B Quantitative RT - PCR detection of microRNA - 34a / b / c - 5p family members in the lung over the course of normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization . C Quantitative RT - PCR detection of microRNA - 34a / b / c - 3p family members in the lung over the course of normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization. C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.


A Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) and miR - 34a - / - mice during normal and aberrant alveolarization ( scale bar , 50 µm ) . B Quantification of total number of alveoli by design - based stereology in wild - type ( WT ) and miR ‑ 34a - / - mice ( 34a - / - ) during normal and aberrant alveolarization . C Quantification of mean septal thickness by design - based stereology in wild - type ( WT ) and miR ‑ 34a - / - mice ( 34a - / - ) during normal and aberrant alveolarization .

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

A Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34a-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). B Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization. C Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization.


D Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) and miR - 34bc - / - mice during normal and aberrant alveolarization ( scale bar , 50 µm ) . E Quantification of total number of alveoli by design - based stereology in wild - type ( WT ) and miR ‑ 34bc - / - mice ( 34bc - / - ) during normal and aberrant alveolarization . F Quantification of mean septal thickness by design - based stereology in wild - type ( WT ) and miR ‑ 34bc - / - mice ( 34bc - / - ) during normal and aberrant alveolarization .

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

D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34bc-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). E Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization. F Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization.


G Localization of miR - 34a expression by β - galactosidase activity staining in the developing lungs of P14 miR - 34a : : lacZ + / + mice that were undergoing normal or aberrant alveolarization ( scale bar , 50 µm ; larger and some additional images are presented in Appendix Fig S2 ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Localization of miR-34a expression by β-galactosidase activity staining in the developing lungs of P14 miR-34a::lacZ+/+ mice that were undergoing normal or aberrant alveolarization (scale bar, 50 µm; larger and some additional images are presented in Appendix Fig S2).


B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA ( SCR ) or a miR - 34a ( MIM34a ) mimic ( n = 3 separate cell cultures for each group ) .

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

B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA (SCR) or a miR-34a (MIM34a) mimic (n = 3 separate cell cultures for each group).


C Quantitative RT - PCR detection of miR - 34a / b / c - 5p levels in MLg cells in vitro , maintained under 21 % O2 or 85 % O2 ( n = 3 separate cell cultures for each group ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O2 or 85% O2 (n = 3 separate cell cultures for each group).


D Immunoblot detection of PDGFRα levels in the lungs of mouse pups ( n = 6 animals for each group ) at post - natal day ( P ) 5 , during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization .

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

D Immunoblot detection of PDGFRα levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P)5, during normal (21% O2) and aberrant (85% O2) alveolarization.


E Immunoblot detection of PDGFRα levels in MLg cells in vitro , maintained under 21 % O2 or 85 % O2 , where cells had been transfected wither with a scrambled ( SCR ) antimiR , or an antimiR directed against miR - 34a ( A34a ) ( n = 3 separate cell cultures for each group ) .

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

E Immunoblot detection of PDGFRα levels in MLg cells in vitro, maintained under 21% O2 or 85% O2, where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group).


F Quantitative RT - PCR detection of miR - 34a - 5p levels in PDGFRα + cells , sorted by FACS from the lungs of mouse pups ( n = 4 animals for each group ; data from an independent repetition are provided in Appendix Fig S5 ) at P5 , maintained under 21 % O2 or 85 % O2 from birth .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O2 or 85% O2 from birth.


H Quantitative RT - PCR detection of miR - 34a - 5p levels in PDGFRα + cells , sorted by FACS from the lungs of either wild - type ( 34awt / wt ) mouse pups , or mouse pups in which miR - 34a expression in Pdgfra ‑ expressing cells ( 34ai ∆ PC / i ∆ PC ) at P5 ( n = 4 animals for each group ) .

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

H Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of either wild-type (34awt/wt) mouse pups, or mouse pups in which miR-34a expression in Pdgfra‑expressing cells (34ai∆PC/i∆PC) at P5 (n = 4 animals for each group).


I Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 during aberrant ( 85 % O2 ) alveolarization , compared with 34ai ∆ PC / i ∆ PC during normal ( 21 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of observations made in four other experiments . J Quantification of total number of alveoli by design - based stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal and aberrant alveolarization ( n = 5 animals for each group ) . K Quantification of mean septal thickness by design - based stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14 during aberrant (85% O2) alveolarization, compared with 34ai∆PC/i∆PC during normal (21% O2) alveolarization (scale bar, 50 µm). Data are representative of observations made in four other experiments. J Quantification of total number of alveoli by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). K Quantification of mean septal thickness by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).


B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR ( SCR ) and miR - 34a ( MIM34a ) mimics , and probing for PDGFRα and c - Kit as TSB ‑ dependent and TSB ‑ independent target readouts , respectively . Protein loading equivalence was controlled by βactin levels . Note : PDGFRα , βactin , and c - Kit were all probed on the same membrane ; hence a single βactin immunoblot is presented . Data are representative of three experiments .

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

B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and c-Kit as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and c-Kit were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.


C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR ( SCR ) and miR - 34a ( MIM34a ) mimics , and probing for PDGFRα and SIRT1 as TSB ‑ dependent and TSB ‑ independent target readouts , respectively . Protein loading equivalence was controlled by βactin levels . Note : PDGFRα , βactin , and SIRT1 were all probed on the same membrane ; hence a single βactin immunoblot is presented . Data are representative of three experiments .

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

C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and SIRT1 as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and SIRT1 were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.


D Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled target site blocker ( SCR ) or a cocktail of both target site blockers ( TSB1 and TSB2 ) during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments . E Quantification of total number of alveoli by design - based stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) . F Quantification of mean septal thickness by design - based stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O2) and aberrant (85% O2) alveolarization (scale bar, 50 µm). Data are representative of three or more experiments. E Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). F Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


G Quantitative analysis of PDGFRα + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

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

G Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


H Quantitative analysis of PDGFRα + / αSMA + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

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

H Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


B Quantitative RT - PCR detection of miR - 34a - 5p levels in wild - type mouse pups at post - natal day ( P ) 14 that had been treated either with a scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization ( n = 4 animals for each group ) .

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

B Quantitative RT-PCR detection of miR-34a-5p levels in wild-type mouse pups at post-natal day (P)14 that had been treated either with a scrambled antimiR (S), or antimiR-34a (A34a), during normal (21% O2) and aberrant (85% O2) alveolarization (n = 4 animals for each group).


C Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments .

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

C Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (scale bar, 50 µm). Data are representative of three or more experiments.


D Quantification of total number of alveoli by design - based stereology in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant lung alveolarization ( n = 5 animals for each group ) .

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

D Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant lung alveolarization (n = 5 animals for each group).


E Quantification of mean septal thickness by design - based stereology in in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

E Quantification of mean septal thickness by design-based stereology in in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


F Quantitative analysis of PDGFRα + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

F Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


G Quantitative analysis of PDGFRα + / αSMA + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

G Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


A Mice expressing nuclear ‑ localized GFP under the control of the Pdgfra promoter were maintained under normoxic ( 21 % O2 ) or hyperoxic ( 85 % O2 ) conditions , and lungs were harvested , processed and immunostained for Ki67 to determine proliferation status . DAPI staining revealed nuclei of all cells present in the section . Low - magnification images from individual channels are presented to the right of the merged ( large ) image first row of images . The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image , as well as visualization of a single Ki67 + , GFP + cell ( white arrowhead ) in all three channels separately .

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

A Mice expressing nuclear‑localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O2) or hyperoxic (85% O2) conditions, and lungs were harvested, processed and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Low-magnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67+,GFP+ cell (white arrowhead) in all three channels separately.


B The number of PDGFRα + cells in four microscopic fields was assessed for co - staining with an anti - Ki67 antibody to reveal proliferating cells . P values were calculated by unpaired Student ' s t ‑ test ( n = 4 fields for each group , trends are representative of those observed in two other experiments ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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 The number of PDGFRα+ cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t‑test (n=4 fields for each group, trends are representative of those observed in two other experiments).


C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild - type mice that were treated with an isotype - matched control IgG used for the Ki67 staining experiments . Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining . Scale bar : 100 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; GFP , green fluorescent protein .

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

C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.


The impact of administration of scrambled antimiR ( S ) or an antimiR directed against miR - 34a ( A34a ) on the abundance of type I alveolar epithelial cells ( marked by aquaporin 5 , Aqp5 ) and type II alveolar epithelial cells ( marked by pro - surfactant protein C , Sftpc ) was assessed in 3 - µm sections of paraffin ‑ embedded lung tissue from P5 mice undergoing normal ( 21 % O2 ) or aberrant ( 85 % O2 ) lung alveolarization . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole . In the DAPI images , white lines delineate tissue from airspaces , and in the 85 % O2 groups demarcate septa . Antibody specificity was validated by rabbit IgG isotype control primary antibodies . The control experiments for the Aqp5 and Sftpc staining runs are illustrated here . Scale bars , 50 µm .

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

The impact of administration of scrambled antimiR (S) or an antimiR directed against miR-34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro-surfactant protein C, Sftpc) was assessed in 3-µm sections of paraffin‑embedded lung tissue from P5 mice undergoing normal (21% O2) or aberrant (85% O2) lung alveolarization. DAPI, 4′,6-diamidino-2-phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 µm.


D ) HEK293 cells were genetically engineered to knockout RIG - I , MDA5 , or both , and subsequently subjected to retroviral transduction to stably express FLAG - LGP2 or an empty vector ( EV ) . Correct gene editing and intact type I IFN responsiveness was validated by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . * , non - specific band .

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

D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.


E ) Cells generated in ( D ) were transfected with siCtrl or siADAR1 . The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post - transfection as in ( B ) . Data are means ± s . d . from a representative of four biological replicate experiments .

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E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments.


F ) Cells generated in ( D ) were transfected with siCtrl ( siC ) or siADAR1 ( siA ) . Protein lysates were prepared 78h post - transfection , followed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) .

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F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).


G ) HEK293 WT cells and FLAG - LGP2 - expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence . Cells were fixed , permeabilized and stained 72h post - transfection with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 450 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of three biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analyses were performed using unpaired two - tailed Mann - Whitney U tests . ns , not significant ; * * * * , p < 0 . 0001 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.


A ) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR / Cas9 . Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness , respectively . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) .

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A) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3).


B ) ADAR1 - knockout HEK293 cells ( clone 1 ) were co - transfected with an empty vector ( EV ) or a FLAG - LGP2 - encoding vector ( LGP2 ) combined with a vector encoding GFP - tagged ADAR1 p110 or p150 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 expression , normalized to ACTB . Data are means ± s . d . from a representative of four biological replicate experiments .

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B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments.


C ) ADAR1 - knockout cells ( clone 1 ) were transfected as in ( B ) . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 4 ) .

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C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).


A ) ADAR1 - knockout HEK293 cells ( clone 1 ) were modified with a lentiviral - based inducible system to express FLAG - LGP2 WT or a FLAG - LGP2 RNA binding mutant ( K138E / R490E / K634E , denoted as " KRK mutant " ) in a doxycycline - regulated manner . Cells were treated 72h with doxycycline ( dox ) . Protein lysates were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 3 ) . iEV = inducible empty vector ; iLGP2 = inducible LGP2 .

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A) ADAR1-knockout HEK293 cells (clone 1) were modified with a lentiviral-based inducible system to express FLAG-LGP2 WT or a FLAG-LGP2 RNA binding mutant (K138E/R490E/K634E, denoted as "KRK mutant") in a doxycycline-regulated manner. Cells were treated 72h with doxycycline (dox). Protein lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). iEV = inducible empty vector; iLGP2 = inducible LGP2.


C ) Cells generated in ( A ) were plated on coverslips and treated with or without doxycycline for 72h . Cells were fixed , permeabilized and stained with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 500 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of two biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analysis was performed using a Kruskal - Wallis test with a Dunn ' s post hoc test for multiple comparisons . ns , not significant ; * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells generated in (A) were plated on coverslips and treated with or without doxycycline for 72h. Cells were fixed, permeabilized and stained with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>500 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of two biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analysis was performed using a Kruskal-Wallis test with a Dunn's post hoc test for multiple comparisons. ns, not significant; ****, p<0.0001.


E ) MDA5 - knockout HEK293 cells , were transfected with an ADAR1 - targeting siRNA ( siADAR1 ) or a control siRNA ( siCtrl ) and 8h later with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of MDA5 . Cells were harvested 72h post - siRNA transfection and the type I IFN response was monitored by RT - qPCR analysis of IFIT1 and ISG15 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of two biological replicate experiments .

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

E) MDA5-knockout HEK293 cells, were transfected with an ADAR1-targeting siRNA (siADAR1) or a control siRNA (siCtrl) and 8h later with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of MDA5. Cells were harvested 72h post-siRNA transfection and the type I IFN response was monitored by RT-qPCR analysis of IFIT1 and ISG15 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments.


B ) HEK293 WT or ADAR1 - knockout cells ( clone 1 ) were transiently transfected with an empty vector ( EV ) or a vector encoding the indicated WT , truncation , or point mutant ( s ) of LGP2 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 transcript expression , normalized to ACTB . Data are means ± s . d . from a representative of three biological replicate experiments .

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B) HEK293 WT or ADAR1-knockout cells (clone 1) were transiently transfected with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of LGP2. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments.


C ) Cells were treated as in ( B ) . Protein lysates were prepared and analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) . The dotted line indicates the juxtaposition of two non - adjacent lanes .

C) Cells were treated as in (B). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). The dotted line indicates the juxtaposition of two non-adjacent lanes.


A ) Kaplan - Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer ( BLCA ; n = 407 ) , breast cancer ( BRCA ; n = 1099 ) and sarcoma ( SARC ; n = 263 ) TCGA datasets . Median cut - offs were used for patient stratification and logrank test P values are shown .

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A) Kaplan-Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer (BLCA; n=407), breast cancer (BRCA; n=1099) and sarcoma (SARC; n=263) TCGA datasets. Median cut-offs were used for patient stratification and logrank test P values are shown.


B ) CAL27 cells transduced with doxycycline - inducible shRNAs targeting ADAR1 or GFP ( negative control ) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 ( siLGP2 # 1 or # 2 ) or a control siRNA ( siCtrl ) . To determine cell confluency at 120h post - transfection , cells were fixed , stained with Crystal Violet , and imaged . Images of a representative experiment are shown ( n = 3 ) . C ) Crystal Violet was extracted from stained cells ( B ) and the dye intensity was quantified using a colorimetric assay ( OD590 ) . OD590 values of doxycycline - treated cells were normalized to the OD 590 values of untreated cells . Quantification of data from three independent experiments is shown as mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 .

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

B) CAL27 cells transduced with doxycycline-inducible shRNAs targeting ADAR1 or GFP (negative control) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine cell confluency at 120h post-transfection, cells were fixed, stained with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3). C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.


A ) HT29 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed by RT - qPCR analysis of IFN - β , IFIT1 , and ISG15 transcript expression , normalized to ACTB . B ) LIM1215 cells were treated with or without 300 nM 5 - AZA - CdR for 2 days and subsequently washed and transfected with the indicated siRNAs . Cells were harvested 72h post - transfection and the type I IFN response was analyzed as in ( A ) . C ) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days . Three days after treatment initiation , cells were transfected with the indicated siRNAs and cultured for an additional 72h . The type I IFN response was monitored as in ( A ) . Data information : Data from three biological independent experiments are shown with mean ± s . d . . Statistical analysis was performed using ordinary two - way ANOVA with Tukey ' s post hoc test . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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

A) HT29 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed by RT-qPCR analysis of IFN-β, IFIT1, and ISG15 transcript expression, normalized to ACTB. B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A). C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN response was monitored as in (A). Data information: Data from three biological independent experiments are shown with mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


ALP assay . The cells expressing Pho8Δ60p were cultured in SD + CA medium ( open bars ) and shifted to SD ( − N ) medium for three hours ( filled bars ) . The ALP activity of the lysates was measured to estimate autophagic activity . Wild - type cells , TN124 ; Δapg8 mutant harboring vector , TK303 ; Δapg8 mutant harboring APG8 on a centromeric vector , TK301 ; Δapg8 mutant harboring 3 × HA - tagged APG8 plasmid , TK307 .

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ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.


Expression of APG8 . A , The lysates prepared from the growing cells were subjected to immunoblotting with the anti - Apg8p antibody . Lane 1 , wild - type cells ( YW5 - 1B ) ; lane 2 , Δapg8 cells harboring APG8 on a multicopy vector ( TK201 ) ; lane 3 , Δapg8 cells ( TK404 ) .

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Expression of APG8. A, The lysates prepared from the growing cells were subjected to immunoblotting with the anti-Apg8p antibody. Lane 1, wild-type cells (YW5-1B); lane 2, Δapg8 cells harboring APG8 on a multicopy vector (TK201); lane 3, Δapg8 cells (TK404).


B , The wild - type ( YW5 - 1B ) cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , 3 , 4 . 5 , and 6 h at 30 ° C . The amount of Apg8p was estimated by immunoblotting with anti - Apg8p antibody . Each lane has 10 μg of total protein .

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B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein.


C , YW5 - 1B cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , and 6 h at 30 ° C , and mRNA was prepared from each culture as described in Materials and Methods . APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe . Each lane has 4 μg of total mRNA .

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C, YW5-1B cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA.


D , YW5 - 1B cells were cultured in YEPD medium until 1 × 107 cells / ml at 30 ° C . Rapamycin was added to the culture at a final concentration of 0 . 2 μg / ml and the cells were further incubated in YEPD medium for two hours at 30 ° C . Western blotting was performed with the anti - Apg8p antibody . Each lane has 20 μg of total protein .

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D, YW5-1B cells were cultured in YEPD medium until 1 × 107 cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.


Subcellular fractionation and solubilization of Apg8p . The wild - type cells ( YW5 - 1B ) growing logarithmically in YEPD medium or starved in SD ( − N ) medium for three hours were lysed . A , The lysates ( T ) were centrifuged at 13 , 000 g for 15 min to generate pellet ( LSP ) and supernatant ( LSS ) . LSS fraction was further centrifuged at 100 , 000 g for one hour and separated to pellet ( HSP ) and supernatant ( HSS ) . LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates . Equal volume of each sample was applied to each well , and immunoblotting was performed with anti - Apg8p antibody .

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Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 g for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 g for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody.


B , The lysates ( T ) were sonicated and centrifuged at 100 , 000 g for one hour to separate pellet ( P ) and supernatant ( S ) . The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody , respectively .

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B, The lysates (T) were sonicated and centrifuged at 100,000 g for one hour to separate pellet (P) and supernatant (S). The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody, respectively.


C , The sonicated lysates were centrifuged at 100 , 000 g for one hour to generate pellet fraction . The pellet fraction was treated with 2 % Triton X - 100 ( TX - 100 ) for 30 min on ice . The sample was centrifuged at 100 , 000 g for one hour and separated to supernatant ( S ) and pellet ( P ) fractions . The distribution of Apg8p was examined by immunoblotting with the anti - Apg8p antibody .

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C, The sonicated lysates were centrifuged at 100,000 g for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 g for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.


Immunofluorescent staining of Δapg8 cells expressing 3 × HA - Apg8p . A , The Δapg8 cells harboring 3 × HA - tagged APG8 plasmid , TK114 cells , were grown until logarithmic phase . The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts . The spheroplasts were permeabilized by 0 . 5 % Triton X - 100 , and incubated with the anti - HA antibody , 16B12 , followed with the FITC - conjugated anti - mouse IgG . Left , Fluorescence image of 3 × HA - Apg8p ; right , Nomarski image of the cells . Arrows show punctate signals that are a little larger than tiny dot signals . Bars , 10 μm . B , Immunofluorescent staining of YW5 - 1B cells before ( 0 h ) and after ( 3 h ) shift to starvation ( negative control ) . Left , Fluorescence image ; right , Nomarski image . Bars , 10 μm . C , The cells were shifted to starvation for 0 , 0 . 5 , and 3 h . Upper panels , Fluorescence images of 3 × HA - Apg8p ; lower panels , Nomarski images of the cell . Bars , 5 μm .

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Immunofluorescent staining of Δapg8 cells expressing 3 × HA-Apg8p. A, The Δapg8 cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti-mouse IgG. Left, Fluorescence image of 3 × HA-Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA-Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.


Change of Apg8p localization in Δpep4 cells after shift to starvation . The Δapg8Δpep4 cells harboring 3 × HA - tagged APG8 plasmid , TK116 cells , were shifted to starvation for 0 , 0 . 5 , 1 , 3 , and 6 h . Immunofluorescence microscopy was performed as described in Fig . 5 . A - E , FITC staining of 3 × HA - Apg8p . F - J , Nomarski images of the cells . Arrows show an autophagic body . Bars , 5 μm .

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Change of Apg8p localization in Δpep4 cells after shift to starvation. The Δapg8Δpep4 cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in Fig. 5. A-E, FITC staining of 3 × HA-Apg8p. F-J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.


Immunostaining image of a cell containing autophagic bodies in the vacuole . The Δapg8Δpep4 cells expressing 3 × HA - Apg8p , TK116 cells , were shifted to SD ( − N ) medium for two hours . Cells were immunolabeled with anti - N mAb , 16B12 , followed by 10 - nm gold - conjugated goat anti - mouse IgG . AB , vacuole ; N , nucleus ; V , vacuole .

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Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δapg8Δpep4 cells expressing 3 × HA-Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-N mAb, 16B12, followed by 10-nm gold-conjugated goat anti-mouse IgG. AB, vacuole; N, nucleus; V, vacuole.


Immuno - EM of autophagosomes . A and B , Mature autophagosome . C and D , Premature autophagosome . Arrows show expanded regions of the intramembrane space of the premature autophagosomes . TK116 cells were incubated in SD ( − N ) medium for one hour ( D ) , two hours ( B and C ) , or three hours ( A ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A and D ) or 10 - nm gold - ( B and C ) conjugated goat anti - mouse IgG . AP , autophagosome ; N , nucleus ; V , vacuole .

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Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.


Possible intermediate structures of autophagosome . A , A membrane sac under construction . Arrows show gold particles . B and C , Isolation membranes . B , Isolation membrane is detected along the arrows . C , The small arrow shows a semicircular isolation membrane and a large arrow marks its open area . D , Apg8p - residing structures gathered in the area close to the vacuole . TK116 cells were incubated in SD ( − N ) medium for one hour ( A and C ) , or two hours ( B and D ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A ) or 10 - nm gold - ( B - D ) conjugated goat anti - mouse IgG . V , vacuole .

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Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.


Fine morphology of Δypt7 and Δapg8 cells and proteinase - K accession to proAPI in these mutants . A , EM image of Δypt7 cell starved in SD ( − N ) medium for four hours . Autophagosomes ( AP , arrows ) were accumulated . B , EM image of the starved Δapg8 cell . C - E , The representatives of the membrane structures detected in the starved Δapg8 cells . C , Autophagosome - like structure indistinguishable from autophagosome . D and E , Aberrant multivesicular structures . N , nucleus ; V , vacuole . F , Proteinase K - sensitivity of proAPI . Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD ( − N ) medium for 4 . 5 h . The lysates were treated with 100 μg / ml proteinase K in the presence or absence of Triton X - 100 . Asterisk shows mature API and digestion product of proAPI by proteinase K .

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Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.


A Mpc1 and Mpc2 are present under fermentative conditions , while Mpc1 and Mpc3 are present under respiratory conditions . MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose ( YPD ) or glycerol ( YPG ) . Mitochondrial matrix protein aconitase ( Aco1 ) , 23 ‐ kDa translocase of the inner membrane subunit ( Tim23 ) , and 70 ‐ kDa translocase of the outer membrane subunit ( Tom70 ) were used as loading controls .

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A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.


B Kinetics of the switch from Mpc2 to Mpc3 . Yeast cells were grown in YPD until mid ‐ log phase and then shifted to YPG medium . Whole cell extracts were taken at the indicated time points after the shift , and HA ‐ tagged Mpc2 and Mpc3 detected by Western blotting . Mpc3 is induced very rapidly by switch from YPD to YPG , while Mpc2 levels decrease only gradually .

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B Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually.


C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN ‐ PAGE . Isolated mitochondria expressing HA ‐ tagged MPC proteins were run on blue native gels , and MPC complexes detected with an HA ‐ specific antibody . MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions , and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions . In addition , a complex of ˜ 300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3 . Non ‐ specific reactivity of the anti ‐ HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk ( * ) .

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C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).


A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant . Growth tests were performed with cells transformed with MPC1 , MPC2 , or MPC3 expression plasmids or with empty vectors in indicated combinations . A serial dilution of yeast cells was spotted on agar plates with glucose ‐ containing synthetic minimal medium with ( SD + AA ) or without ( SD − AA ) amino acids .

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A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.


B Plasmid ‐ encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co ‐ expressed with either Mpc2 or Mpc3 . Cells were grown in selective medium containing glycerol as a carbon source . MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti ‐ HA or anti ‐ Flag antibodies . Mitochondrial enzymes aconitase ( Aco1 ) and malate dehydrogenase ( Mdh1 ) were used as loading controls .

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B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls.


C BN ‐ PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid ‐ based protein expression . Non ‐ specific reactivity of the antibody is denoted by an asterisk ( * ) . It has to be noted that all complexes migrate slightly faster than in Fig C , most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA ‐ tag in Fig C .

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C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.


D Mpc1 is co ‐ immunoprecipitated with Mpc2 or Mpc3 . Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol ‐ containing medium were lysed in 1 % digitonin , and either Mpc2 ‐ Flag or Mpc3 ‐ Flag was immunoprecipitated . Mpc1 ‐ HA was detected by Western blot with an antibody directed against the epitope tag . The matrix protein aconitase ( Aco1 ) and 40 ‐ kDa translocase of the outer membrane subunit ( Tom40 ) were not co ‐ immunoprecipitated .

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D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.


E , F Stoichiometry of Mpc1 complexes . Chemical cross ‐ linking indicates that Mpc1 / Mpc2 and Mpc1 / Mpc3 form heterodimers of the predicted molecular weight . DSG , disuccimidyl glutarate . Cross ‐ linking products with unknown partners are labeled with a question mark ( ? ) .

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E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).


A , B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 ( A ) and Mpc3 ( B ) . Mitochondria were treated with IASD as indicated . Negative [ quenching by pretreatment with DTT ( pre ‐ DTT ) ] and positive [ lysis with 0 . 5 % Triton X ‐ 100 ( T ‐ X100 ) ] controls for the labeling reaction are shown . As a further control , also translocase of the inner membrane subunit Tim23 is detected by Western blot , which contains three endogenous cysteines , all of which are located in membrane ‐ embedded regions . Labeling of an IMS ‐ exposed cysteine with IASD is indicated by a mobility shift on SDS - PAGE corresponding to the molecular weight of IASD ( ˜ 500 Da ) .

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A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da).


C Protease protection assay in mitochondria expressing Mpc1 ‐ GFP , Mpc2 ‐ GFP , or Mpc3 ‐ GFP . Intact mitochondria ( M ) , mitoplasts with a ruptured outer membrane after hypo ‐ osmotic swelling ( Sw ) , or mitochondrial lysates with 0 . 5 % Triton X ‐ 100 ( Tr ) were treated with proteinase K ( PK ) . Loading controls are Tom70 ( outer membrane ) , Tim23 ( inner membrane ) , and Mdh1 ( matrix ) . GFP ‐ fused Mpc2 and Mpc3 are degraded in mitoplasts , whereas Mpc1 ‐ GFP is not .

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C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not.


A MPCOX has higher transport activity than MPCFERM . The uptake of 14C ‐ labeled pyruvate into intact mitochondria was measured in vitro . Mitochondria had been isolated from cells grown in glycerol ‐ containing medium and expressing no subunit ( vectors ) , MPCFERM , MPCOX , Mpc2 , or Mpc3 . Imported pyruvate was quantified by re ‐ isolation of mitochondria and subsequent scintillation counting after 1 , 2 , or 5 min of incubation with [ 14C ] ‐ pyruvate . B Imported [ 14C ] ‐ pyruvate as in ( A ) after 5 ‐ min incubation . The difference between MPCOX and MPCFERM ( * * P = 0 . 0037 ) was significant ( unpaired t ‐ test ) .

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A MPCOX has higher transport activity than MPCFERM. The uptake of 14C‐labeled pyruvate into intact mitochondria was measured in vitro. Mitochondria had been isolated from cells grown in glycerol‐containing medium and expressing no subunit (vectors), MPCFERM, MPCOX, Mpc2, or Mpc3. Imported pyruvate was quantified by re‐isolation of mitochondria and subsequent scintillation counting after 1, 2, or 5 min of incubation with [14C]‐pyruvate.B Imported [14C]‐pyruvate as in (A) after 5‐min incubation. The difference between MPCOX and MPCFERM (**P = 0.0037) was significant (unpaired t‐test).


C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit ( vectors ) , MPCFERM , or MPCOX .

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C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit (vectors), MPCFERM, or MPCOX.


The chimeric construct Mpc2C3 is expressed in isolated mitochondria , while Mpc3C2 is not . Mpc1 ‐ HA and the Flag ‐ tagged chimeric proteins were detected by Western blotting in isolated mitochondria .

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The chimeric construct Mpc2C3 is expressed in isolated mitochondria, while Mpc3C2 is not. Mpc1‐HA and the Flag‐tagged chimeric proteins were detected by Western blotting in isolated mitochondria.


Mpc2C3 forms a carrier complex with Mpc1 , as analyzed by BN ‐ PAGE . When co ‐ expressed with Mpc1 , Mpc2C3 shows the same complex pattern as Mpc3 , including the 300K complex .

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Mpc2C3 forms a carrier complex with Mpc1, as analyzed by BN‐PAGE. When co‐expressed with Mpc1, Mpc2C3 shows the same complex pattern as Mpc3, including the 300K complex.


Pyruvate transport activity of the Mpc1 / Mpc2C3 complex reaches ˜ 85 % of MPCOX activity . Uptake of [ 14C ] ‐ pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro . Difference between MPCFERM and Mpc1 / Mpc2C3 is significant ( unpaired t ‐ test ; * P = 0 . 02 ) . Means of n = 4 experiments are shown , with error bars representing SEM .

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Pyruvate transport activity of the Mpc1/Mpc2C3 complex reaches ˜85% of MPCOX activity. Uptake of [14C]‐pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro. Difference between MPCFERM and Mpc1/Mpc2C3 is significant (unpaired t‐test; *P = 0.02). Means of n = 4 experiments are shown, with error bars representing SEM.


( C ) Interaction analysis between hCAP - G and hCAP - H . Bacterial cell lysates co - expressing hCAP - G ( residues 1 - 478 , 554 - 900 ) and hCAP - H ( residues 394 - 515 ) , either wild type ( WT ; lanes 2 , 10 and 13 ) , 3Q ( F463Q , F469Q and F473Q ; lane 3 ) , 5Q ( F463Q , F469Q , F473Q , F501Q and Y503Q ; lane 4 ) , 3A ( F463A , F469A and F473A ; lane 5 ) , 5A ( F463A , F469A , F473A , F501A and Y503A ; lane 6 ) or 2A ( F501A and Y503A ; lane 11 ) , or a C - terminal deletion mutant ( 506 - 514 residues were deleted from 394 - 515 ; lane 14 ) were applied to Ni - NTA agarose resin , and the bound fraction was analyzed by SDS - PAGE . Alternatively , a cell lysate co - expressing mutant hCAP - G ( D647K ) and wild - type hCAP - H was examined ( lane 7 ) . The uninduced cell lysate was also used as a negative control ( lane 9 ) .

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(C) Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1-478, 554-900) and hCAP-H (residues 394-515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6) or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506-514 residues were deleted from 394-515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS-PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9).


( A ) Expression of condensin I subunits in insect cells . The wild - type ( WT ) or IV - 5Q mutant CAP - H subunit was co - expressed with the other four subunits ( GST - SMC4 , SMC2 , CAP - D2 and CAP - G ) in insect cells . Cell lysates were prepared and subjected to SDS - PAGE , followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 ( left panel ) or against CAP - D2 , - G , and - H ( right panel ) .

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(A) Expression of condensin I subunits in insect cells. The wild-type (WT) or IV-5Q mutant CAP-H subunit was co-expressed with the other four subunits (GST-SMC4, SMC2, CAP-D2 and CAP-G) in insect cells. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting with a mixture of antibodies against SMC2 and SMC4 (left panel) or against CAP-D2, -G, and -H (right panel).


( B ) Purification of the WT and IV - 5Q mutant condensin I complexes . Protein samples purified through glutathione - affinity chromatography were subjected to SDS - PAGE and analyzed by CBB staining ( left panel ) or immunoblotting with a mixture of antibodies as described above ( middle and right panels ) .

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(B) Purification of the WT and IV-5Q mutant condensin I complexes. Protein samples purified through glutathione-affinity chromatography were subjected to SDS-PAGE and analyzed by CBB staining (left panel) or immunoblotting with a mixture of antibodies as described above (middle and right panels).


( C ) Add - back assay using the WT and mutant condensin I complexes . Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes ( from top to bottom ; WT , IV - 5Q , ∆ G , ∆ G [ IV - 5Q ] ) . The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes . The samples were fixed and labeled with an antibody against mSMC4 ( red ) . DNA was counterstained with DAPI ( blue ) . The data from a single representative experiment out of two repeats are shown . In the experiment shown here , multiple images were collected for condensin - depleted extracts supplemented with the WT ( n = 17 ) , IV - 5Q ( n = 22 ) , ∆ G ( n = 20 ) and ∆ G ( IV - 5Q ) ( n = 25 ) complexes . The scale bar represents 10 μm .

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(C) Add-back assay using the WT and mutant condensin I complexes. Xenopus extracts depleted of endogenous condensin complexes were supplemented with the purified complexes (from top to bottom; WT, IV-5Q, ∆G, ∆G[IV-5Q]). The supplemented extracts were then incubated with sperm nuclei to assemble mitotic chromosomes. The samples were fixed and labeled with an antibody against mSMC4 (red). DNA was counterstained with DAPI (blue). The data from a single representative experiment out of two repeats are shown. In the experiment shown here, multiple images were collected for condensin-depleted extracts supplemented with the WT (n = 17), IV-5Q (n = 22), ∆G (n = 20) and ∆G(IV-5Q) (n = 25) complexes. The scale bar represents 10 μm.


( A ) Purification of hCAP - G and hCAP - G - H subcomplexes : wild type ( WT ) , CAP - G K60D / R848E double mutant ( K60D / R848E ) and CAP - G R168E mutant ( R168E ) . Purified protein samples were subjected to SDS - PAGE and analyzed by CBB staining .

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(A) Purification of hCAP-G and hCAP-G-H subcomplexes: wild type (WT), CAP-G K60D/R848E double mutant (K60D/R848E) and CAP-G R168E mutant (R168E). Purified protein samples were subjected to SDS-PAGE and analyzed by CBB staining.


( B ) Double - stranded DNA ( dsDNA ) and single - stranded DNA binding assay of the hCAP - G . 30 - bp dsDNA was incubated with no protein ( lanes 1 ) or increasing amounts of WT hCAP - G ( WT ; lanes 2 - 4 ) . 30 - mer ssDNA was incubated with no protein ( lanes 5 ) or increasing amounts of WT hCAP - G ( WT ; lanes 6 - 8 ) .

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(B) Double-stranded DNA (dsDNA) and single-stranded DNA binding assay of the hCAP-G. 30-bp dsDNA was incubated with no protein (lanes 1) or increasing amounts of WT hCAP-G (WT; lanes 2-4). 30-mer ssDNA was incubated with no protein (lanes 5) or increasing amounts of WT hCAP-G (WT; lanes 6-8).


( C ) The dsDNA binding assay for the hCAP - G - H subcomplexes . 30 - bp dsDNA was incubated with no protein ( lanes 1 , 5 and 9 ) , increasing amounts of WT hCAP - G - H subcomplex ( WT ; lanes 2 - 4 ) , CAP - G K60D / R848E double mutant ( K60D / R848E ; lanes 6 - 8 ) or CAP - G R168E mutant ( R168E ; lanes 10 - 12 ) .

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(C) The dsDNA binding assay for the hCAP-G-H subcomplexes. 30-bp dsDNA was incubated with no protein (lanes 1, 5 and 9), increasing amounts of WT hCAP-G-H subcomplex (WT; lanes 2-4), CAP-G K60D/R848E double mutant (K60D/R848E; lanes 6-8) or CAP-G R168E mutant (R168E; lanes 10-12).


( D ) The ssDNA binding assay for the hCAP - G - H

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(D) The ssDNA binding assay for the hCAP-G-H subcomplexes


W0 , W6 and W12 refer to the week of study . Serum FGF - 21 and GDF - 15 levels before , during and after treatment ( two - sided Wilcoxon signed - rank test , P - value = 0 . 031 in all significant cases ) ( see Fig . EV2 for details where P1 - P6 refer to the individual patients ) .

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W0, W6 and W12 refer to the week of study. Serum FGF-21 and GDF-15 levels before, during and after treatment (two-sided Wilcoxon signed-rank test, P-value = 0.031 in all significant cases) (see Fig. EV2 for details where P1 - P6 refer to the individual patients).


Volcano plot showing differences in metabolite levels between 0 and 6 & 12 weeks of treatment . Metabolites in histidine , alanine , aspartate and glutamine metabolism , and the TCA cycle pathways are annotated at 10 % False Discovery Rate ( see Dataset EV1 for the whole list of differentially regulated metabolites . Although some other metabolites showed a more pronounced difference , these did not fit into a recognised pathway ) . Metabolite labels are colour - coded according to the identified KEGG pathways ( dark red = " Alanine , aspartate and glutamate metabolism " , dark green = " Histidine metabolism " and dark blue = " Citrate cycle ( TCA cycle ) " , with transition colour - code if the metabolite belongs to more than one KEGG pathways ) .

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Volcano plot showing differences in metabolite levels between 0 and 6&12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% False Discovery Rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour-coded according to the identified KEGG pathways (dark red = "Alanine, aspartate and glutamate metabolism", dark green = "Histidine metabolism" and dark blue = "Citrate cycle (TCA cycle)", with transition colour-code if the metabolite belongs to more than one KEGG pathways).


Scatterplot depicting P - values from the metabolites Pathway Enrichment Analysis ( y - axis ) and impact values from Pathway Topology Analysis ( x - axis ) ( see Dataset EV2 for details ) . KEGG pathways at 10 % adjusted Holm - Bonferroni P - value are highlighted .

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Scatterplot depicting P-values from the metabolites Pathway Enrichment Analysis (y-axis) and impact values from Pathway Topology Analysis (x-axis) (see Dataset EV2 for details). KEGG pathways at 10% adjusted Holm-Bonferroni P-value are highlighted.


Volcano plot of acyl - carnitine metabolites between 0 and 6 & 12 weeks of treatment with highlighted differential metabolites at 10 % FDR .

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Volcano plot of acyl-carnitine metabolites between 0 and 6&12 weeks of treatment with highlighted differential metabolites at 10% FDR.


Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment . No significant differences were detected before and after 12 weeks of treatment by using the two - sided Wilcoxon signed - rank test with an empirical level of significance .

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Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment. No significant differences were detected before and after 12 weeks of treatment by using the two-sided Wilcoxon signed-rank test with an empirical level of significance.


P1 - P6 refer to individual patients . Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment . The box in the top right - hand corner indicates the normal range , using two antibodies to NDUFB8 and COX I .

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P1 - P6 refer to individual patients. Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment. The box in the top right-hand corner indicates the normal range, using two antibodies to NDUFB8 and COX I.


Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease ( two - sided Wilcoxon signed - rank test with an empirical level of significance ) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment ( empirical P - value = 0 . 048 ) .

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Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease (two-sided Wilcoxon signed-rank test with an empirical level of significance) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment (empirical P-value = 0.048).


Hierarchical cluster analysis of skeletal muscle RNA - seq transcripts before and after treatment showing no clear separation between untreated and treated patients .

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Hierarchical cluster analysis of skeletal muscle RNA-seq transcripts before and after treatment showing no clear separation between untreated and treated patients.


Volcano plot depicting differentially expressed RNA - seq genes at 10 % FDR after 12 weeks of treatment . Significantly different mammalian mitochondrial genes ( MitoCarta 2 . 0 ) are annotated .

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Volcano plot depicting differentially expressed RNA-seq genes at 10% FDR after 12 weeks of treatment. Significantly different mammalian mitochondrial genes (MitoCarta 2.0) are annotated.


Percentage level m . 3243A > G in blood , urinary sediment and skeletal muscle . No differences were detected by using the two - sided Wilcoxon signed - rank test with an empirical level of significance . W = week of the study .

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Percentage level m.3243A>G in blood, urinary sediment and skeletal muscle. No differences were detected by using the two-sided Wilcoxon signed-rank test with an empirical level of significance. W = week of the study.


Cardiac parameters before and after treatment ( two - sided Wilcoxon signed - rank test with an empirical level of significance ) . ES = end - systolic volume ( ml ) ( empirical P - value = 0 . 047 ) and index ( ml / m2 ) ( empirical P - value = 0 . 046 ) , measured by MRI . Horizontal lines on ( B ) , ( F ) and ( G ) refer to statistical comparisons of the mean values , where ns = non - significant , * P - value ≤ 0 . 05 . For normal reference ranges see ( Hollingsworth et al . , 2012 )

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Cardiac parameters before and after treatment (two-sided Wilcoxon signed-rank test with an empirical level of significance). ES = end-systolic volume (ml) (empirical P-value = 0.047) and index (ml/m2) (empirical P-value = 0.046), measured by MRI. Horizontal lines on (B), (F) and (G) refer to statistical comparisons of the mean values, where ns = non-significant, *P-value ≤ 0.05. For normal reference ranges see (Hollingsworth et al., 2012)


B - C ' ) Immunofluorescence analysis of active - caspase 3 in the ONL of P347S and P347S / miR - 181a / b + / - . D , E ) TUNEL analysis in the ONL of P347S and P347S / miR - 181a / b + / - . F ) Quantification of immunofluorescence analysis in ( B - C ' ) ( N = 4 eyes P347S and N = 6 eyes P347S / miR - 181a / b + / ) . G ) Quantification of TUNEL analysis in ( D , E ) ( N = 3 eyes / genotype ) .

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B-C') Immunofluorescence analysis of active-caspase 3 in the ONL of P347S and P347S/miR-181a/b+/-. D, E) TUNEL analysis in the ONL of P347S and P347S/miR-181a/b+/-. F) Quantification of immunofluorescence analysis in (B-C') (N=4 eyes P347S and N=6 eyes P347S/ miR-181a/b+/). G) Quantification of TUNEL analysis in (D, E) (N=3 eyes/genotype).


D - P ) Immunofluorescence analysis showed amelioration of Rhodopsin localization ( D - F ' ; white arrowheads ) ; C - arrestin expression ( G - I ' ) and PR outer segment ( OS ) and inner segment ( IS ) structures , as determined , respectively , by PNA ( J - L ' ) ; and Phalloidin staining ( M - O ' ' ) in P347S / miR - 181a / b + / - vs P347S eyes at p30 . M ' ' - O ' ' show higher magnification of M - O . Scale bars 25 μm in D - O ' ; 5μm in M ' ' - O ' ' . Fluorescence densitometry quantification of each staining is reported in ( P ) , N = 3 eye / genotype for each staining . WT vs P347S p values are reported in red , P347S vs P347S / miR - 181a / b + / - p values are reported in black .

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D-P) Immunofluorescence analysis showed amelioration of Rhodopsin localization (D-F'; white arrowheads); C-arrestin expression (G-I') and PR outer segment (OS) and inner segment (IS) structures, as determined, respectively, by PNA (J-L'); and Phalloidin staining (M-O'') in P347S/miR-181a/b+/- vs P347S eyes at p30. M''-O'' show higher magnification of M-O. Scale bars 25 μm in D-O'; 5μm in M''-O''. Fluorescence densitometry quantification of each staining is reported in (P), N=3 eye/genotype for each staining. WT vs P347S p values are reported in red, P347S vs P347S/miR-181a/b+/- p values are reported in black.


D - L ) Immunofluorescence analysis of Rhodopsin ( D - E ' ) , C - arrestin ( F - G ' ) , PNA ( H - I ' ) ; and Phalloidin staining ( J - K ' ' ) in P347S and P347S / miR - 181a / b + / - retinas at p90 . J ' ' - K ' ' show higher magnification of J - K . Scale bars 25 μm in D - K ' ; 10μm in J ' ' - K ' ' .

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D-L) Immunofluorescence analysis of Rhodopsin (D-E'), C-arrestin (F-G'), PNA (H-I'); and Phalloidin staining (J-K'') in P347S and P347S/miR-181a/b+/- retinas at p90. J''-K'' show higher magnification of J-K. Scale bars 25 μm in D-K'; 10μm in J''-K''.