUM159 - LM1 tumor - bearing mice treated with JNKi . Quantification of lung metastases . FOV , field of view ; LS , lung section . Each value represents the mean ± SEM . For metastatic foci per FOV , 8 - 10 fields were quantified per mouse . MDA231 - LM2 tumor - bearing mice : n = 6 mice per group . SUM159 - LM1 tumor - bearing mice , vehicle n = 8 mice ; JNKi n = 7 mice .

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Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Quantification of lung metastases. FOV, field of view; LS, lung section. Each value represents the mean ± SEM. For metastatic foci per FOV, 8-10 fields were quantified per mouse. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice, vehicle n = 8 mice; JNKi n = 7 mice.


Lung metastatic burden in MDA231 - LM2 or SUM159 - LM1 tumor - bearing mice treated with JNKi . Representative histology examples of metastasis in lungs , detected by expression of human vimentin . Scale bar , 100 μm .

Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Representative histology examples of metastasis in lungs, detected by expression of human vimentin. Scale bar, 100 μm.


Metastatic burden determined by photon flux in lungs of MDA231 - LM2 - injected NSG mice treated with vehicle and JNKi . Boxes show the median with upper and lower quartiles . Whiskers represent minimum and maximum values . Vehicle control , n = 8 ; JNKi - treated , n = 8 . Examples of bioluminescence in

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Metastatic burden determined by photon flux in lungs of MDA231-LM2-injected NSG mice treated with vehicle and JNKi. Boxes show the median with upper and lower quartiles. Whiskers represent minimum and maximum values. Vehicle control, n = 8; JNKi-treated, n = 8. Examples of bioluminescence in mice


Scatter plot elucidating fold change ( FC ) in gene expression between samples treated with JNKi compared to vehicle ( Y - axis ) and expression of constitutively active JNK ( MKK7 - JNK ) versus vector control ( X - axis ) from biological triplicates . Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red .

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Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red.


GSEA graphs showing ( C ) enrichment of a mammary stem cell signature ( Pece et al , 2010 ) in MDA231 - LM2 cancer cells expressing MKK7 - JNK NES , normalized enrichment score .

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GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK NES, normalized enrichment score.


GSEA graphs showing under - representation of the same mammary stem cell signature in cancer cells treated with JNKi . NES , normalized enrichment score .

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GSEA graphs showing under-representation of the same mammary stem cell signature in cancer cells treated with JNKi. NES, normalized enrichment score.


GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231 - LM2 cells either expressing MKK7 - JNK or treated with JNKi . Mammary stem cell signatures are indicated as follows : a , up in mammary stem cells b , up in fetal mammary stem cells ; c , up in adult mammary stem cells and d , up in mammary repopulating

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GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells b, up in fetal mammary stem cells ; c, up in adult mammary stem cells and d, up in mammary repopulating units


Heat map of the JNK signature composed of 68 genes that are induced by expression of MKK7 - JNK and repressed by JNKi ,

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Heat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi,


Proportion of JNK signature - positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK - S , JNK signature as in panel F . Subtype patient numbers : Luminal , n = 1213 ; HER2 , n = 240 ; Basal - like , n = 331 .

Proportion of JNK signature-positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK-S, JNK signature as in panel F. Subtype patient numbers: Luminal, n = 1213; HER2, n = 240; Basal-like, n = 331.


Gene expression profile of MDA231 - LM2 cells grown as oncospheres or as a monolayer . Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red .

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Gene expression profile of MDA231-LM2 cells grown as oncospheres or as a monolayer. Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red.


GSEA graph showing enrichment of JNK signature in MDA231 - LM2 cancer cells grown as oncospheres . NES , normalized enrichment score .

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GSEA graph showing enrichment of JNK signature in MDA231-LM2 cancer cells grown as oncospheres. NES, normalized enrichment score.


Immunoblot of phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) and c - Jun in oncospheres from SUM159 - LM1 cells . Tubulin was included as a loading control .

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Immunoblot of phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in oncospheres from SUM159-LM1 cells. Tubulin was included as a loading control.


GSEA of a basal - like breast cancer signature induced in MDA231 - LM2 oncospheres .

GSEA of a basal-like breast cancer signature induced in MDA231-LM2 oncospheres.


Normalized enrichment score ( GSEA ) of mammary stem cell signatures in oncospheres from MDA231 - LM2 cells .

Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells.


Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK . Values are mean from 10 wells per group ± SD .

Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD.


Oncosphere formation in SUM159 - LM1 cells following treatment with JNKi or vehicle control . Values are mean from 10 wells per condition ± SD . Representative images of

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Oncosphere formation in SUM159-LM1 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD. Representative images of spheres


Oncosphere formation in JNKi - treated cancer cells from breast cancer patients ( pleural effusions or ascites ) . ( I ) Quantification of oncospheres per well in 4 different patients . Values are mean from 10 wells ± SD . ( J ) Representative oncospheres from patient samples . Scale bar , 50 μm .

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Oncosphere formation in JNKi-treated cancer cells from breast cancer patients (pleural effusions or ascites). (I) Quantification of oncospheres per well in 4 different patients. Values are mean from 10 wells ± SD. (J) Representative oncospheres from patient samples. Scale bar, 50 μm.


Matrigel invasion assay of MDA231 - LM2 cells treated with JNKi . Left , examples of invading cancer cells stained with crystal violet ( arrows ) . Scale bar , 50 μm . Right , quantification of invading cells per field of view ( FOV ) showing mean from 8 fields ± SEM .

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Matrigel invasion assay of MDA231-LM2 cells treated with JNKi. Left, examples of invading cancer cells stained with crystal violet (arrows). Scale bar, 50 μm. Right, quantification of invading cells per field of view (FOV) showing mean from 8 fields ± SEM.


SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi .

SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi.


SPP1 and TNC expression in MDA231 - LM2 cells transduced with pLVX - puro lentivirus without a transgene ( control ) or expressing constitutively - active JNK ( MKK7 - JNK ) or mutated inactive JNK ( MKK7 - JNK ( mut ) ) .

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SPP1 and TNC expression in MDA231-LM2 cells transduced with pLVX-puro lentivirus without a transgene (control) or expressing constitutively-active JNK (MKK7-JNK) or mutated inactive JNK (MKK7-JNK(mut)).


SPP1 and TNC expression in pleural effusion ( 1 and 2 ) and ascites ( 3 and 4 ) samples from four different patients treated with JNKi ( 4 μM , 48h ) in culture . Values are mean ± SD from triplicates . The results from all four patient samples were included to calculate significance of JNKi on gene expression . SPP1 , P = 0 . 0079 ; TNC , P = 0 . 0002 .

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SPP1 and TNC expression in pleural effusion (1 and 2) and ascites (3 and 4) samples from four different patients treated with JNKi (4 μM, 48h) in culture. Values are mean ± SD from triplicates. The results from all four patient samples were included to calculate significance of JNKi on gene expression. SPP1, P = 0.0079; TNC, P = 0.0002.


Analysis of SPP1 and TNC mRNA levels in SUM159 - LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi . For panels A - D , values represent the mean of qPCR triplicates ± SD .

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Analysis of SPP1 and TNC mRNA levels in SUM159-LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi. For panels A-D, values represent the mean of qPCR triplicates ± SD.


ChIP analysis of c - Jun binding to sequences in SPP1 or TNC promoters . Analysis of SPP1 and TNC promoter fragments pulled down by c - Jun and IgG antibodies . Values represent the mean of qPCR triplicates ± SD .

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ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. Analysis of SPP1 and TNC promoter fragments pulled down by c-Jun and IgG antibodies. Values represent the mean of qPCR triplicates ± SD.


Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients ( GSE14020 ) . N = 65 metastasis samples .

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Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients (GSE14020). N = 65 metastasis samples.


Lung metastasis in Spp1 + / - mice implanted with MDA231 - LM2 cells expressing control shRNA ( control ) , and Spp1 - / - mice implanted with MDA231 - LM2 cells independently expressing one of two different SPP1 shRNAs ( SPP1 - deficient ) . Ex vivo lung luminescence was analyzed , panel H . Boxes show the median value with upper and lower quartiles . Whiskers represent minimum and maximum values . Control n = 16 mice , SPP1 deficient ( 1 ) n = 14 mice and SPP1 deficient ( 2 ) n = 15 mice . P values were determined by a two - tailed Mann - Whitney test . ( I ) Representative histological images of metastatic foci ( top ) and ex vivo lung bioluminescence ( bottom ) . In histological samples , immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin ( arrows ) . Scale bar , 100 µm .

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Lung metastasis in Spp1+/- mice implanted with MDA231-LM2 cells expressing control shRNA (control), and Spp1-/- mice implanted with MDA231-LM2 cells independently expressing one of two different SPP1 shRNAs (SPP1-deficient). Ex vivo lung luminescence was analyzed, panel H. Boxes show the median value with upper and lower quartiles. Whiskers represent minimum and maximum values. Control n = 16 mice, SPP1 deficient (1) n = 14 mice and SPP1 deficient (2) n = 15 mice. P values were determined by a two-tailed Mann-Whitney test. (I) Representative histological images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). In histological samples, immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin (arrows). Scale bar, 100 µm.


SUM159 - LM1 cells expressing either control shRNA ( control ) , or one of two different TNC shRNAs ( shTNC 1 and 2 ) , were injected intravenously into NSG mice . ( J ) Metastatic colonization was quantified by lung bioluminescence . Control and shTNC ( 1 ) n = 8 mice per group , shTNC ( 2 ) n = 7 mice . P value was determined by a two - tailed Mann - Whitney test . ( K ) Representative images of metastatic foci ( top ) and ex vivo lung bioluminescence ( bottom ) . Arrows denote vimentin - expressing cancer cells in metastases .

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SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases.


Kaplan - Meier analysis of breast cancer patients ( combined GSE2603 and GSE5327 datasets ) showing a link between high SPP1 and TNC expression and poor lung metastasis - free survival . Patient samples , SPP1 and TNC low n = 70 ; SPP1 and TNC high n = 70 . HR , Hazard ratio .

Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio.


Gene expression profile of MDA231 - LM2 cells treated with paclitaxel ( PAX ) for 48 hours . Highlighted are examples of JNK signature genes that are induced by PAX treatment .

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Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.


GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231 - LM2 cells .

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GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.


Immunofluorescence analysis of p - c - Jun + cancer cells in lung metastases from mice injected intravenously with MDA231 - LM2 cells and treated with PAX at week 3 post injection . Lungs were harvested 72h after PAX treatment . Cancer cells express GFP as a marker . DAPI was used for nuclear staining . Scale bar , 50 µm .

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Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.


Quantification of cancer cells expressing p - c - Jun in panel D . Values are mean from three mice ± SEM . Nine metastatic foci were analyzed per mouse .

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Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse.


Western blot analyzing phosphorylated JNK ( p - JNK ) , JNK , phosphorylated c - Jun ( p - c - Jun ) and c - Jun in MDA231 - LM2 cells treated with PAX for 48 hours . Tubulin was used as a loading control .

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Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.


SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.


SPP1 and TNC expression in SUM159 - LM1 cells treated with increasing concentrations of doxorubicin ( DOXO ) . qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.


SPP1 and TNC expression in SUM159 - LM1 cancer cells treated with 4 μM 5 - Fluorouracil or 4 μM methotrexate . Cells were treated for 48 hours . qPCR triplicates ± SD .

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SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD.


SPP1 and TNC expression in SUM159 - LM1 cells treated with paclitaxel and JNKi . qPCR triplicates ± SD . SPP1 and TNC repression by JNKi , P < 0 . 05 for both vehicle and PAX - treated cells .

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SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.


Growth curves of mammary tumors in vehicle - or PAX - treated Spp1 + / - mice implanted with the indicated cells expressing control shRNA , and Spp1 - / - mice implanted with the indicated cells expressing SPP1 shRNA . MDA231 - LM2 : control , PAX and SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 14 tumors . SUM159 - LM1 : control , PAX and SPP1 def . + PAX n = 16 tumors per group , SPP1 def . n = 15 tumors .

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Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors.


Quantification of lung metastasis from the animals described in panels A and B . The number of metastatic foci per field of view ( FOV ) or per lung section ( LS ) was determined . MDA231 - LM2 tumor - bearing mice : control , PAX and SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice . SUM159 - LM1 tumor - bearing mice : control , PAX and SPP1 def . + PAX n = 8 mice per group , SPP1 def . n = 7 mice .

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Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice.


Tumor weight in mice at day 40 after implantation of MDA231 - LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin ( Adriamycin ) and cyclophosphamide ( AC regimen ) . Control , AC and SPP1 def . n = 16 tumors per group , SPP1 def . + AC n = 14 tumors .

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Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors.


Lung metastasis was quantified in mice that were implanted with MDA231 - LM2 tumors and subjected to AC chemotherapy . Each value represents the mean ± SEM ; n = 5 mice per group . Ten random fields per lung section were analyzed for percentage of metastatic area . Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E . Metastases were determined by expression of human vimentin ( arrows ) . Scale bar , 100 µm .

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Lung metastasis was quantified in mice that were implanted with MDA231-LM2 tumors and subjected to AC chemotherapy. Each value represents the mean ± SEM; n = 5 mice per group. Ten random fields per lung section were analyzed for percentage of metastatic area. Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E. Metastases were determined by expression of human vimentin (arrows). Scale bar, 100 µm.


Mammary tumor growth curves in PAX - treated NSG mice bearing control or TNC knockdown MDA231 - LM2 tumors . Values are means ± SEM , n = 16 tumors per group . * * * * P < 0 . 0001 . Lung metastases quantified in tumor bearing

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Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001. Lung metastases quantified in tumor bearing mice


Representative histology of human vimentin - positive lung metastases ( arrows )

Representative histology of human vimentin-positive lung metastases (arrows)


Mammary tumor growth curves in NSG mice implanted with MDA231 - LM2 or SUM159 - LM1 cells and treated with paclitaxel as single agent or in combination with JNKi . Each value represents the mean ± SEM . MDA231 - LM2 : n = 12 tumors per group . SUM159 - LM1 : vehicle , JNKi and PAX n = 12 tumors per group ; PAX + JNKi n = 10 tumors . * * P < 0 . 01 , * * * P < 0 . 001 .

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Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.


Metastatic burden in lungs of MDA231 - LM2 and SUM159 - LM1 tumor - bearing mice treated with paclitaxel as single therapy or in combination with JNKi . Each value represents the mean ± SEM . MDA231 - LM2 tumor - bearing mice : n = 6 mice per group . SUM159 - LM1 tumor - bearing mice : vehicle , JNKi and PAX n = 6 mice per group ; PAX + JNKi n = 5 . Ten fields were quantified per lung section . FOV , field of view , LS , lung section . PAX , paclitaxel . Representative histological images of metastatic foci ( arrows ) in lungs of the MDA231 - LM2 - implanted mice described in panel C , stained for human vimentin expression in differentially treated groups . Scale bar , 100 μm .

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Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.


Apoptosis was determined by quantification of TUNEL - positive cells per field of view ( FOV ) in MDA231 - LM2 tumors ( from the animals described in ( C ) ) treated with JNKi and / or PAX . Values are means from 6 mice per condition ± SEM .

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Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM.


GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial ( GSE16446 ) , stratified according to pathological response to neoadjuvant chemotherapy . NES , normalized enrichment score . pCR , pathological complete response .

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GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response.


Kaplan - Meier analyses of chemotherapy - treated breast cancer patients associating JNK signature ( JNK - S ) with metastasis - free survival ( TOP trial data set , JNK - S low n = 54 patients ; JNK - S high n = 53 patients ( G ) ) or overall survival ( METABRIC data set , JNK - S low n = 135 patients ; JNK - S high n = 135 patients ( H ) ) . HR , hazard ratio . P values were determined by log - rank 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 O O

Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test.


( E ) . Relative luciferase activity of the sensor ( + ) reporter or sensor ( - ) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293 - FT cells . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

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(E). Relative luciferase activity of the sensor (+) reporter or sensor (-) reporter induced by expression vectors of the top 5 genes identified in the screen or the GFP vector in HEK293-FT cells. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


( F ) . Heat map of relative miRNA expression of various miRNAs in HEK - 293FT cells transfected with expression vectors of the top 5 genes from the screen , or the ctrl ( GFP ) vector . Red color represents suppressed expression compared with ctrl ( GFP ) . 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 O O O O

(F). Heat map of relative miRNA expression of various miRNAs in HEK-293FT cells transfected with expression vectors of the top 5 genes from the screen, or the ctrl (GFP) vector. Red color represents suppressed expression compared with ctrl (GFP). n=3.


( A ) . Volcano plot of TaqMan array showing the mean average expression and p - value of miRNA expression profiles between TruB1 KD and ctrl ( scramble ) . N = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test . MiRNAs suppressed ( Relative expression TruB1 KD / ctrl < 0 . 8 and p - value < 0 . 1 ) are enclosed by the red square .

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(A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. Significance was assessed using 2‐tailed Student's t‐test. MiRNAs suppressed (Relative expression TruB1 KD / ctrl <0.8 and p-value < 0.1) are enclosed by the red square.


( D ) . Relative expression of let - 7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl ( siRNA ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( E ) . Relative expression of primary - let - 7 family miRNAs as in ( D ) determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( C ) . Relative miRNA expression of let - 7a in HEK - 293 cells infected with tetracycline - inducible expressing lentiviruses for TruB1 , mt1 , mt2 or GFP 5 days after doxycycline treatment , as determined by qRT - PCR . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( D ) . Northern blotting for let - 7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP , 5 days after doxycycline treatment .

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(D). Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment.


( G ) . Location of pseudouridine sites detected by the CMC primer extension method . Total RNA purified from HEK - 293FT cells were treated with CMC . CMC treated RNA were reverse - transcribed with RI - labelled specific primers for tRNAphe or pri - let - 7b . ddATP was used for sequence control . Pseudouridines are indicated by black arrows .

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

(G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.


( A ) . RIP analysis of pri - let - 7a1 and Flag - TruB1 from HEK - 293FT cells . RNA was extracted from IP material and analyzed by qRT - PCR . HEK - 293FT cells were infected with lentiviruses encoding tetracycline - inducible expression of TruB1 , mt1 , mt2 , or GFP and treated with doxycycline . Error bars show SD ; n = 3 . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

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(A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


( B ) . EMSA of 32p - ATP - labelled pri - let - 7a1 or pri - let - 7a1 loop mt mixed with recombinant TruB1 , mt1 or mt2 at several doses . RNP : Ribonucleoprotein complexes .

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(B). EMSA of 32p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes.


( E ) . Tag densities represented as dots . Density of miRNA clusters in HITS - CLIP were normalized by miRNA expression as determined by TaqMan array .

O O O O O 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). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array.


( A ) . In vitro processing assay for RI - labelled pri - let - 7a1 . Autoradiographs of gels showing pri - let - 7a1 treated with whole cell lysate ( WCL ) from HEK - 293FT cells transfected with GFP , TruB1 , mt1 , or mt2 ( overexpression , left ) , and TruB1 KD or ctrl ( siRNA , right ) . Dicer KD was used to show the height of pre - let - 7a1 . ( B ) RI intensities of ( A ) were quantified and normalized to ctrl . Relative processing rate of pri - let - 7a1 into pre - and mature - are shown . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). Dicer KD was used to show the height of pre-let-7a1. (B) RI intensities of (A) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( C ) . RIP assay of pri - let - 7a1 and DGCR8 from HEK - 293FT cells . Western blotting for input or immunoprecipitate ( IP ) using anti - DGCR8 antibody and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . ( D ) . RIP assay of pri - let - 7a1 and Flag - tagged TruB1 from HEK - 293FT cells with Lin28B KD or ctrl ( siRNA ) . Western blotting for input or IP material using anti - Flag antibody , anti - Lin28B antibody , and anti - actin antibody are shown on top . RNA was extracted from IP material and analyzed by qRT - PCR ( bottom ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). RIP assay of pri-let-7a1 and DGCR8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. (D). RIP assay of pri-let-7a1 and Flag-tagged TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show


( B ) . Relative luciferase activity of KRAS reporter or Ctrl ( empty ) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline - inducible TruB1 , mt1 , mt2 , or GFP 5 days after doxycycline treatment . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

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(B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( D ) . Relative expression of let - 7 families determined by qRT - PCR in HEK - 293FT cells with KD of let - 7 family members or ctrl ( using 2 ' - O - methylated antisense inhibitor ) . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * . Data information : All experiments were performed in triplicate . Error bars show SD .

(D). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2'-O-methylated antisense inhibitor). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*. Data information: All experiments were performed in triplicate. Error bars show SD.


( E ) . Real - time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline - inducible TruB1 or GFP , 5 days after doxycycline treatment , with or without KD of let - 7 family members . GFP : GFP without let - 7 KD . WT : overexpression of wild type TruB1 without let - 7 KD . WT + let7 KD : WT : overexpression of wild type TruB1 with let - 7 KD . GFP + let - 7KD : GFP with let - 7 KD . Significance was assessed using 2 ‐ tailed Student ' s t ‐ test , < 0 . 05 * .

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(E). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. GFP: GFP without let-7 KD. WT: overexpression of wild type TruB1 without let-7 KD. WT + let7 KD: WT: overexpression of wild type TruB1 with let-7 KD. GFP + let-7KD: GFP with let-7 KD. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.


RT - qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR + / + and LXR − / − primary BMMs . Fold changes are shown relative to LXR + / + 0 h . Error bars represent ± SEM for n = 4 - 8 ( * * P = 0 . 001 at 8 h and * * P = 0 . 00004 at 16 h versus LXR − / − ; * * P = 0 . 0003 at 0 h versus LXR + / + ) ; bottom , proposed model explaining the ligand ‐ dependent induction of Abca1 . Following ligand stimulation , LXR - corepressor complexes are exchanged for LXR - coactivator complexes , which promote gene expression . The LXR - cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown .

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

RT-qPCR of Abca1 transcripts following 1 μM T0901317 stimulation of LXR+/+ and LXR−/− primary BMMs. Fold changes are shown relative to LXR+/+ 0 h. Error bars represent ± SEM for n = 4-8 (**P = 0.001 at 8 h and **P = 0.00004 at 16 h versus LXR−/−; **P = 0.0003 at 0 h versus LXR+/+); bottom, proposed model explaining the ligand‐dependent induction of Abca1. Following ligand stimulation, LXR-corepressor complexes are exchanged for LXR-coactivator complexes, which promote gene expression. The LXR-cofactor interactions responsible for the attenuation of expression beginning after 16 h remain unknown.


Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length SND1 , SART1 , or HMBOX1 . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . A diagram of the reporter construct is shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 9 ( * * P = 0 . 001 , * P = 0 . 011 for SND1 , * P = 0 . 046 for SART1 ) .

O O O O O O O O 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

Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length SND1, SART1, or HMBOX1. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. A diagram of the reporter construct is shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 9 (**P = 0.001, *P = 0.011 for SND1, *P = 0.046 for SART1).


Luciferase reporter assays from RAW 264 . 7 macrophages expressing reporter alone , or together with full ‐ length NCOA5 or an NCOA5 mutant lacking the NH2 ‐ terminus . LXR ligand stimulations with 1 μM T0901317 were performed for 18 h . Diagrams of the reporter constructs are shown above . Fold changes are shown relative to vehicle ‐ stimulated reporter alone ( first lane ) . Error bars represent ± SEM for n = 6 - 12 ( * * P 0 . 001 , * P = 0 . 035 ) .

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

Luciferase reporter assays from RAW 264.7 macrophages expressing reporter alone, or together with full‐length NCOA5 or an NCOA5 mutant lacking the NH2‐terminus. LXR ligand stimulations with 1 μM T0901317 were performed for 18 h. Diagrams of the reporter constructs are shown above. Fold changes are shown relative to vehicle‐stimulated reporter alone (first lane). Error bars represent ± SEM for n = 6-12 (**P 0.001, *P = 0.035).


RT - qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus . LXR ligand stimulations were performed with 1 μM T0901317 for 18 h . Fold changes are shown relative to vehicle ‐ stimulated control . Error bars represent ± SEM for n = 4 ( * P = 0 . 029 ) .

O 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

RT-qPCR of Abca1 transcripts following infection of primary BMMs with Ncoa5 or control retrovirus. LXR ligand stimulations were performed with 1 μM T0901317 for 18 h. Fold changes are shown relative to vehicle‐stimulated control. Error bars represent ± SEM for n = 4 (*P = 0.029).


In vitro pulldown assays using recombinant GST ‐ LXRα to isolate the indicated in vitro translated NCOA5 constructs . Assays were performed for 2 h in the presence or absence of 2 μM T0901317 . Samples were immunoblotted as indicated . Note the requirement of the NCOA5 NH2 ‐ terminus for interacting with LXRα .

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In vitro pulldown assays using recombinant GST‐LXRα to isolate the indicated in vitro translated NCOA5 constructs. Assays were performed for 2 h in the presence or absence of 2 μM T0901317. Samples were immunoblotted as indicated. Note the requirement of the NCOA5 NH2‐terminus for interacting with LXRα.


Immunoprecipitation of Protein C ‐ tagged LXRα from stable RAW 264 . 7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h , followed by immunoblotting for endogenous NCOA5 or over ‐ expressed LXRα . Protein immunoblots of nuclear extracts are shown below .

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

Immunoprecipitation of Protein C‐tagged LXRα from stable RAW 264.7macrophagenuclear extracts stimulated with 1 μM T0901317 or vehicle control for 18 h, followed by immunoblotting for endogenous NCOA5 or over‐expressed LXRα. Protein immunoblots of nuclear extracts are shown below.


NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the ligand ‐ stimulated association of NCOA5 . Error bars represent ± SEM for n = 4 - 9 ( * P = 0 . 02 ) .

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NCOA5 ChIP time course from primary BMMs stimulated with 1 μM T0901317 or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the ligand‐stimulated association of NCOA5. Error bars represent ± SEM for n = 4-9 (*P = 0.02).


NCOA5 ChIP assays from LXR − / − BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 ( * * P = 0 . 0003 for vehicle and * * P = 0 . 002 for T0901317 ) .

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NCOA5 ChIP assays from LXR−/− BMMs stimulated with 1 μM T0901317 or vehicle control for 18 h. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6 (**P = 0.0003 for vehicle and **P = 0.002 for T0901317).


RT - qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non ‐ silencing or Ncoa5 ‐ specific shRNA . Fold changes are shown relative to shControl 0 h . Note the elevated Abca1 expression at 24 - 30 h following loss of NCOA5 . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0007 at 24 h and * * P = 0 . 004 at 30 h , * P = 0 . 02 ) .

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RT-qPCR of Abca1 expression following 1 μM T0901317 treatment in primary BMMs infected with a non‐silencing or Ncoa5‐specific shRNA. Fold changes are shown relative to shControl 0 h. Note the elevated Abca1 expression at 24-30 h following loss of NCOA5. Error bars represent ± SEM for n = 4-6 (**P = 0.0007 at 24 h and **P = 0.004 at 30 h, *P = 0.02).


Quantification of ABCA1 immunoblots from primary BMMs infected as in ( A ) . Representative immunoblot is shown in Supplementary Fig S7E . LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h . Error bars represent ± SEM for n = 2 ( * * P = 0 . 007 ) .

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Quantification of ABCA1 immunoblots from primary BMMs infected as in (A). Representative immunoblot is shown in Supplementary Fig S7E. LXR ligand stimulation with 1 μM T0901317 or vehicle control was performed for 8 h or 30 h. Error bars represent ± SEM for n = 2 (**P = 0.007).


A , B RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 alone or together with 6 μg / ml PolyIC ( A ) or 10 ng / ml LPS ( B ) . Fold changes are shown relative to vehicle ‐ stimulated shControl . Note only the loss of TLR3 ‐ mediated repression following Ncoa5 silencing . Error bars represent ± SEM for n = 4 - 10 ( * * P = 0 . 0003 for A , * * P = 0 . 0004 for shControl in B , * * P = 0 . 0002 for shNcoa5 in B versus T0901317 ) .

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

A, B RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317 alone or together with 6 μg/ml PolyIC (A) or 10 ng/ml LPS (B). Fold changes are shown relative to vehicle‐stimulated shControl. Note only the loss of TLR3‐mediated repression following Ncoa5 silencing. Error bars represent ± SEM for n = 4-10 (**P = 0.0003 for A, **P = 0.0004 for shControl in B, **P = 0.0002 for shNcoa5 in B versus T0901317).


C , D RT - qPCR of Abca1 expression from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h unstimulated , or with 6 μg / ml PolyIC ( C ) or 10 ng / ml LPS ( D ) . Fold changes are shown relative to unstimulated shControl . Error bars represent ± SEM for n = 4 - 6 ( * * P = 0 . 0006 for C , * * P = 0 . 00000001 for shControl in D , * * P = 0 . 000001 for shNcoa5 in D , * P = 0 . 013 versus T0901317 ) .

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C, D RT-qPCR of Abca1 expression from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h unstimulated, or with 6 μg/ml PolyIC (C) or 10 ng/ml LPS (D). Fold changes are shown relative to unstimulated shControl. Error bars represent ± SEM for n = 4-6 (**P = 0.0006 for C, **P = 0.00000001 for shControl in D, **P = 0.000001 for shNcoa5 in D, *P = 0.013 versus T0901317).


E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 proximal LXRE . Note the increased occupancy only in the presence of both lipid and inflammatory ligands . Error bars represent ± SEM for n = 8 - 12 ( * * P = 0 . 0007 versus T0901317 ) .

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E NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 proximal LXRE. Note the increased occupancy only in the presence of both lipid and inflammatory ligands. Error bars represent ± SEM for n = 8-12 (**P = 0.0007 versus T0901317).


F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg / ml PolyIC or left unstimulated . qPCR was performed for the Abca1 proximal LXRE . Error bars represent ± SEM for n = 5 - 6 .

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F NCOA5 ChIP assays from primary BMMs stimulated for 3 h with 6 μg/ml PolyIC or left unstimulated. qPCR was performed for the Abca1 proximal LXRE. Error bars represent ± SEM for n = 5-6.


G Cholesterol efflux assays to APOA1 from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Agonist stimulations were performed for 6 h . Error bars represent ± SEM for n = 3 ( * P = 0 . 02 versus T0901317 ) .

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G Cholesterol efflux assays to APOA1 from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Agonist stimulations were performed for 6 h. Error bars represent ± SEM for n = 3 (*P = 0.02 versus T0901317).


A , B Unmodified / pSer5 RNAPII ( A ) or RNAPII pSer2 ( B ) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317 , 1 μM T0901317 + 6 μg / ml PolyIC , or vehicle control . qPCR was performed for the Abca1 TSS . Error bars represent ± SEM for n = 6 - 9 ( * * P = 0 . 0001 in A , * * P = 0 . 002 in B versus T0901317 ) .

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A, B Unmodified/pSer5 RNAPII (A) or RNAPII pSer2 (B) ChIP assays from primary BMMs stimulated for 4 h with 1 μM T0901317, 1 μM T0901317 + 6 μg/ml PolyIC, or vehicle control. qPCR was performed for the Abca1 TSS. Error bars represent ± SEM for n = 6-9 (**P = 0.0001 in A, **P = 0.002 in B versus T0901317).


C , D RNAPII ChIP assays as in ( A , B ) but performed from LXR − / − BMMs . Error bars represent ± SEM for n = 5 - 9 ( * * P = 0 . 000000001 , * P = 0 . 02 versus T0901317 ) .

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C, D RNAPII ChIP assays as in (A, B) but performed from LXR−/− BMMs. Error bars represent ± SEM for n = 5-9 (**P = 0.000000001, *P = 0.02 versus T0901317).


E RT - qPCR of Abca1 expression from LXR + / + versus LXR − / − BMMs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 together with 6 μg / ml PolyIC . Fold changes are shown relative to vehicle ‐ stimulated LXR + / + . Error bars represent ± SEM for n = 4 ( * * P = 0 . 004 , * P = 0 . 012 versus T0901317 ) .

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E RT-qPCR of Abca1 expression from LXR+/+ versus LXR−/− BMMs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 together with 6 μg/ml PolyIC. Fold changes are shown relative to vehicle‐stimulated LXR+/+. Error bars represent ± SEM for n = 4 (**P = 0.004, *P = 0.012 versus T0901317).


F RNAPII pSer2 ChIP assays from primary BMMs infected with non ‐ silencing or Ncoa5 ‐ specific shRNAs . Ligand stimulations were performed for 4 h with vehicle control , 1 μM T0901317 , or 1 μM T0901317 + 6 μg / ml PolyIC . qPCR was performed for the Abca1 TSS . Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs ( * * P = 0 . 004 versus baseline ) . Error bars represent ± SEM for n = 3 from 11 mice .

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F RNAPII pSer2 ChIP assays from primary BMMs infected with non‐silencing or Ncoa5‐specific shRNAs. Ligand stimulations were performed for 4 h with vehicle control, 1 μM T0901317, or 1 μM T0901317 + 6 μg/ml PolyIC. qPCR was performed for the Abca1 TSS. Note RNAPII pSer2 returns to baseline occupancy following TLR3 stimulation in shControl but not shNcoa5 BMMs (**P = 0.004 versus baseline). Error bars represent ± SEM for n = 3 from 11 mice.


The C - terminal domains of IFT52 and IFT46 co - purify with the IFT81460 - C / 74460 - C / 27 / 251 - 136 protein complex ( C ) The SEC fractions indicated on the right with 1 - 3 were analysed by SDS - PAGE and stained with Coomassie to evaluate the protein composition .

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The C-terminal domains of IFT52 and IFT46 co-purify with the IFT81460-C/74460-C/27/251-136 protein complex (C) The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.


The C - terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623 - C of IFT81 and 615 - C of IFT74 ( D ) . The SEC fractions indicated on the right with 1 - 3 were analysed by SDS - PAGE and stained with Coomassie to evaluate the protein composition .

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The C-terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623-C of IFT81 and 615-C of IFT74 (D). The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.


( G ) Coomassie stained SDS PAGE gel of a GST - pulldown using the CrIFT88His - GST / 70 / 521 - 430 / 46 complex immobilized on GST beads as bait and the CrIFT811 - 387 - GFP / 74135 - 420 complex used as prey . The band highlighted by an * corresponds to pulled down CrIFT74135 - 420 . CrIFT811 - 387 - GFP runs on top of IFT70 and is not visible in the pull - down .

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(G) Coomassie stained SDS PAGE gel of a GST-pulldown using the CrIFT88His-GST/70/521-430/46 complex immobilized on GST beads as bait and the CrIFT811-387-GFP/74135-420 complex used as prey. The band highlighted by an * corresponds to pulled down CrIFT74135-420. CrIFT811-387-GFP runs on top of IFT70 and is not visible in the pull-down.


( B ) Pull - down experiments with purified IFT80Venus immobilized on GFP - binder beads and either IFT172 or IFT172968 - C . IFT80 pulls down full length IFT172 but not the truncated version lacking the N - terminal 967 residues .

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(B) Pull-down experiments with purified IFT80Venus immobilized on GFP-binder beads and either IFT172 or IFT172968-C. IFT80 pulls down full length IFT172 but not the truncated version lacking the N-terminal 967 residues.


( F ) GST tagged CrIFT1721 - 968 pulls down the CH - domain of CrIFT571 - 234 but not the mutated CH - domain of CrIFT57 where E77 and K84 residues were replaced by alanines .

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(F) GST tagged CrIFT1721-968 pulls down the CH-domain of CrIFT571-234 but not the mutated CH-domain of CrIFT57 where E77 and K84 residues were replaced by alanines.


( a ) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV - OAT at the doses of 1x1013 ( n = 9 ) and 3x1013 genome copies ( gc ) / Kg ( n = 6 ) , or AAV - GFP at the dose of 1x1013 gc / Kg ( n = 9 ) . Normal range was established on wild - type mice ( n = 7 ) and average ± 2SD was calculated . Data are shown as averages ± SEM . * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA test ) .

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(a) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV-OAT at the doses of 1x1013 (n=9) and 3x1013 genome copies (gc)/Kg (n=6), or AAV-GFP at the dose of 1x1013 gc/Kg (n=9). Normal range was established on wild-type mice (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVA test).


( c ) Plasma ornithine and lysine concentrations at 12 months of age ( 10 - months post - injection ) in Oatrhg mice injected with AAV - OAT ( n = 3 ) or AAV - GFP ( n = 3 ) ; WT mouse controls ( n = 5 ) are also shown ; each sample was analyzed as triplicates , data are shown as averages ± SEM . One - way ANOVA test with Tukey correction ; * p < 0 . 05 ; * * * * p < 0 . 0001 .

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(c) Plasma ornithine and lysine concentrations at 12 months of age (10-months post-injection) in Oatrhg mice injected with AAV-OAT (n=3) or AAV-GFP (n=3); WT mouse controls (n=5) are also shown; each sample was analyzed as triplicates, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.


( e ) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV - GFP or AAV - OAT . A WT mouse liver was included as control . Vinculin was used as loading control .

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(e) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV-GFP or AAV-OAT. A WT mouse liver was included as control. Vinculin was used as loading control.


( g ) Liver immunohistochemistry showing approximately 10 % of OAT - positive cells in Oatrhg mice injected with AAV - OAT . Livers of WT and Oatrhg mice injected with AAV - GFP are shown as controls . 5x ( images on the left side ) and 20x ( images on the right side ) fields of view are shown .

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(g) Liver immunohistochemistry showing approximately 10% of OAT-positive cells in Oatrhg mice injected with AAV-OAT. Livers of WT and Oatrhg mice injected with AAV-GFP are shown as controls. 5x (images on the left side) and 20x (images on the right side) fields of view are shown.


( h ) Representative H & E staining of the retinas of 11 - month - old Oatrhg mice injected with AAV - GFP or AVV - OAT at the doses of 1x1013 or 3x1013 gc / Kg ; age - matched WT mice and uninjected Oatrhg mice were included as controls ( 40X ) . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; INL , inner nuclear layer .

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(h) Representative H&E staining of the retinas of 11-month-old Oatrhg mice injected with AAV-GFP or AVV-OAT at the doses of 1x1013 or 3x1013 gc/Kg; age-matched WT mice and uninjected Oatrhg mice were included as controls (40X). Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


( a ) Representative electron micrographs of the mouse retina of an AAV - GFP injected Oatrhg mouse showing retinal pigmented epithelium ( RPE ) and adjacent abnormal outer segment ( OS ) disk membranes ( black arrows ) . Phagolysosomes containing undigested OS disc membranes are present ( asterisks ) . ( b ) Retina of AAV - OAT injected Oatrhg mice showing photoreceptor disk membranes ( white arrows ) and RPE ultrastructure comparable to age matched WT mice ( c , white arrows ) . ( c ) Control retina of WT mouse is shown as comparison .

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(a) Representative electron micrographs of the mouse retina of an AAV-GFP injected Oatrhg mouse showing retinal pigmented epithelium (RPE) and adjacent abnormal outer segment (OS) disk membranes (black arrows). Phagolysosomes containing undigested OS disc membranes are present (asterisks). (b) Retina of AAV-OAT injected Oatrhg mice showing photoreceptor disk membranes (white arrows) and RPE ultrastructure comparable to age matched WT mice (c, white arrows). (c) Control retina of WT mouse is shown as comparison.


( a ) Plasma ornithine concentrations in AAV - GFP - ( n = 3 ) and AAV - OAT - injected ( n = 3 ) OatΔ mice . Normal range of wild - type mice is also shown . Each value plotted represents mean ± SEM ; Two - way ANOVA test , * p < 0 . 05 , * * p < 0 . 01 .

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(a) Plasma ornithine concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) OatΔ mice. Normal range of wild-type mice is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.


( c ) Retinal H & E ( 40X ) of OatΔ mice injected with either AAV - GFP or AAV - OAT . Representative of three mice per groups are shown ; WT mice were included as control . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; INL , inner nuclear layer .

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(c) Retinal H&E (40X) of OatΔ mice injected with either AAV-GFP or AAV-OAT. Representative of three mice per groups are shown; WT mice were included as control. Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


( A ) WT or Mfn2 KO MEFs ( Mfn2 KO cells ) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology . Scale bar : 1 μm .

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(A) WT or Mfn2 KO MEFs (Mfn2 KO cells) were treated with 1 μM Tg for 12 h and then processed for EM visualization of the ER morphology. Scale bar: 1 μm.


( B ) EM images of Tg ‐ treated Mfn2 KO cells show accumulation of ER membrane stacking . Scale bar : 1 μm .

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(B) EM images of Tg‐treated Mfn2 KO cells show accumulation of ER membrane stacking. Scale bar: 1 μm.


( C ) WT , Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β ‐ GFP plasmid and treated with 1 μM Tg for 24 h . Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg . Scale bar : 10 μm . Insets show × 10 zoomed images . Scale bar : 5 μm .

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(C) WT, Mfn2 KO or Mfn1 KO cells were transfected with the Sec61β‐GFP plasmid and treated with 1 μM Tg for 24 h. Confocal microscopy images show ER vacuolization in Mfn2 KO cells treated with Tg. Scale bar: 10 μm. Insets show × 10 zoomed images. Scale bar: 5 μm.


( D ) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A ‐ bodipy to stain ER and Golgi . Representative flow ‐ cytometry histograms ( upper panel ) . Mean fluorescence intensity was used to quantify ER expansion ( n = 5 ) ( lower panel ) . Data are mean ± s . e . m . * P0 . 05 versus WT group .

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(D) WT and Mfn2 KO cells were treated with Tg 1 μM for 3 h and then incubated with brefeldin A‐bodipy to stain ER and Golgi. Representative flow‐cytometry histograms (upper panel). Mean fluorescence intensity was used to quantify ER expansion (n=5) (lower panel). Data are mean±s.e.m. *P0.05 versus WT group.


( A ) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h . Total and cleaved caspase 3 levels were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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(A) WT and Mfn2 KO cells were treated with 1 μM Tg for 12 or 24 h. Total and cleaved caspase 3 levels were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( B , C ) WT and Mfn2 KO cells were treated with 0 . 5 μg / ml tunicamycin ( Tm ) , 100 ng / ml brefeldin A ( Bref ) , or 1 μM Tg for 24 h . Total and cleaved caspase 3 levels were detected by western blot ( B ) and caspase activity ( C ) by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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(B, C) WT and Mfn2 KO cells were treated with 0.5 μg/ml tunicamycin (Tm), 100 ng/ml brefeldin A (Bref), or 1 μM Tg for 24 h. Total and cleaved caspase 3 levels were detected by western blot (B) and caspase activity (C) by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( D ) Flow ‐ cytometry analysis of the Sub G1 DNA fragmentation in methanol ‐ fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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(D) Flow‐cytometry analysis of the Sub G1 DNA fragmentation in methanol‐fixed WT and Mfn2 KO cells after incubation with or without 1 μM Tg for 24 h. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.