b , Validation targets in lung at 4 dpi . Horizontal lines indicate medians . p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney . Data information : M : hamsters ; F : hamsters . Data were obtained from two independent experiments for each sex .

O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O

b, Validation targets in the lung at 4 dpi. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test. Data information: M: male hamsters; F: female hamsters. Data were obtained from two independent experiments for each sex.


a , representative histopathology photomicrographies of lungs according to different groups : mock _ saline , CoV _ saline CoV _ ivermectin . Top panels : whole lung sections . Bottom panels : high magnification . CoV _ saline section exhibits important congestion ( * ) , edema associated with few mononuclear ( arrowheads ) . Note thickening of alveolar walls . CoV _ ivermectin section exhibits important amounts of mononuclear ( black arrowheads ) less marked signs of congestion or edema . Hematoxylin Eosin . Scale bars = 1 mm ( top panels ) 20 μm ( bottom panels ) .

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

a, representative histopathology photomicrographies of lungs according to the different groups: mock_saline, CoV_saline and CoV_ivermectin. Top panels: whole lung sections. Bottom panels: high magnification. CoV_saline section exhibits important congestion (*), edema associated with few mononuclear cells (white arrowheads). Note the thickening of the alveolar walls. CoV_ivermectin section exhibits important amounts of mononuclear cells (black arrowheads) and less marked signs of congestion or edema. Hematoxylin and Eosin. Scale bars = 1 mm (top panels) and 20 μm (bottom panels).


b , representative immunofluorescence photomicrographies of neutrophils ( Ly - 6G ) , monocytes / macrophages ( Iba1 ) , M2 macrophages ( Arg1 ) SARS - CoV - 2 ( NP ) in lung . Scale bars = 50 μm .

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

b, representative immunofluorescence photomicrographies of neutrophils (Ly-6G), monocytes/macrophages (Iba1), M2 macrophages (Arg1) and SARS-CoV-2 (NP) in the lung. Scale bars = 50 μm.


c , of Iba1 + , Arg1 + Iba1 + Arg1 + in lungs . mock _ saline n = 3 ( ) , mock _ ivermectin n = 4 ( ) , CoV _ saline n = 9 ( 6 3 ) , CoV _ ivermectin n = 6 ( 4 2 ) . Data information : M : hamsters ; F : hamsters . Horizontal lines indicate medians . p value is indicated in bold when significant at a 0 . 05 threshold . Mann - Whitney

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

c, quantification of Iba1+ cells, Arg1+ cells and Iba1+Arg1+ cells in the lungs. mock_saline n=3 (males), mock_ivermectin n=4 (males), CoV_saline n=9 (6 males and 3 females), CoV_ivermectin n=6 (4 males and 2 females). Data information: M: male hamsters; F: female hamsters. Horizontal lines indicate medians. The p value is indicated in bold when significant at a 0.05 threshold. Mann-Whitney test


B . qRT - PCR expression of lncMN2 isoforms ( left panel ) embedded miRNAs ( right panel ) in mESC EBs differentiated for 6 days ( EB6 ) . Data , that are expressed as mean ( error bars represent SD ) , were normalized over Atp50 mRNA ( left ) snoRNA U25 ( right ) reported as fold over control ( mESC ) , set as 1 . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

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

B. qRT-PCR expression analysis of lncMN2 isoforms (left panel) and embedded miRNAs (right panel) in mESC and EBs differentiated for 6 days (EB6). Data, that are expressed as mean (error bars represent SD), were normalized over Atp50 mRNA (left) and snoRNA U25 (right) levels and reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


C . qRT - PCR expression of lncMN2 - 202 / 203 / 204 isoforms ( left panel ) , miR - 325 - 3p ( middle panel ) miR - 384 - 5p ( right panel ) in WT , KO1 KO2 EB6 . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

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

C. qRT-PCR expression analysis of lncMN2-202/203/204 isoforms (left panel), miR-325-3p (middle panel) and miR-384-5p (right panel) in WT, KO1 and KO2 EB6. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


D . Cytofluorimetric of from dissociated EB6 . left diagram reports distribution of ( of peak ) according to GFP , from WT ( gray ) , KO1 ( orange ) KO2 ( blue ) EB . In histogram aside , of GFP + in WT , KO1 KO2 is reported . Data are expressed as mean error bars represent SD . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cytofluorimetric analysis of cells from dissociated EB6. The left diagram reports the distribution of cells (percentage of the peak) according to GFP levels, from WT (gray), KO1 (orange) and KO2 (blue) EB. In the histogram aside, the percentage of GFP+ cells in WT, KO1 and KO2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


E . Left : representative immunofluorescence of MN marker Isl1 ( red signals ) in maintained in vitro for 14 days after EB6 dissociation ( DIV14 ) . DAPI counterstaining in blue . Scale bar = 100 mm . . Right : box plot shows distribution of Isl1 + nuclei with respect to total number marked with DAPI in WT in lncMN2 KO mutants ( clone 1 clone 2 ) . Data are expressed as mean error bars represent SD . central band represents median whiskers represent distribution that goes from minimum value to lower quartile from upper quartile to maximum value . N = 3 biological replicates 10 fields were analyzed for each replicate . Scale bar = 100 mm . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm.. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in lncMN2 KO mutants (clone 1 and clone 2). Data are expressed as mean and error bars represent SD. The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Scale bar=100 mm. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


B . qRT - PCR expression of lncMN2 - 203 isoform , lncMN2 - 202 / 204 , miR - 325 - 3p miR - 384 - 5p embedded miRNAs in WT , ∆ SP1 ∆ SP2 EB6 . Data , that are expressed as mean ( error bars represent SD ) , are normalized over Atp5o mRNA ( for lncRNA isoform ) or over snoRNA U25 ( for miRNA ) are reported as fold over control ( mESC ) , set as 1 . N = 3 biological replicates . Data are expressed as mean error bars represent SD . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

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

B. qRT-PCR expression analysis of lncMN2-203 isoform, lncMN2-202/204, miR-325-3p and miR-384-5p embedded miRNAs in WT, ∆SP1 and ∆SP2 EB6. Data, that are expressed as mean (error bars represent SD), are normalized over Atp5o mRNA levels (for lncRNA isoform quantification) or over snoRNA U25 levels (for miRNA quantification) and are reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


C . Cytofluorimetric of from dissociated EB6 . right diagram reports distribution ( of peak ) according to GFP , from WT ( gray ) , ∆ SP1 ( purple ) ∆ SP2 ( blue ) EB6 . In histogram aside , of GFP + in WT , ∆ SP1 ∆ SP2 is reported . Data are expressed as mean error bars represent SD . N = 3 biological replicates . Data are expressed as mean error bars represent SD . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-TISSUE O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Cytofluorimetric analysis of cells from dissociated EB6. The right diagram reports the cell distribution (percentage of the peak) according to GFP levels, from WT (gray line), ∆SP1 (purple line) and ∆SP2 (blue line) EB6. In the histogram aside, the percentage of GFP+ cells in WT, ∆SP1 and ∆SP2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


D . Left : representative immunofluorescence of MN marker Isl1 ( red signals ) in maintained in vitro for 14 days after EB6 dissociation ( DIV14 ) . DAPI counterstaining in blue . Scale bar = 100 mm . Right : box plot shows distribution of Isl1 + nuclei with respect to total number marked with DAPI in WT in ∆ SP mutants ( clone 1 clone 2 ) . central band represents median whiskers represent distribution that goes from minimum value to lower quartile than from upper quartile to maximum value . N = 3 biological replicates 10 fields were analyzed for each replicate . Data are expressed as mean error bars represent SD . Scale bar = 100 mm . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - , unpaired Student ' s t - ) .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆SP mutants (clone 1 and clone 2). The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Data are expressed as mean and error bars represent SD. Scale bar=100 mm. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


B - E ) Representaitve pictures showing that withdrawal of dietary leu but not his significantly reduced follicle proliferation ( pH3 , red ) in adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm .

O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O

B-E) Representaitve pictures showing that the withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.


withdrawal of dietary leu but not his significantly reduced follicle proliferation ( pH3 , red ) in adult ovary after 10 days , quantified in ( E ) ( n = 13 , 14 , 13 ) , scale bar = 100 μm .

O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-CELL_TYPE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O

withdrawal of dietary leu but not his significantly reduced stem cells and follicle cell proliferation (pH3, red) in the adult ovary after 10 days, quantified in (E) (n=13, 14,13), scale bar = 100 μm.


wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , significantly increased by dietary histidine depletion ( 25 % his ) , quantified in ( F ) ( n = 28 , 22 , 7 ) , scale bar = 50

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

wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his), quantified in (F) (n=28,22,7), scale bar = 50 μm


F - H ) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion ( 25 % leu ) , significantly increased by dietary histidine depletion ( 25 % his ) ,

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

F-H) Representaitve pictures showing that wildtype larval NB clonal growth was significantly reduced after 6 days of dietary leucine depletion (25% leu), and significantly increased by dietary histidine depletion (25% his),


I - K ) Representaitve pictures showing that adult nerfin - 1 clonal growth was significantly reduced on - his diet compared to CDD ( measured at day 0 , day 9 ) quantified in ( K ) ( n = 27 , 110 , 136 ) . Scale bar = 75μm . * * * p < 0 . 001 L - N ) Representaitve pictures showing that larval nerfin - 1 clonal growth was significantly reduced on 25 % his diet compared to CDD ( after 6 days ) , quantified in ( N ) ( n = 53 , 36 ) . Scale bar = 50 μm .

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

I-K) Representaitve pictures showing that adult nerfin-1 clonal growth was significantly reduced on -his diet compared to CDD (measured at day 0, and day 9) quantified in (K) (n= 27, 110, 136). Scale bar = 75μm. ***p<0.001 L-N) Representaitve pictures showing that larval nerfin-1 clonal growth was significantly reduced on 25% his diet compared to CDD (after 6 days), quantified in (N) (n=53,36). Scale bar = 50 μm.


O - Q ) Representaitve pictures showing that adult pros clonal growth was not significantly altered on - his diet compared to CDD ( after 7 days ) , quantified in ( Q ) ( n = 12 , 23 , 16 ) . Scale bar = 75μm . R - T ) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction ( 25 % his ) compared to CDD ( after 6 days ) , quantified in ( T ) ( n = 19 , 13 ) . Scale bar = 50 μm .

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

O-Q) Representaitve pictures showing that adult pros clonal growth was not significantly altered on -his diet compared to CDD (after 7 days), quantified in (Q) (n = 12, 23,16). Scale bar = 75μm. R-T) Representaitve pictures showing that larval pros clonal growth was not significantly altered by dietary histidine reduction (25% his) compared to CDD (after 6 days), quantified in (T) (n=19,13). Scale bar = 50 μm.


U - W ) Representative pictures showing that growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction ( 25 % his ) compared to CDD , quantified in ( W ) ( n = 27 , 19 ) . Scale bar = 100 μm .

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

U-W) Representative pictures showing that the growth of larval type II lineages overexpressing NACT was significantly reduced dietary histidine reduction (25% his) compared to CDD, quantified in (W) (n=27,19). Scale bar = 100 μm.


B ) of histidine 3 - methyl - L - histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or - his diet ( n = 3 , 3 ) . Both compounds are reduced to below detection ( < 0 . 3mM ) upon dietary his depletion .

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

B) Concentration of histidine and 3-methyl-L-histidine from whole Drosophila adults extracts measured by 1H NMR after 3 days of feeding on either CDD or -his diet (n=3,3). Both compounds are reduced to below detection levels (<0.3mM) upon dietary his depletion.


C ) Dietary supplementation of 3 - methyl - L - his for 4 days significantly increased adult nerfin - 1 clonal growth , rescued nerfin - 1 growth inhibition due to his dietary withdrawal ( n = 18 , 14 , 7 , 9 ) .

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

C) Dietary supplementation of 3-methyl-L-his for 4 days significantly increased adult nerfin-1 clonal growth, and rescued nerfin-1 growth inhibition due to his dietary withdrawal (n =18,14, 7,9).


D ) Dietary supplementation of histamine does not significantly increase adult nerfin - 1 clone , but can rescue nerfin - 1 clonal growth inhibition due to his dietary withdrawal ( n = 42 , 14 , 23 , 25 ) .

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

D) Dietary supplementation of histamine does not significantly increase adult nerfin-1 clone size, but can rescue nerfin-1 clonal growth inhibition due to his dietary withdrawal (n =42,14,23,25).


E ) Larval nerfin - 1 clonal growth is significantly decreased upon overexpression of HDC RNAi ( n = 23 , 36 ) .

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

E) Larval nerfin-1 clonal growth is significantly decreased upon overexpression of HDC RNAi (n=23,36).


G ) Hdc inhibition significantly reduced of Dpn + NBs ( n = 16 , 17 ) , significantly increased of Elav + neurons per clone ( n = 16 , 17 ) .

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

G) Hdc inhibition significantly reduced the percentage of Dpn+ NBs (n=16,17), and significantly increased the percentage of Elav+ neurons per clone (n=16,17).


H ) HdcRi overexpression reduced growth of nerfin - 1 NBs ( n = 17 , 19 ) neurons ( n = 36 , 19 ) measured as of nucleolus / volume .

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

H) HdcRi overexpression reduced cellular growth of nerfin-1 NBs (n=17,19) and neurons (n=36,19) measured as the ratio of nucleolus/nuclear volume.


A - E ) Representative of nerfin - 1 clones in adult VNC after 6 days of feeding on CDD or - his diet , scale bar = 100μm . Histidine dietary withdrawal resulted in clones significantly altering amount of per clone quantified in ( C ) ( n = 6 , 7 ) .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

A-E) Representative images of nerfin-1 clones in the adult VNC after 6 days of feeding on CDD or -his diet, scale bar = 100μm. Histidine dietary withdrawal resulted in clones significantly altering the amount of cell death per clone quantified in (C) (n = 6, 7).


Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav + neurons ( red ) quantified in ( D ) ( n = 16 , 17 ) significantly fewer Mira + NBs ( blue ) , quantified in ( E ) ( n = 12 , 22 ) ,

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

Histidine dietary withdrawal resulted in clones containing significantly more differentiated Elav+ neurons (red) quantified in (D) (n = 16,17) and significantly fewer Mira+ NBs (blue), quantified in (E) (n = 12,22),


F - G ) Larval nerfin - 1 clones consisted of significantly greater proportion of differentiated neurons ( n = 10 , 7 ) significantly reduced proportion of NBs per clone upon his dietary depletion ( 25 % his , n = 9 , 11 ) .

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

F-G) Larval nerfin-1 clones consisted of significantly greater proportion of differentiated neurons (n=10,7) and significantly reduced proportion of NBs per clone upon his dietary depletion (25% his, n=9,11).


Representative of adult nerfin - 1 ( I - J ' , green ) pros clones ( K - L ' , green ) , scale bar = 5μm . In nerfin - 1 clones , of NBs ( Ase + , blue ) was significantly reduced upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 33 , 20 ) . In pros clones , of NBs ( Ase + blue ) , growth measured as of nucleolus ( Fib , ) / ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( H ) ( n = 9 , 8 )

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

Representative images of adult nerfin-1 (I-J', green ) and pros clones (K-L', green), scale bar = 5μm. In nerfin-1 clones, the size of the NBs (Ase+, blue) was significantly reduced upon 6 days of histidine dietary withdrawal, quantified in (H) (n=33,20). In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (H) (n=9,8)


growth measured as of nucleolus ( Fib , ) / ( Ase , blue ) volume was also significantly reduced , quantified in ( M ) ( n = 33 , 20 ) .

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

Cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was also significantly reduced, quantified in (M) (n=33,20).


growth measured as of nucleolus / volume was significantly reduced in larval nerfin - 1 NBs after 6 days his dietary reduction ( 25 % his compared to CDD , n = 14 , 6 ) quantified in ( N ) .

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

Cellular growth measured as the ratio of nucleolus /nuclear volume was significantly reduced in larval nerfin-1 NBs after 6 days his dietary reduction (25% his compared to CDD, n=14,6) quantified in (N).


In pros clones , of NBs ( Ase + blue ) , growth measured as of nucleolus ( Fib , ) / ( Ase , blue ) volume was not significantly altered upon 6 days of histidine dietary withdrawal , quantified in ( O ) ( n = 9 , 7 ) .

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

In pros clones, the size of the NBs (Ase+ blue), and cellular growth measured as the ratio of nucleolus (Fib, white)/nuclear (Ase, blue) volume was not significantly altered upon 6 days of histidine dietary withdrawal, quantified in (O) (n=9,7).


Panel ( P ) shows that larval nerfin - 1 neuron to NB reversion requires 15 - fold increase in volume ( n = 10 ) pros GMC to NB reversion requires approximately 3 . 5 - fold increase in volume ( n = 31 ) .

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

Panel (P) shows that the larval nerfin-1 neuron to NB reversion requires 15-fold increase in cellular volume (n=10) and pros GMC to NB reversion requires approximately 3.5-fold increase in cellular volume (n=31).


A - D ) Representative pictures showing that pros NBs GMCs are smaller than wildtype ( NBs GMCs are distinguished at telophase , as a doublet of unequal ) , quantified in C ( n = 25 , 31 , 21 , 16 ) . pros NBs exhibit similar nucleolus / ( arrows , nucleolus marked by Fib ) compared to wild type pros ; TorDN NBs , quantified in D ( n = 10 , 18 , 15 ) .

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

A-D) Representative pictures showing that pros NBs and GMCs are smaller than wildtype (NBs and GMCs are distinguished at telophase, as a doublet of unequal size), quantified in C (n=25,31,21,16). pros NBs exhibit similar nucleolus /nuclear ratio (white arrows, nucleolus marked by Fib) compared to wild type and pros;TorDN NBs, quantified in D (n=10,18,15).


E - F ) Representative pictures showing pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / ( E - F )

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

E-F) Representative pictures showing pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F)


pros clones ( green ) generated in myc hypomorphic background did not significantly alter pros nucleolus / ( E - F ) , nucleolus labelled with Fib ( red ) [ quantified in G ( n = 44 , 43 ) ] , clone [ quantified in H ( n = 41 , 34 ) ] , or proportion of Dpn + NBs Elav + neurons per clone [ quantified in I ( n = 15 , 15 , 15 , 15 ) ] .

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

pros clones (green) generated in myc hypomorphic background did not significantly alter pros nucleolus/nuclear ratio (E-F), nucleolus labelled with Fib (red) [quantified in G (n=44,43)], clone size [quantified in H (n=41,34)], or the proportion of Dpn+ NBs and Elav+ neurons per clone [quantified in I (n=15,15,15,15)].


J - K ) Representative pictures showing that nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K )

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

J-K) Representative pictures showing that nerfin-1 clones (green) generated in myc hypomorphic background (J-K)


nerfin - 1 clones ( green ) generated in myc hypomorphic background ( J - K ) exhibited significantly reduced nucleolus / , [ nucleolus labelled with Fib ( red ) ] reduced clone , quantified in L ( n = 34 , 27 ) M ( n = 106 , 75 ) , displayed an increased proportion of differentiated Elav + neurons , reduced proportion of Dpn + NBs per clone , quantified in N ( n = 20 , 20 , 20 , 20 ) . scale bar =

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

nerfin-1 clones (green) generated in myc hypomorphic background (J-K) exhibited significantly reduced nucleolus/nuclear ratio, [nucleolus labelled with Fib (red)] and reduced clone size, quantified in L (n=34,27) and M (n= 106,75), and displayed an increased proportion of differentiated Elav+ neurons, and reduced proportion of Dpn+ NBs per clone, quantified in N (n=20,20,20,20). scale bar =10μm


O ) Reducing Myc dosage with hypomorphic Myc allele , mycP0 , abolished increase in nerfin - 1 clone mediated by 2x histidine dietary intake , 2x his ( n = 41 , 18 , 10 , 57 ) .

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

O) Reducing Myc dosage with the hypomorphic Myc allele, mycP0, abolished the increase in nerfin-1 clone size mediated by 2x histidine dietary intake, 2x his (n=41,18,10,57).


P ) Overexpression of myc restored growth of nerfin - 1 clones inhibited by histidine dietary reduction , 25 % his ( n = 51 , 36 , 39 , 29 ) .

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

P) Overexpression of myc restored the growth of nerfin-1 clones inhibited by histidine dietary reduction, 25% his (n=51,36,39,29).


Q , R ) pros ( Q ) nerfin - 1 ( R ) clones are reduced upon Tor inhibition ( n = 22 , 63 ) compared to control ( n = 51 , 65 ) , but their growth differentially depend on acid transporter Slif ( n = 12 , 32 ) downstream growth regulators eIF4E ( n = 21 , 47 ) S6K ( n = 39 , 37 ) .

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

Q, R) pros (Q) and nerfin-1 (R) clones are reduced upon Tor inhibition (n=22,63) compared to control (n=51,65), but their growth differentially depend on amino acid transporter Slif ( n=12,32) and downstream growth regulators eIF4E (n= 21,47) and S6K (n=39,37).


, Maximum projections of confocal z - stacks taken from apical cochlear region of control ( A ) mice at postnatal day 11 . Inner hair ( IHCs ) were stained with antibodies against Kir2 . 1 ( green ) hair marker MYO7a ( blue ) . At least 3 mice for each genotype were used .

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

, Maximum intensity projections of confocal z-stacks taken from the apical cochlear region of control (A) mice at postnatal day 11. Inner hair cells (IHCs) were stained with antibodies against Kir2.1 (green) and the hair cell marker MYO7a (blue). At least 3 mice for each genotype were used.


C , D , Currents from IHCs of control ( C , P9 ) Kir2 . 1 - OE ( D , P10 ) pre - hearing mice . Currents were elicited by using depolarizing hyperpolarizing voltage steps , with a nominal increment of 10 mV , from a holding potential of − 84 mV . potentials are shown next to some of traces . Note that large inward rectifier Kir2 . 1 current is only present in of Kir2 . 1 - OE mouse ( D ) . outward current is primarily carried by a delayed rectifier current IK . IK1 identifies small inwardly rectifying K + current normally expressed in IHCs . E , Steady - state current - voltage curves obtained from IHCs of control ( P9 - P11 ) Kir2 . 1 - OE ( P9 - P11 ) mice .

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

C, D, Currents from IHCs of control (C, P9) and Kir2.1-OE (D, P10) pre-hearing mice. Currents were elicited by using depolarizing and hyperpolarizing voltage steps, with a nominal increment of 10 mV, from a holding potential of −84 mV. Test potentials are shown next to some of the traces. Note that the large inward rectifier Kir2.1 current is only present in the IHC of the Kir2.1-OE mouse (D). The outward current is primarily carried by a delayed rectifier current IK. IK1 identifies the small inwardly rectifying K+ current normally expressed in IHCs. E, Steady-state current-voltage curves obtained from IHCs of control (P9-P11) and Kir2.1-OE (P9-P11) mice.


F , G , Representative ∆ F / F0 traces from IHCs GER of P7 - P9 control ( F ) Kir2 . 1 - OE ( G ) mice in presence of 0 . 3 mM Ca2 + . Spontaneous ATP - dependent Ca2 + waves from GER ( green traces ) were eliciting coordinated Ca2 + signals in IHCs from both controls Kir2 . 1 - OE mice . For each genotype , two separate sets of recordings from 2 mice are shown ( top bottom right ) , with top traces being linked to on left : before [ 1 ] , during [ 2 ] after [ 3 ] ) generation of a large Ca2 + wave from GER . For details about frequency duration of Ca2 + waves , number of mice recordings see Fig EV3 . recordings were obtained at . Traces are computed as pixel averages of regions of interest centred on IHCs .

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

F, G, Representative ∆F/F0 traces from the IHCs and GER of P7-P9 control (F) and Kir2.1-OE (G) mice in the presence of 0.3 mM Ca2+. Spontaneous ATP-dependent Ca2+ waves from the GER (green traces) were eliciting coordinated Ca2+ signals in the IHCs from both controls and Kir2.1-OE mice. For each genotype, two separate sets of recordings from 2 mice are shown (top and bottom right), with the top traces being linked to the images on the left: before [1], during [2] and after [3]) the generation of a large Ca2+ wave from the GER. For details about the frequency and duration of the Ca2+ waves, and the number of mice and recordings see Fig EV3. All recordings were obtained at body temperature. Traces are computed as pixel averages of regions of interest centred on IHCs.


C , Peak - to - peak MET current - voltage curves from P6 - P7 apical - coil IHCs of 7 control ( 12 IHCs ) 3 Kir2 . 1 - OE mice ( 7 IHCs ) . Recordings were obtained by mechanically stimulating hair bundles of IHCs at same time as stepping their potential from − 124 mV to + 96 mV in 20 mV increments . two sets of data are not significantly different : P = 0 . 6320 , 2 - way ANOVA .

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

C, Peak-to-peak MET current-voltage curves from P6-P7 apical-coil IHCs of 7 control (12 IHCs) and 3 littermates Kir2.1-OE mice (7 IHCs). Recordings were obtained by mechanically stimulating the hair bundles of IHCs at the same time as stepping their membrane potential from −124 mV to +96 mV in 20 mV increments. The two sets of data are not significantly different: P = 0.6320, 2-way ANOVA.


C , Peak - to - peak MET current - voltage curves from P8 - P9 apical - coil IHCs of 12 control ( 17 IHCs ) 13 Kir2 . 1 - OE mice ( 24 IHCs ) . two sets of data are significantly different : P < 0 . 0001 , 2 - way ANOVA .

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

C, Peak-to-peak MET current-voltage curves from P8-P9 apical-coil IHCs of 12 control (17 IHCs) and 13 littermates Kir2.1-OE mice (24 IHCs). The two sets of data are significantly different: P < 0.0001, 2-way ANOVA.


J , Example of FM1 - 43 uptake by IHCs from P11 control ( top ) Kir2 . 1 - OE ( bottom ) mice , showing lack of labelling in latter , which is an indication of lack of MET channels open at at this stage in IHCs overexpressing Kir2 . 1 channels . At least 3 mice for each genotype were used .

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

J, Example of FM1-43 uptake by IHCs from P11 control (top) and Kir2.1-OE (bottom) mice, showing the lack of fluorescence labelling in the latter, which is an indication of the lack of MET channels open at rest at this stage in the IHCs overexpressing the Kir2.1 channels. At least 3 mice for each genotype were used.


A , B , Scanning electron microscope ( ) showing hair bundle structure in apical coil of cochlea of P11 control ( A ) P8 - P11 Kir2 . 1 - OE ( B ) mice . Control IHCs large majority of P8 IHCs from Kir2 . 1 - OE mice show a normal hair bundle structure composed of three rows of stereocilia : tall , intermediate short ( arrows ) . From about P9 in Kir2 . 1 - OE mice , some IHCs start to lose third row of stereocilia ( arrowheads ) some already exhibit some fusion of stereocilia ( asterisk ) . These changes in hair - bundle structure became more prominent at P11 . At least 3 mice for each genotype were used . In these panels those below , asterisks are used to define some of abnormal hair bundles .

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

A, B, Scanning electron microscope (SEM) images showing the IHC hair bundle structure in the apical coil of the cochlea of P11 control (A) and P8-P11 Kir2.1-OE (B) mice. Control IHCs and the large majority of P8 IHCs from Kir2.1-OE mice show a normal hair bundle structure composed of three rows of stereocilia: tall, intermediate and short (arrows). From about P9 in Kir2.1-OE mice, some IHCs start to lose the third row of stereocilia (arrowheads) and some already exhibit some fusion of the stereocilia (asterisk). These changes in hair-bundle structure became more prominent at P11. At least 3 mice for each genotype were used. In these panels and those below, asterisks are used to define some of the abnormal hair bundles.


C , D , of both IHCs OHCs from cochlea of P26 control ( C , upper panel ) P26 Kir2 . 1 - OE ( D , upper panel ) mice . Lower panels show a higher - magnification view of hair bundle of IHCs from both genotypes , highlighting profound disruption of stereocilia in IHCs overexpressing Kir2 . 1 channels . At least 3 mice for each genotype were used .

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

C, D, SEM images of both IHCs and OHCs from the cochlea of P26 control (C, upper panel) and P26 Kir2.1-OE (D, upper panel) mice. Lower panels show a higher-magnification view of the hair bundle of IHCs from both genotypes, highlighting the profound disruption of the stereocilia in IHCs overexpressing Kir2.1 channels. At least 3 mice for each genotype were used.


A , B , Maximum projections of confocal z - stacks showing of hair bundles from apical - coil IHCs of P6 P11 control ( A ) Kir2 . 1 - OE ( B ) mice immunostained with antibodies against MYOSIN VI ( blue ) EPS8 ( magenta ) . At least 3 mice for each genotype were used . In panels ( A - D ) , stereocilia are labelled with phalloidin ( green ) . Note that despite disrupted hair - bundle structure in IHCs overexpressing Kir2 . 1 channels ; stereocilia retained a normal distribution of these bundle . At least 3 mice for each genotype were used .

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

A,B, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P6 and P11 control (A) and Kir2.1-OE (B) mice immunostained with antibodies against MYOSIN VI (blue) and EPS8 (magenta). At least 3 mice for each genotype were used. In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.


C , D , Confocal of hair bundles of P11 IHCs from control ( C ) Kir2 . 1 - OE ( D ) mice immunostained with antibodies against MYOSIN XV isoform 1 ( blue ) WHIRLIN ( magenta ) . In panels ( A - D ) , stereocilia are labelled with phalloidin ( green ) . Note that despite disrupted hair - bundle structure in IHCs overexpressing Kir2 . 1 channels ; stereocilia retained a normal distribution of these bundle . At least 3 mice for each genotype were used .

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

C,D, Confocal images of the hair bundles of P11 IHCs from control (C) and Kir2.1-OE (D) mice immunostained with antibodies against MYOSIN XV isoform 1 (blue) and WHIRLIN (magenta). In all panels (A-D), stereocilia are labelled with phalloidin (green). Note that despite the disrupted hair-bundle structure in the IHCs overexpressing Kir2.1 channels; the stereocilia retained a normal distribution of these bundle proteins. At least 3 mice for each genotype were used.


A , B , Maximum projections of confocal z - stacks showing IHCs of apical cochlear region from control ( A ) Kir2 . 1 - OE ( B ) pups with being in continuous presence of DOX in drinking water from conception up to when pups were P5 ( upper panels ) . Middle bottom panels show IHCs at P7 P14 following removal of DOX at P5 for both control ( A ) Kir2 . 1 - OE mice ( B ) . IHCs were stained with antibodies against K + channel Kir2 . 1 ( green ) Myosin 7a ( Myo7a , blue : marker ) . Note that after 2 days following removal of DOX , Kir2 . 1overexpression was already largely downregulated . At least 3 mice for each genotype were used .

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

A,B, Maximum intensity projections of confocal z-stacks showing the IHCs of the apical cochlear region from control (A) and Kir2.1-OE (B) pups with the females being in the continuous presence of DOX in the drinking water from conception up to when the pups were P5 (upper panels). Middle and bottom panels show IHCs at P7 and P14 following the removal of DOX at P5 for both control (A) and Kir2.1-OE mice (B). IHCs were stained with antibodies against the K+ channel Kir2.1 (green) and Myosin 7a (Myo7a, blue: cell marker). Note that after 2 days following the removal of DOX, Kir2.1overexpression was already largely downregulated. At least 3 mice for each genotype were used.


H , I , Maximum projections of confocal z - stacks showing of hair bundles from apical - coil IHCs of P14 control ( H ) Kir2 . 1 - OE ( I ) mice stained with phalloidin . DOX was removed from mother ' s drinking water when pups were P5 . At least 3 mice for each genotype were used .

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

H,I, Maximum intensity projections of confocal z-stacks showing images of the hair bundles from apical-coil IHCs of P14 control (H) and Kir2.1-OE (I) mice stained with phalloidin. DOX was removed from the mother's drinking water when the pups were P5. At least 3 mice for each genotype were used.


J , K , showing normal structure of hair bundles of IHCs in apical coil of cochlea of P14 control ( J ) P14 Kir2 . 1 - OE ( K ) mice . DOX was removed from mother ' s drinking water when pups were P5 . Note that morphological profile of hair bundles in IHCs is comparable between control Kir2 . 1 - OE mice , indicating that removal of intrinsic Ca2 + action potentials prior to second postnatal week has no effect on mechanoelectrical transduction apparatus . At least 3 mice for each genotype were used .

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

J,K, SEM images showing the normal structure of the hair bundles of the IHCs in the apical coil of the cochlea of P14 control (J) and P14 Kir2.1-OE (K) mice. DOX was removed from the mother's drinking water when the pups were P5. Note that the morphological profile of the hair bundles in IHCs is comparable between control and Kir2.1-OE mice, indicating that the removal of the intrinsic Ca2+ action potentials prior to the second postnatal week has no effect on the mechanoelectrical transduction apparatus. At least 3 mice for each genotype were used.


D , of for each - library for 118 genes associated with microtubule or cytoskeletal processes . Each row is z - scored . 118 genes were differentially expressed as per previous .

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D, Heatmap of the counts for each RNA-library for the 118 genes associated with microtubule or cytoskeletal processes. Each row is z-scored. 118 genes were all differentially expressed as per previous analysis.


( A ) ADAR2 is most strongly expressed in vascular tissues . Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues . Note highest expression of ADAR2 in tibial artery followed by colon esophagus . Boxes represent 25th 75th percentile with median represented by black in box . whiskers depict minimum maximum value .

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

(A) ADAR2 is most strongly expressed in vascular tissues. Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues. Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( B ) FLNA gene expression in few representative human tissues from > 500 different donors using GTEx data . Tibial artery , colon esophagus show highest gene expression similar to ADAR2 expression . Boxes represent 25th 75th percentile with median represented by black in box . whiskers depict minimum maximum value .

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

(B) FLNA gene expression in few representative human tissues from >500 different donors using GTEx data. Tibial artery, colon and esophagus show highest gene expression similar to ADAR2 expression. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( C ) Bar graph shows FLNA editing ( % ) among few representative human tissues . Note very high editing in arterial system ( red bars ) .

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(C) Bar graph shows FLNA RNA editing (%) among few representative human tissues. Note very high editing levels in the arterial system (red bars).


( D ) Scatter plot shows log fold change in editing of several candidates between ventricles of healthy donors heart samples of cardiomyopathy . FLNA marked in graph is one significant discriminator . Y axis is - log 10 of p value for difference between healthy sick . colors ( red vs green ) reflect threshold cutoffs randomly assigned to distinguish sites , which demonstrate large , highly significant differences between two groups . Fold change cutoff of 0 . 9 was used .

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(D) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients. FLNA marked in the graph is one significant discriminator. Y axis is the -log 10 of the p value for the difference between healthy and sick. The colors (red vs green) reflect the threshold cutoffs randomly assigned to distinguish the sites, which demonstrate large, highly significant differences between the two groups. Fold change cutoff of 0.9 was used.


( E ) Scatter plot shows FLNA editing in control diseased human tibial arteries aorta . At least 4 control 7 diseased human donors were used in each case . P value < 0 . 05 measured by t was considered significant .

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(E) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta. At least 4 control and 7 diseased human donors were used in each case. P value <0.05 measured by t test was considered significant.


( A ) Scheme showing FLNA wild - type ( wt ) allele , targeting vector FLNAΔECS neo - allele . loxP flanked PGK - neo cassette in targeting vector replaced editing complementary ( ECS ) using homologous recombination , which was then deleted using Cre recombinase . Right , southern blotting screened for positive clones shown . positions of loxP sites , restriction enzyme ( EcoRI ) Southern blotting probe are also indicated .

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

(A) Scheme showing the FLNA wild-type (wt) allele, the targeting vector and the FLNAΔECS neo-allele. The loxP flanked PGK-neo cassette in the targeting vector replaced the editing complementary sequence (ECS) using homologous recombination, which was then deleted using Cre recombinase. Right, southern blotting analysis screened for positive clones shown. The positions of loxP sites, restriction enzyme (EcoRI) and Southern blotting probe are also indicated.


( B ) Sequencing electropherograms show average FLNA editing in wt FLNAΔECS tissues ( Stomach , colon , heart , lung tibial artery ) . Editing were checked in tissues from 3 independent mice value below chromatogram depict average value of 3 replicates . Data information : Arrows indicate FLNA editing site .

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

(B) Sequencing electropherograms show the average FLNA editing levels in wt and FLNAΔECS tissues (Stomach, colon, heart, lung and tibial artery). Editing levels were checked in tissues from 3 independent mice and the value below the chromatogram depict the average value of 3 replicates. Data information: Arrows indicate the FLNA editing site.


( C ) FLNA mRNA expression measured by qPCR in wt FLNAΔECS colon tissue showed no difference . Data information : data shown as mean ± SD from three independent experiments . * * P < 0 .

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(C) FLNA mRNA expression levels measured by qPCR in wt and FLNAΔECS colon tissue showed no difference. Data information: data shown as mean ± SD from three independent experiments. ** P<0.05


( D ) FLNA measured by western blotting are identical in wt FLNAΔECS stomach tissue . Y - axis represents FLNA expression normalized to tubulin . Data information : data shown as mean ± SD from three independent experiments . * * P < 0 .

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(D) FLNA protein levels measured by western blotting are identical in wt and FLNAΔECS stomach tissue. Y-axis represents FLNA expression normalized to tubulin levels. Data information: data shown as mean ± SD from three independent experiments. ** P<0.05


( E ) FLNA editing ( % ) in wt whole dorsal aorta , tunica media tunica adventitia . Tunica media consisting of smooth muscle show highest FLNA editing . Data information : Arrows indicate FLNA editing site . data shown as mean ± SD from three independent experiments . * * P < 0 .

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

(E) FLNA editing levels (%) in wt whole dorsal aorta, tunica media and tunica adventitia. Tunica media consisting of smooth muscle cells show the highest FLNA RNA editing. Data information: Arrows indicate the FLNA editing site. data shown as mean ± SD from three independent experiments. ** P<0.05


A , B ) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to ( A ) hypercontraction ( B ) increased contraction force in FLNAΔECS aortae in myograph chambers . Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae . For each condition , 10 - 12 aortic rings from at least 4 wild - type ( wt ) 4 FLNAΔECS mice were used . Data shown as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t )

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

A,B) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to (A) hypercontraction and (B) increased contraction force in FLNAΔECS aortae in myograph chambers. Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae. For each condition, 10-12 aortic rings from at least 4 wild-type (wt) and 4 FLNAΔECS mice were used. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001 (Student's t test)


( C ) Graph shows normalized contraction measured by Xcelligence Real time analyzer in wt ( red ) FLNAΔECS ( green ) vascular smooth muscle ( vSMC ) indicating hypercontraction in FLNAΔECS in response to 10μM U46619 . Data shown as mean ± SD from three independent experiments .

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(C) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt (red) and FLNAΔECS (green) primary vascular smooth muscle cells (vSMC) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619. Data shown as mean ± SD from three independent experiments.


( D ) of index measurements plotted as of contraction following different concentrations of U46619 . Data shown as mean ± SD from three independent experiments . P value < 0 . 05 ( Student ' s t ) was considered significant . difference between wt FLNAΔECS was significant at every of U46619 .

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(D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.


( A ) Myosin chain phosphorylation detected by a phospho - specific antibody ( pMLC ) normalized to total myosin chain ( MLC ) . Tubulin served as a loading control . shows a significant increase in pMLC in FLNAΔECS vSMCs . Data information data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t )

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

(A) Myosin light chain phosphorylation detected by a phospho-specific antibody (pMLC) and normalized to total myosin light chain (MLC). Tubulin served as a loading control. Quantification shows a significant increase in pMLC in FLNAΔECS vSMCs. Data information data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( B ) Rhotekin Pull down activation was performed on wt FLNAΔECS vSMC lysates activated ( GTP ) bound RhoA was compared between 2 genotypes . FLNAΔECS vSMCs show more GTP - loaded RhoA than wt vSMCs . Tubulin was used as a loading control . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t )

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

(B) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated (GTP) bound RhoA was compared between the 2 genotypes. FLNAΔECS vSMCs show more GTP-loaded RhoA than wt vSMCs. Tubulin was used as a loading control. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( C ) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin chain phosphatase ( pMYPT1 ) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor - specific MYPT1 antibody . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t )

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

(C) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase (pMYPT1) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor-specific MYPT1 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( D ) CPI17 phosphorylation were measured in wt FLNAΔECS vSMCs in untreated in presence of ROCK PKC inhibitor using pCPI17 antibody . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t )

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(D) CPI17 phosphorylation levels were measured in wt and FLNAΔECS vSMCs in untreated cells and in the presence of ROCK and PKC inhibitor using pCPI17 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


A - C ) Telemetry measurements were done on mice continuously for 72 hrs reading was recorded every minute to analyse blood pressure in wt FLNAΔECS mice . Scatter plots show diastolic blood pressure over 72 hrs ( A ) , diastolic blood pressure during inactive phase ( B ) diastolic pressure during active phase ( C ) . Note : Significant increase in diastolic blood pressure in FLNAΔECS mice during inactive phase . 9 mice ( 5 marked as square + 4 marked as round ) were analyzed for each genotype . Data shown as mean ± . P value < 0 . 05 ( Student ' s t ) was considered significant . Boxes represent 25th 75th percentile with median represented by black in box . whiskers depict minimum maximum value .

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

A-C) Telemetry measurements were done on mice continuously for 72 hrs and reading was recorded every minute to analyse blood pressure in wt and FLNAΔECS mice. Scatter plots show diastolic blood pressure over 72 hrs (A), diastolic blood pressure during inactive phase (B) and diastolic pressure during active phase (C). Note: Significant increase in diastolic blood pressure in FLNAΔECS mice during the inactive phase. 9 mice (5 males marked as square + 4 females marked as round) were analyzed for each genotype. Data shown as mean ± SEM. P value <0.05 (Student's t test) was considered significant. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( A ) Dorsal aortae were fixed , cross - sectioned then stained with Elastica van Gieson stain to visualize collagen around blood vessel . 6 independent mice ( 5 - 6 months ) of each genotype were used for . Scale bar : 50 μm . ( B ) Plot shows data points ( black - wt , red - mutant ) of adventitial in both genotypes . Measurements show increased adventitial in FLNAΔECS aortae . P value < 0 . 05 was considered significant ( Student ' s t )

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

(A) Dorsal aortae were fixed, cross-sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel. 6 independent mice (5-6 months) of each genotype were used for the analysis. Scale bar: 50 μm. (B) Plot shows individual data points (black-wt, red-mutant) of adventitial area in both the genotypes. Measurements show increased adventitial area in FLNAΔECS aortae. P value <0.05 was considered significant (Student's t test)


( C ) Representative heart sections of a 5 - 6 months old mice stained with Masson Trichrome shows increased collagen ( blue ) around coronary vessels ( marked by arrowheads ) Scale bar : 50 μm . ( D ) shows a significant increase in perivascular fibrosis in FLNAΔECS hearts . Four sections from each heart were measured . Four mice analyzed for each genotype . P value < 0 . 05 considered significant . ( mixed model approach )

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

(C) Representative heart sections of a 5-6 months old mice stained with Masson Trichrome shows increased collagen (blue) around coronary vessels (marked by arrowheads) Scale bar: 50 μm. (D) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts. Four sections from each heart were measured. Four mice analyzed for each genotype. P value <0.05 considered significant. (mixed model approach)


( E ) Representative longitudinal heart sections stained with H & E ( RV : right ventricle , LV : left ventricle ) . Black lines represent thickness of interventricular ( IVS ) lateral left ventricular wall ( LLVH ) . Scale bar : 1mm . ( F ) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts . Six mice ( 6months old ) analyzed for each genotype . P value < 0 . 05 was considered significant . ( Student ' s t )

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

(E) Representative longitudinal heart sections stained with H&E staining (RV: right ventricle, LV: left ventricle). Black lines represent thickness of interventricular (IVS) and lateral left ventricular wall (LLVH). Scale bar: 1mm. (F) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts. Six male mice (6months old) analyzed for each genotype. P value <0.05 was considered significant. (Student's t test)


( G ) Heart cross - sections were stained with FITC - WGA ( Wheat germ agglutinin ) to visualize membranes of cardiomyocytes . Scale bar : 20 μm . ( H ) Respective areas measured by Image J software represented by box plots for each ( six per genotype ) . Boxes represent 25th 75th percentile with median represented by black in box . whiskers depict minimum maximum value . P value < 0 . 05 ( mixed model approach ) was considered significant .

O O O B-TISSUE O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heart cross-sections were stained with FITC-WGA (Wheat germ agglutinin) to visualize cell membranes of cardiomyocytes. Scale bar: 20 μm. (H) Respective cell areas measured by Image J software and represented by box plots for each animal (six per genotype). Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. P value <0.05 (mixed model approach) was considered significant.


( A ) Graph showing blood velocity profile determined by MRI over time in 4 wt mice ( shades of grey ) 5 FLNAΔECS mice ( shades of red ) . mouse shown in dotted purple is clearly an outlier within FLNAΔECS cohort . Aortic velocity waveform represents velocity during different stages of cardiac cycle . A significant reduction of velocity was observed at 40ms in systolic cycle .

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

(A) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice (shades of grey) and 5 FLNAΔECS mice (shades of red). The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort. Aortic velocity waveform represents velocity during different stages of cardiac cycle. A significant reduction of velocity was observed at 40ms in the systolic cycle.


( B ) Representative wt ( top ) FLNAΔECS ( below ) mouse showing blood flow just above aortic valve . Note : Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring . red circle represents time window at which are shown .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-TISSUE O O O B-TISSUE I-TISSUE O O O O B-TISSUE O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O

(B) Representative wt (top) and FLNAΔECS (below) mouse showing the blood flow just above the aortic valve. Note: Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring. The red circle represents the time window at which the images are shown.


( A - C ) Bone marrow derived mouse macrophages ( BMDMs ) from wild type C57BL / 6 mice were treated with 4µ8c ( 100 µM ) or vehicle ( dimethyl sulfoxide ; DMSO ) for 24 hours prior to sample processing for lipidomics : ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP are shown . Data represent mean values ± standard error of mean ( ) of peak areas normalized to internal standards ( n = 3 biological replicates ) .

(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).


( D ) BMDMs were treated with 4µ8c ( 100 µM ) or DMSO for 24 hours lysates were analyzed by western blotting using specific antibodies against , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 β - actin . Western blot quantifications are shown next to figure ( n = 3 biological replicates ; a representative blot is shown ) .

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

(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).


( E ) Wild type C57BL / 6 mice were injected with tunicamycin ( 1 mg / kg ) 4µ8c ( 10 µg / kg ) for 8 hours prior to sacrifice collection of thiogylcolate - elicited peritoneal macrophages . lysates were analyzed by western blotting using specific antibodies against , pIRE1S742 , IRE1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 β - actin ( n = 3 biological replicates ; a representative blot is shown ) . Western blot quantifications are shown next to figure .

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

(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.


( F ) IRE1 + / + IRE1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours lysates were analyzed by western blotting using specific antibodies against pIRE1S742 , IRE1 , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 β - actin . Western blot quantifications are shown next to figure ( n = 3 biological replicates , a representative blot is shown ) .

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

(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).


IRE1 - / - IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to isolation for miRNA using a micro - array platform . ( B ) lysates from same experiment were analyzed by qRT - PCR for miR - 2137 expression .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.


IRE1 - / - IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to isolation for miRNA using a micro - array platform . ( C ) lysates were analyzed by western blotting using specific antibodies for pIRE1S742 , IRE1 β - actin ( n = 3 biological replicates ) .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).


IRE1 - / - IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to isolation for miRNA using a micro - array platform . ( D ) lysates were analyzed by qRT - PCR for sXBP1 expression ( n = 3 biological replicates ) .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).


IRE1 - / - IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to isolation for miRNA using a micro - array platform . ( E - F ) lysates from same experiment were analyzed by qRT - PCR for ( E ) BLOC1S1 ( F ) SCARA3 expression ( n = 3 biological replicates ) .

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

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).


( A - B ) BMDMs were treated with PA , steric acid ( SA ) , palmitoleic acid ( PAO ) or oleic acid ( OA ) ( 500 µM ) for 16 hours . lysates were analyzed by qRT - PCR for expression of ( A ) miR - 2137 ( B ) sXBP1 ( n = 3 biological replicates ) .

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

(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).


( C , D ) ( C ) C57BL / 6 ( WT ) ( D ) Apoe - / - mice were fed with chow or WD for 16 followed by peritoneal macrophage isolation . lysates were analyzed by qRT - PCR for miR - 2137 expression ( WT ; n = 4 - 4 mice per group , Apoe - / - ; n = 5 - 5 mice per group ) .

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

(C, D) (C) C57BL/6 (WT) and (D) Apoe-/- mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (WT; n=4-4 mice per group, Apoe-/-; n=5-5 mice per group).


( E - F ) Apoe - / - mice were fed with chow or WD for 12 injected with 4µ8c ( 10 mg / kg / day ) or vehicle ( DMSO ) in final 6 of diet ( n = 5 - 7 mice per group ) : ( E ) lysates from bone marrow were analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) . ( F ) Total isolated from aortic root plaques was analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) .

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

(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).


( A ) BMDMs were transfected with scrambled ( Scr ) or IRE1 - specific siRNA ( 40 nM ) treated with palmitate ( PA , 500 µM ) or vehicle for 6 9 hours ( hr ) . lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( B ) BMDMs were treated with PA ( 500 µM ) 4µ8c ( 100 µM ) or vehicle for 9 hours . lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( C ) BMDM were treated with tunicamycin ( Tun ; 5 µg / ml ) 4µ8c ( 100 µM ) or vehicle ( DMSO ) for 9 hours . lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(B) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( D ) IRE1 - / - MEFs were transfected with vector ( control ) , WT - IRE1 or K907A - IRE1 ( RNase dead mutant ) plasmids for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( E ) BMDMs were transfected with scrambled or XBP - 1 specific siRNA ( 70 nM ) for 24 hours , followed by treatment with PA ( 500 µM ) for 16 hours . lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

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

(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( F ) XBP1 - / - MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . lysates were analyzed for miR - 2137 expression by qRT - PCR ( n = 3 biological replicates ) .

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

(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).


( G ) IRE1 cleavage performed using synthetic pre - miR - 2137 ( 50 nM ) as substrate with active , recombinant IRE1 ( 100 ng ) at 37 ℃ for 2 hours , followed by sample separation in Urea - PAGE detection with SYBR gold . M indicates micro marker .

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

(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.


( H - I ) BMDMs isolated from WT ( C57BL6 ) DICER - / - mice were treated with PA ( 500 µM ) for 12 hours . lysates were analyzed by qRT - PCR for ( H ) miR - 2137 ( I ) sXBP1 expression ( n = 3 biological replicates ) .

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

(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).


( B ) HEK293 were co - transfected with an INPPL1 3 ` UTR - luciferase plasmid scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours . lysates were analyzed for luciferase activity normalized to secreted embryonic alkaline phosphatase ( SEAP ) reporter activity ( n = 3 biological replicates ) .

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

(B) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates).


( C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) miR - 205 mimic ( 40 nM ) for 48 hours lysates were analyzed by western blotting using specific antibodies for INPPL1 β - actin . quantifications are displayed in graph next to blots ( n = 3 biological replicates ) .

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

(C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates).


( D ) BMDMs were transfected with negative control antagomir ( 200 nM ) or miR - 2137 antagomir ( 200 nM ) for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . lysates were analyzed by western blotting using specific antibodies for INPPL1 β - actin . quantifications shown in graph next to blots ( n = 3 biological replicates ) .

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

(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).


( A - C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours were processed for lipidomics as described in materials methods section . of lipids from this is shown for ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 . ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP . Data from LC / MS / MS using a Waters Xevo TQ - S MS / MS in MRM mode represent mean values ± of peak areas normalized to internal standards as described in Methods . ( n = 3 biological replicates )

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

(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5)P3 to PIP2.; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates)


BMDMs were transfected with ( D ) scrambled miRNA mimic ( 40 nM ) or miR - 2137 mimic ( 40 nM ) for 48 hours lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR β - actin . quantifications are shown next to western blots ( n = 3 biological replicates ) .

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

BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).


BMDMs were transfected ( E ) with control antagomir ( 200 nM ) or miR - 2137 antagomir for 24 hours lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR β - actin . quantifications are shown next to western blots ( n = 3 biological replicates ) .

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

BMDMs were transfected (E) with control antagomir (200 nM) or miR-2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).