Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( C ) Representative photomicrographs of β - galactosidase staining of hippocampal slices derived from young ( 3 months ) , middle - aged ( 12 months ) and aged ( 22 - 24 months ) of IRE1WT or IRE1cKO animals . Magnification : 20x ; scale bar : 200 μm . Arrows indicate β - galactosidase staining .

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Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (C) Representative photomicrographs of β-galactosidase staining of hippocampal slices derived from young (3 months), middle-aged (12 months) and aged (22-24 months) of IRE1WT or IRE1cKO animals. Magnification: 20x; scale bar: 200 μm. Arrows indicate β-galactosidase staining.


Young ( 3 - 6 months ) , middle aged ( 12 - 14 months ) and aged ( 18 - 20 months ) conditional knockout for IRE1 ( IRE1cKO ) and their littermates flox / flox control animals ( IRE1WT ) were evaluated ( D ) Mean and SEM of percentage of β - galactosidase staining in CA3 region ( n = 3 - 5 animals / group ) .

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Young (3-6 months), middle aged (12-14 months) and aged (18-20 months) conditional knockout for IRE1 (IRE1cKO) and their littermates flox/flox control animals (IRE1WT) were evaluated (D) Mean and SEM of percentage of β-galactosidase staining in CA3 region (n = 3-5 animals/group).


( F ) Representative confocal images of dendritic spines ( arrows ) in the CA1 region of middle - aged IRE1WT animals injected with AAV - Mock or AAV - Cre . 60x magnification , ( G ) Mean and SEM of spine density per μm ( n = 3 , 3 ; 4 , 4 animals ) . P computed by unpaired Student ' s t test within each age - matched group .

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(F) Representative confocal images of dendritic spines (arrows) in the CA1 region of middle-aged IRE1WT animals injected with AAV-Mock or AAV-Cre. 60x magnification, (G) Mean and SEM of spine density per μm (n = 3, 3; 4, 4 animals). P computed by unpaired Student's t test within each age-matched group.


( E ) Representative images of β - galactosidase staining of hippocampal slices derived from aged ( 22 - 24 months ) TgXBP1s and WT animals . ( F ) Mean and SEM of percentage of area stained by β - galactosidase in the hippocampus of aged ( 20 - 24 months ) TgXBP1s compared to littermate controls .

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(E) Representative images of β-galactosidase staining of hippocampal slices derived from aged (22-24 months) TgXBP1s and WT animals. (F) Mean and SEM of percentage of area stained by β-galactosidase in the hippocampus of aged (20-24 months) TgXBP1s compared to littermate controls.


( D ) Left panel : Representative images showing dendritic spines of aged ( 22 - 24 months ) animals injected with AAV2 - eGFP ( mock ) or with AAV2 - XBP1s ( arrows indicate dendritic spines ) . Young animal dendrite is shown for reference . 60x magnification . Scale bar : 5μm . Right panel : Mean and s . e . m . of spine density per μm for indicated experimental groups ( n = 24 , 53 and 35 dendrites from 3 , 5 and 6 animals ,

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(D) Left panel: Representative images showing dendritic spines of aged (22-24 months) animals injected with AAV2-eGFP (mock) or with AAV2-XBP1s (arrows indicate dendritic spines). Young animal dendrite is shown for reference. 60x magnification. Scale bar: 5μm. Right panel: Mean and s.e.m. of spine density per μm for indicated experimental groups (n = 24, 53 and 35 dendrites from 3, 5 and 6 animals, respectively


( E ) Left panel : Representative images of β - galactosidase staining of hippocampal slices derived from aged mice injected with AAV2 - Mock or AAV2 - XBP1s ( arrow indicate β - galactosidase staining ) . Magnification 20x ; scale bar , 200 μm . Right panel : Histograms show mean and s . e . m . of percentage of β - galactosidase - stained area . n = 6 , 6 ; unpaired Student ' s t test .

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

(E) Left panel: Representative images of β-galactosidase staining of hippocampal slices derived from aged mice injected with AAV2-Mock or AAV2-XBP1s (arrow indicate β-galactosidase staining). Magnification 20x; scale bar, 200 μm. Right panel: Histograms show mean and s.e.m. of percentage of β-galactosidase-stained area. n = 6, 6; unpaired Student's t test.


( F ) Left panel : Immunofluorescence of γH2aX ( red ) counterstained with nuclei ( blue ) comparing aged mice injected with AAV - Mock or AAV - XBP1s . Magnification : 40x ; Scale bar : 50μm . Right panel : Histograms show mean and s . e . m . of percentage of γH2A - positive cells ; unpaired Student ' s t test comparing the aged groups ( n = 6 , 6 animals / group ; unpaired Student ' s t test ) .

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(F) Left panel: Immunofluorescence of γH2aX (red) counterstained with nuclei (blue) comparing aged mice injected with AAV-Mock or AAV-XBP1s. Magnification: 40x; Scale bar: 50μm. Right panel: Histograms show mean and s.e.m. of percentage of γH2A- positive cells; unpaired Student's t test comparing the aged groups ( n = 6, 6 animals/group; unpaired Student's t test).


A . Whole chromosomes imaged by conventional TEM . Data information : Scale bars :

A. Whole chromosomes imaged by conventional TEM. Data information: Scale bars: 1µm


B . Slice from a large tomographic volume ; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk ; the inset illustrates that the slice of a plate corresponds to a line in the x - y plane ( perpendicular to the direction of the electron beam ) . Data information : Scale bars : 200

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B. Slice from a large tomographic volume; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk; the inset illustrates that the slice of a plate corresponds to a line in the x-y plane (perpendicular to the direction of the electron beam). Data information: Scale bars: 200 nm


C - G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes . In addition to a typical slice through the x - y plane ( yellow ) , two slices through the x - z ( red ) and y - z ( green ) planes ( orthogonal to the x - y plane ) are shown in ( C ) . Data information : Scale bars : 200 nm D ) , 100 nm ( E - G ) , 50 nm ( C ) .

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C-G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes. In addition to a typical slice through the x-y plane (yellow), two slices through the x-z (red) and y-z (green) planes (orthogonal to the x-y plane) are shown in (C). Data information: Scale bars: 200 nm D), 100 nm (E-G), 50 nm (C).


A . Nucleosomes ( N ) decorating a relaxed plate ( P1 ) ; insets in the upper right show additional examples . Nucleosomes are not visible as individual units in typical compact plates ( P2 ) . Short compact interdigitated solenoids are shown in the main image ( S ) and in the bottom - left insets . Data information : Scale bars : 50

A. Nucleosomes (N) decorating a relaxed plate (P1); insets in the upper right show additional examples. Nucleosomes are not visible as individual units in typical compact plates (P2). Short compact interdigitated solenoids are shown in the main image (S) and in the bottom-left insets. Data information: Scale bars: 50 nm


B - E Slice ( B ) and segmentations in three different orientations ( C ) of a large relaxed plate decorated with many nucleosomes on its right side . Slice ( D ) and segmentation ( E ) of a relaxed plate forming a tube decorated with nucleosomes . Data information : Scale bars : 50 nm insets , B - E )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Slice (B) and segmentations in three different orientations (C) of a large relaxed plate decorated with many nucleosomes on its right side. Slice (D) and segmentation (E) of a relaxed plate forming a tube decorated with nucleosomes. Data information: Scale bars: 50 nm insets, B-E)


A , B Plates with several layers that are not closely appressed . C - E Large multilayer plates having the size of human metaphase chromatids [ ~ 0 . 6 µm diameter ] ( C ) ; the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin - plate model In other slices ( D , E ) , the multilayer structures shown in ( C ) are more compact and the individual layers are not visible as separate elements . Data information : Scale bars : 50 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plates with several layers that are not closely appressed. C-E Large multilayer plates having the size of human metaphase chromatids [~0.6 µm diameter ] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin-plate model In other slices (D,E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements. Data information: Scale bars: 50 nm.


A - C Slices showing lateral association between two plates and the corresponding 3D segmentations . D . Edge - to - edge interactions form cylindrical structures . Data information : Scale bars : 50 nm .

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A-C Slices showing lateral association between two plates and the corresponding 3D segmentations. D. Edge-to-edge interactions form cylindrical structures. Data information: Scale bars: 50 nm.


A - C The cation concentrations used are indicated for each experiment ; AU , arbitrary units .

A-C The cation concentrations used are indicated for each experiment; AU, arbitrary units.


Dox - induced prolongation of Nanog expression in Nanogtg embryos up to E9 . 5 results in lack of blood ( left ) and downregulation of erythropoietic gene expression . The center and right panels show whole - mount in situ hybridization for Hbb - bh1 ( in embryos with intact yolk sacs ) and for the long non - coding RNA Redrum . Asterisks mark the aorta - gonad - mesonephros region ( AGM ) and arrows the tailbud . Embryos of the same genotype but not treated with dox were used as controls ( - dox ) . Scale bars , 500 µm .

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

Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.


Endomucin staining of vessels in control ( - dox ) or treated ( + dox ) E9 . 5 Nanogtg embryos . On the right , higher magnifications of the boxed areas . Scale bar , 500 µm .

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Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.


Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox - treated E9 . 5 Nanogtg embryos . Quantification of the CD71 + Ter119 + population in controls ( - dox , black dots ; n = 8 ) and Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 yolk sacs . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg embryos . * * * P . < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars standard deviation ( SD ) .

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Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).


Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls ( - dox ) and Nanog expressing ( + dox ) E9 . 5 Nanogtg embryos . Quantification of different progenitor populations in yolk sacs from control ( - dox , black dots ; n = 8 ) and Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 embryos . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg embryos . Horizontal line represents mean values and error bars SD .

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

Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD.


Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71 + Ter119 + population of control ( - dox ; n = 7 ) and Nanog - expressing ( + dox ; n = 4 ) E9 . 5 embryos . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Freshly dissected dox - treated Nanogtg E10 . 5 embryos without ( control ) and with ( chimera ) contribution of wt - ESGFP cells ( left , brightfield ; right , GFP ) . Arrows mark the presence of blood in chimeric embryos that is absent from controls . Scale bar , 500 µm .

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Freshly dissected dox-treated Nanogtg E10.5 embryos without (control) and with (chimera) contribution of wt-ESGFP cells (left, brightfield; right, GFP). Arrows mark the presence of blood in chimeric embryos that is absent from controls. Scale bar, 500 µm.


Representative FACS plots showing of red blood cell maturation as determined by CD71 / Ter119 staining in single dox - treated E10 . 5 control ( left ) and chimeric ( right ) embryos .

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Representative FACS plots showing of red blood cell maturation as determined by CD71/Ter119 staining in single dox-treated E10.5 control (left) and chimeric (right) embryos.


Quantification of the CD71 + Ter119 + population in single dox - treated E10 . 5 embryos ( D ; control , n = 18 ; chimera , n = 10 ) , untreated embryos ( E ; control , n = 7 ; chimera , n = 12 ) , and in GFP - cells ( not derived from wild type ES cells ) from dox - treated embryos ( F ; control , n = 18 ; chimera , n = 10 ) . * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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Quantification of the CD71+ Ter119+ population in single dox-treated E10.5 embryos (D; control, n=18; chimera, n=10), untreated embryos (E; control, n=7; chimera, n=12), and in GFP- cells (not derived from wild type ES cells) from dox-treated embryos (F; control, n=18; chimera, n=10). ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Quantification of colony - forming units generated by wild type ( wt ) and knockout ( Nanog - / - ) ES cells after culture of EBs for 5 ( D5 ) , 6 ( D6 ) , or 7 ( D7 ) days and plating disaggregated cells in different hemogenic - promoting conditions ) . Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media . CFU - GEMM , progenitors giving rise to granulocytes , erythrocytes , monocytes , and megakaryocytes ; BFU - E , burst forming units - erythroid ; Ery - P , colony forming primitive erythroid ; CFU - GM , granulocyte - monocyte precursors ; CFU - M , monocyte precursors ; CFU - G , granulocyte precursors . No CFU - GEMM are detected at D5 and no BFU - E at D7 . For both wt and knockout cells , n = 3 each with 3 technical replicates . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 00005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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Quantification of colony-forming units generated by wild type (wt) and knockout (Nanog-/-) ES cells after culture of EBs for 5 (D5), 6 (D6), or 7 (D7) days and plating disaggregated cells in different hemogenic-promoting conditions). Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media. CFU-GEMM, progenitors giving rise to granulocytes, erythrocytes, monocytes, and megakaryocytes; BFU-E, burst forming units-erythroid; Ery-P, colony forming primitive erythroid; CFU-GM, granulocyte-monocyte precursors; CFU-M, monocyte precursors; CFU-G, granulocyte precursors. No CFU-GEMM are detected at D5 and no BFU-E at D7. For both wt and knockout cells, n=3 each with 3 technical replicates. *P < 0.05, **P < 0.005, ***P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control ( wt , right ) and knockout ( Nanog - / - , left ) ES cells ( n = 3 ) during 10 days of EB differentiation in hematopoietic - cytokine - enriched medium . Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic - gene expression .

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RT-qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control (wt, right) and knockout (Nanog-/-, left) ES cells (n=3) during 10 days of EB differentiation in hematopoietic-cytokine-enriched medium. Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic-gene expression.


Representative FACS plots showing the distribution of different populations distinguished by CD71 / Ter119 staining in whole bone marrow from untreated ( - dox ) or treated ( + dox ) adult mice . S0 ( double negative cell ) , S1 ( proerythroblast ) , S2 ( basophylic erythroblast ) , S3 ( polychromatic erythroblast ) , and S4 ( orthochromatic erythroblast ) are different stages of blood maturation . Quantification of the S1 - S4 erythroid populations ( - dox , n = 4 ; + dox , n = 5 ) . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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Representative FACS plots showing the distribution of different populations distinguished by CD71/Ter119 staining in whole bone marrow from untreated (-dox) or treated (+dox) adult mice. S0 (double negative cell), S1 (proerythroblast), S2 (basophylic erythroblast), S3 (polychromatic erythroblast), and S4 (orthochromatic erythroblast) are different stages of blood maturation. Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Representative FACS plots showing the distribution of CD16 / 32 and CD34 hematopoietic precursors sorted from the cKit + Sca1 - LIN - bone marrow of untreated ( - dox ) or treated ( + dox ) adult Nanogtg mice .

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Representative FACS plots showing the distribution of CD16/32 and CD34 hematopoietic precursors sorted from the cKit+Sca1-LIN- bone marrow of untreated (-dox) or treated (+dox) adult Nanogtg mice.


Quantification of precursor populations based on CD16 / 32 and CD34 sorting , as total number of cells per individual femur ( - dox , n = 5 ; + dox , n = 6 ) . * P < 0 . 05 , * * P < 0 . 005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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Quantification of precursor populations based on CD16/32 and CD34 sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *P < 0.05, **P < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte - erythroid progenitors ( MEP ) ( - dox , n = 8 ; + dox , n = 5 ) . * P < 0 . 05 , * * * * P < 0 . 00005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

RT-qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *P < 0.05, ****P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before ( left ) and after ( right ) dox treatment . Percentage of host derived cells ( CD45 . 1 + ) are shown in black , and of donor derived cells ( CD45 . 1 / CD45 . 2 double + ) in red . Individual mice are indicated on the x - axis ( n = 7 ) .

Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before (left) and after (right) dox treatment. Percentage of host derived cells (CD45.1+) are shown in black, and of donor derived cells (CD45.1/CD45.2 double +) in red. Individual mice are indicated on the x-axis (n=7).


Contribution of Nanogtg transplanted cells to LSK , CMP , GMP and MEP populations purified from bone marrow . Percentage of host derived cells ( CD45 . 1 + ) are show in black , and of donor derived cells ( CD45 . 1 / CD45 . 2 double + ) in blue . Individual mice are indicated on the x - axis ( n = 7 ) .

Contribution of Nanogtg transplanted cells to LSK, CMP, GMP and MEP populations purified from bone marrow. Percentage of host derived cells (CD45.1+) are show in black, and of donor derived cells (CD45.1/CD45.2 double +) in blue. Individual mice are indicated on the x-axis (n=7).


Expected and observed number of mesodermal ( Flk1 + ) cells of the E7 . 0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression , based on single cell RNA - seq data ( Scialdone et al . , 2016 ) . Statistical significance was calculated with a hypergeometric test .

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Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test.


PCA showing the distribution of Flk1 + E7 . 0 mesoderm cells expressing Nanog ( green ) or Tal1 ( red ) . The single cell expressing both genes is shown in yellow and indicated by an arrow .

PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing Nanog (green) or Tal1 (red). The single cell expressing both genes is shown in yellow and indicated by an arrow.


E6 . 5 Nanogtg embryos after 8 hours ex - utero culture in the presence ( + dox ) or absence ( - dox ) of doxycycline . Scale bar , 100 µm .

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E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.


RT - qPCR quantification of the relative expression of Nanog , Tal1 , and Klf1 in individual untreated embryos ( - dox ) or treated embryos ( + dox ) ( n = 5 ) . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Whole mount in situ hybridization of Tal1 in E7 . 5 untreated ( - dox ) or in utero treated ( + dox ) Nanogtg embryos . Scale bar , 100 µm .

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Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm.


RT - qPCR determination of relative expression in wild type and CRISPR - deleted embryos ( n = 5 ) of Tal1 ( wt , n = 19 ; deleted , n = 13 ) , Klf1 ( wt , n = 3 ; deleted , n = 6 ) , Gfi1b ( wt , n = 10 ; deleted , n = 8 ) , Runx1 ( wt , n = 13 ; deleted , n = 5 ) and Stil ( wt , n = 19 ; deleted , n = 13 ) . * * P < 0 . 005 , Student ' s t - test . Horizontal line represents mean values and error bars SD .

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RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of Tal1 (wt, n=19; deleted, n=13), Klf1 (wt, n=3; deleted, n=6), Gfi1b (wt, n=10; deleted, n=8), Runx1 (wt, n=13; deleted, n=5) and Stil (wt, n=19; deleted, n=13). **P < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD.


Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones ( Nanogtg ; dTal1del # 1 and Nanogtg ; dTal1del # 2 ) where the binding site for NANOG distal to Tal1 has been deleted ( left ) . On the right , relative expression of Tal1 determined by RT - qPCR for each ES cell line ( ESC ; n = 9 for all three lines ) and EpiL cells without ( EpiLC ; Nanogtg and Nanogtg ; dTal1del # 1 , n = 8 ; Nanogtg ; dTal1del # 2 , n = 6 ) or with dox treatment ( EpiLC + dox ; n = 9 for all three lines ) . The genotype of the cell lines is indicated below . Values were normalized to Nanogtg ESC . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ; ANOVA with Fisher post - test . Horizontal line represents mean values and error bars standard error of the mean ( SEM ) .

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Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).


B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths . Yeast cells expressing clogger proteins ( cytochrome b2 - DHFR and cytochrome b2 ∆ 19 - DHFR ) or cytosolic DHFR for comparison under control of the galactose - inducible promoter were grown to mid - log phase on lactate medium . Ten - fold serial dilutions were dropped on lactate ( no induction ) or lactate with 0 . 5 % galactose medium ( induction ) . MTS , matrix - targeting signal .

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B Competitive inhibition of mitochondrial protein import by clogger proteins inhibits cell growths. Yeast cells expressing clogger proteins (cytochrome b2-DHFR and cytochrome b2∆19-DHFR) or cytosolic DHFR for comparison under control of the galactose-inducible promoter were grown to mid-log phase on lactate medium. Ten-fold serial dilutions were dropped on lactate (no induction) or lactate with 0.5% galactose medium (induction). MTS, matrix-targeting signal.


E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates . Cells were grown in lactate medium and clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before samples were harvested and lysed . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

E Levels of the proteasome protein Pre6 were detected upon expression of cytosolic DHFR or clogger by Western blotting and quantified from three replicates. Cells were grown in lactate medium and clogger expression was induced for 4.5 h with 0.5% galactose before samples were harvested and lysed. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


A The clogger was expressed in wild type ( WT ) and ∆ rpn4 ( ∆ ) cells for 4 . 5 h with medium containing 0 . 5 % galactose . Medium was exchanged for a non - inducing lactate medium . Precursor ( pre ) and mature ( m ) forms of the b2 ∆ - DHFR clogger were visualized by Western blotting using a DHFR - specific antibody .

A The clogger was expressed in wild type (WT) and ∆rpn4 (∆) cells for 4.5 h with medium containing 0.5% galactose. Medium was exchanged for a non-inducing lactate medium. Precursor (pre) and mature (m) forms of the b2∆-DHFR clogger were visualized by Western blotting using a DHFR-specific antibody.


G Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆ rpn4 cells . The cytosolic soluble DHFR protein was expressed for control . The strains were grown in lactate medium and clogger expression was induced with 0 . 5 % galactose for 6 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Signals of Hsp104 and Hsp42 before and after clogger induction in wild type and ∆rpn4 cells. The cytosolic soluble DHFR protein was expressed for control. The strains were grown in lactate medium and clogger expression was induced with 0.5% galactose for 6 h.


A Cytosolic DHFR ( control ) or the clogger protein b2 - DHFR were expressed in wild type and ∆ rpn4 cells for 4 . 5 h . The distribution of constitutively expressed Hsp104 - GFP was visualized by fluorescence microscopy . Diameter of Hsp104 - GFP - bound aggregates were quantified . . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates . Significance was assessed using two - sided , paired Student ' s t - test . P values are indicated as asterisks * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . See also Fig . EV4A for quantification of the number of aggregates per cell .

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A Cytosolic DHFR (control) or the clogger protein b2-DHFR were expressed in wild type and ∆rpn4 cells for 4.5 h. The distribution of constitutively expressed Hsp104-GFP was visualized by fluorescence microscopy. Diameter of Hsp104-GFP-bound aggregates were quantified. . Data are displayed as mean ± standard deviations from n = 36 independent biological replicates. Significance was assessed using two-sided, paired Student's t-test. P values are indicated as asterisks ** p ≤ 0.01, **** p ≤ 0.0001. See also Fig. EV4A for quantification of the number of aggregates per cell.


B Hsp104 - GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures . SN , supernatant representing the non - bound fraction . The signals for Ssa1 and Sod1 are shown for control .

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B Hsp104-GFP was purified on nanotrap sepharose in wild type cells that were grown at the indicated temperatures. SN, supernatant representing the non-bound fraction. The signals for Ssa1 and Sod1 are shown for control.


A The mitochondrial proteins Pdb1 , Mam33 and Aim17 were co - expressed for 4 . 5 h with clogger as fusions with the red fluorescent protein RFP ( mCherry ) . Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4 . 5 h after induction of the clogger or cytosolic DHFR for control . For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17 - expressing plasmids and grown to mid - log phase in lactate medium . Clogger expression was induced for 4 . 5 h with 0 . 5 % galactose before microscopy was perfomed . Please note that in the absence of Hsp42 and Hsp104 , no Aim17 - RFP - containing granules are formed . C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N - terminally truncated version of Mam33 - RFP ( both under control of GAL promoter ) together with constitutively expressed Hsp104 - GFP ( TPI promoter ) . The cells were grown on lactate plus 0 . 5 % galactose - containing medium for 4 . 5 h . The distribution of the fluorescent proteins was visualized by microscopy .

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

A The mitochondrial proteins Pdb1, Mam33 and Aim17 were co-expressed for 4.5 h with clogger as fusions with the red fluorescent protein RFP (mCherry). Fluorescence microscopic images of the indicated mitochondrial proteins visualized 4.5 h after induction of the clogger or cytosolic DHFR for control. For quantification of colocalization B Mutants lacking Hsp104 and Hsp42 were transformed with Aim17-expressing plasmids and grown to mid-log phase in lactate medium. Clogger expression was induced for 4.5 h with 0.5 % galactose before microscopy was perfomed. Please note that in the absence of Hsp42 and Hsp104, no Aim17-RFP-containing granules are formed. C Wild type cells were transformed with plasmids for the simultaneous expression of the clogger and an N-terminally truncated version of Mam33-RFP (both under control of GAL promoter) together with constitutively expressed Hsp104-GFP (TPI promoter) . The cells were grown on lactate plus 0.5% galactose-containing medium for 4.5 h. The distribution of the fluorescent proteins was visualized by microscopy.


D Wild type cells expressing Hsp104 - GFP were grown on glucose or lactate medium to log phase . Cells were diluted and visualized by life cell imaging . Please note , that in glucose media , Hsp104 - GFP is initially dispersed in cells and only aggregates when cells become stationary . In contrast , Hsp104 - GFP - containing granules are frequent on lactate medium even at log phase . See movie EV1 for life cell imaging of glucose - grown cells .

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D Wild type cells expressing Hsp104-GFP were grown on glucose or lactate medium to log phase. Cells were diluted and visualized by life cell imaging. Please note, that in glucose media, Hsp104-GFP is initially dispersed in cells and only aggregates when cells become stationary. In contrast, Hsp104-GFP-containing granules are frequent on lactate medium even at log phase. See movie EV1 for life cell imaging of glucose-grown cells.


E Clogger and Mam33 - HA were co - expressed for 4 . 5 h in wild type and ∆ hsp42 cells . Mitochondria were isolated and subjected to Western blotting to detect mature ( m ) and precursor ( pre ) forms of Mam33 - HA . The matrix protein Tim16 served as loading control .

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E Clogger and Mam33-HA were co-expressed for 4.5 h in wild type and ∆hsp42 cells. Mitochondria were isolated and subjected to Western blotting to detect mature (m) and precursor (pre) forms of Mam33-HA. The matrix protein Tim16 served as loading control.


F The mitochondria used for E were treated with proteinase K ( PK ) to remove surface - exposed proteins . Mitochondrial membranes were lysed with NP - 40 and soluble ( S ) and aggregated ( P ) proteins were separated by centrifugation . T , total .

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F The mitochondria used for E were treated with proteinase K (PK) to remove surface-exposed proteins. Mitochondrial membranes were lysed with NP-40 and soluble (S) and aggregated (P) proteins were separated by centrifugation. T, total.


G To measure the toxicity of clogger expression , clogger proteins were induced for 24 h with lactate medium containing 0 . 5 % galactose in the mutants indicated . Aliquots were removed and the number of living cells were assessed by a plating assay on glucose - containing plates . The resistance of ∆ rpn4 to clogger expression depends on Hsp104 and Hsp42 . Data are displayed as mean ± standard deviations from n = 3 independent biological replicates .

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G To measure the toxicity of clogger expression, clogger proteins were induced for 24 h with lactate medium containing 0.5 % galactose in the mutants indicated. Aliquots were removed and the number of living cells were assessed by a plating assay on glucose-containing plates. The resistance of ∆rpn4 to clogger expression depends on Hsp104 and Hsp42. Data are displayed as mean ± standard deviations from n = 3 independent biological replicates.


( B ) Expression of CH25H in heathy donors and COVID - 19 patients . The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors , three moderate COVID - 19 patients and six severe COVID - 19 patients by scRNA - seq analysis ( Liao et al . , 2020 ) .

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(B) Expression of CH25H in heathy donors and COVID-19 patients. The box plot shows the expression of CH25H in macrophages of bronchoalveolar lavage fluids from three healthy donors, three moderate COVID-19 patients and six severe COVID-19 patients by scRNA-seq analysis (Liao et al., 2020).


Characterization of SARS - CoV - 2 pseudovirus . ( C ) SARS - CoV - 2 , SARS - CoV and VSV pseudovirus ( VSVG ) were concentrated by ultracentrifuge . Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS - CoV and SARS - CoV - 2 . VSV Matrix protein served as a loading control for viral particles . Black arrows indicate full length S and cleaved spike ( S2 subunit ) , respectively .

Characterization of SARS-CoV-2 pseudovirus. (C) SARS-CoV-2, SARS-CoV and VSV pseudovirus (VSVG) were concentrated by ultracentrifuge. Incorporation of spike protein on viral membrane was analyzed by western blot using an antibody recognizing S2 subunit of SARS-CoV and SARS-CoV-2. VSV Matrix protein served as a loading control for viral particles. Black arrows indicate full length S and cleaved spike (S2 subunit), respectively.


25HC inhibits membrane fusion mediated by SARS - CoV - 2 S ( F ) 293FT or Vero cells were treated with EtOH or 25HC ( 5 µM ) for 16h prior to transfection with pLVX plasmids encoding SARS - CoV - 2 S ( F ) in the absence of EtOH or 25HC . At 24h post - transfection , syncytium formation was visualized by fluorescence microscopy . White arrows indicate syncytia . Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells .

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

25HC inhibits membrane fusion mediated by SARS-CoV-2 S (F) 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding SARS-CoV-2 S (F) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.


25HC inhibits membrane fusion mediated by , SARS - CoV S ( G , left ) , and MERS - CoV S ( G , right ) . 293FT or Vero cells were treated with EtOH or 25HC ( 5 µM ) for 16h prior to transfection with pLVX plasmids encoding , SARS - CoV S ( G , left ) , and MERS - CoV S ( G , right ) in the absence of EtOH or 25HC . At 24h post - transfection , syncytium formation was visualized by fluorescence microscopy . Scale bar , 50 μm . White arrows indicate syncytia . Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells .

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

25HC inhibits membrane fusion mediated by , SARS-CoV S (G, left), and MERS-CoV S (G, right). 293FT or Vero cells were treated with EtOH or 25HC (5 µM) for 16h prior to transfection with pLVX plasmids encoding , SARS-CoV S (G, left), and MERS-CoV S (G, right) in the absence of EtOH or 25HC. At 24h post-transfection, syncytium formation was visualized by fluorescence microscopy. Scale bar, 50 μm. White arrows indicate syncytia. Quantification of membrane fusion was performed by calculating the percentages of nuclei involved in syncytia formation from all nuclei in GFP positive cells.


( B - C ) 25HC depletes accessible cholesterol on the plasma membrane . ( B ) Calu - 3 cells were treated with indicated concentrations of 25HC for 1h , and subsequently incubated with ALOD4 for 30 min . Cell were washed twice with PBS , lysed , and the amounts of bound ALOD4 were examined by western blotting . ( C ) Calu - 3 cells were treated with 5 µM 25HC for 1h , and subsequently incubated with AF488 labeled ALOD4 for 30 min . Cells were washed twice and examined by fluorescence microscopy .

O O O O O B-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O 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

(B-C) 25HC depletes accessible cholesterol on the plasma membrane. (B) Calu-3 cells were treated with indicated concentrations of 25HC for 1h, and subsequently incubated with ALOD4 for 30 min. Cell were washed twice with PBS, lysed, and the amounts of bound ALOD4 were examined by western blotting. (C) Calu-3 cells were treated with 5 µM 25HC for 1h, and subsequently incubated with AF488 labeled ALOD4 for 30 min. Cells were washed twice and examined by fluorescence microscopy.


( D ) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC - treated cells . Calu - 3 cells were pretreated with ethanol or 5 µM 25HC for 16h . Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h . After further incubation with ALOD4 , cells were washed with PBS twice and lysed . The amount of bound ALOD4 was examined by western blotting .

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

(D) Cholesterol rescues the level of accessible cholesterol on the plasma membrane of 25HC-treated cells. Calu-3 cells were pretreated with ethanol or 5 µM 25HC for 16h. Cells were washed and incubated with PBS or 20 µM cholesterol for 1 h. After further incubation with ALOD4, cells were washed with PBS twice and lysed. The amount of bound ALOD4 was examined by western blotting.


Addition of cholesterol rescues entry of SARS - CoV - 2 pseudovirus , SARS - CoV pseudovirus into 25HC - treated Calu - 3 cells . Cells were pretreated with ethanol or 5 µM 25HC for 16h , washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS - CoV - 2 pseudovirus . Entry of SARS - CoV - 2 pseudovirus ( E and F ) was assessed by luciferase assay or by fluorescence microscopy at 24h post - infection .

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

Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. Entry of SARS-CoV-2 pseudovirus (E and F) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.


Addition of cholesterol rescues entry of SARS - CoV - 2 pseudovirus , SARS - CoV pseudovirus , and MERS - CoV pseudovirus into 25HC - treated Calu - 3 cells . Cells were pretreated with ethanol or 5 µM 25HC for 16h , washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS - CoV - 2 pseudovirus . , SARS - CoV pseudovirus ( G ) , and MERS - CoV pseudovirus ( H ) was assessed by luciferase assay or by fluorescence microscopy at 24h post - infection .

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Addition of cholesterol rescues entry of SARS-CoV-2 pseudovirus, SARS-CoV pseudovirus, and MERS-CoV pseudovirus into 25HC-treated Calu-3 cells. Cells were pretreated with ethanol or 5 µM 25HC for 16h, washed and incubated with PBS or 80 µM cholesterol for 1h prior to infection with SARS-CoV-2 pseudovirus. , SARS-CoV pseudovirus (G), and MERS-CoV pseudovirus (H) was assessed by luciferase assay or by fluorescence microscopy at 24h post-infection.


( C ) SZ58 - 035 rescues surface accessible cholesterol in 25HC - treated cells . Calu - 3 cells were treated with vehicle or 40 µM SZ58 - 035 for 16 h , prior to treatment with ethanol or 2 . 5 µM 25HC for 1h . Cells were then incubated with ALOD4 for 30 min . The amount of bound ALOD4 was examined by western blotting .

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

(C) SZ58-035 rescues surface accessible cholesterol in 25HC-treated cells. Calu-3 cells were treated with vehicle or 40 µM SZ58-035 for 16 h, prior to treatment with ethanol or 2.5 µM 25HC for 1h. Cells were then incubated with ALOD4 for 30 min. The amount of bound ALOD4 was examined by western blotting.


E Reproducibility of 2 independent experiments - correlation of observed changes in expression . Blue line indicates the linear fit with shaded areas as confidence intervals .

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E Reproducibility of 2 independent experiments - correlation of observed changes in expression. Blue line indicates the linear fit with shaded areas as confidence intervals.


A Number of target genes identified using differential expression for each assayed TF . * genes identified as essential for self - renewal of Hoxb8 - FL cells either in ( Basilico et al , 2020 ) .

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A Number of target genes identified using differential expression for each assayed TF. *genes identified as essential for self-renewal of Hoxb8-FL cells either in (Basilico et al, 2020).


A A representation of the pairwise degree of overlap in targets ( size of the circle ) and correlation in gene expression changes ( colour ) among overlapping targets for indicated TF perturbations . Red indicates positive correlations and blue negative correlations .

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A A representation of the pairwise degree of overlap in targets (size of the circle) and correlation in gene expression changes (colour) among overlapping targets for indicated TF perturbations. Red indicates positive correlations and blue negative correlations.


D Identification of target gene modules - groups of genes with common regulatory patterns by TFs . Colour indicates the fold change ( adjusted for significance ) of gene expression following each TF perturbation . Rows ( perturbed TFs ) and columns ( target genes ) are hierarchically clustered . 44 modules for target genes are shown . Below examples . Selected modules with highlighted overall regulation pattern and example genes listed below .

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D Identification of target gene modules - groups of genes with common regulatory patterns by TFs. Colour indicates the fold change (adjusted for significance) of gene expression following each TF perturbation. Rows (perturbed TFs) and columns (target genes) are hierarchically clustered. 44 modules for target genes are shown. Below examples. Selected modules with highlighted overall regulation pattern and example genes listed below.


D Changes in expression for genes co - regulated by separate Cebpa / Meis1 / Spi1 and Hoxb8 perturbations ( FDR < 0 . 1 and | log2 ( FoldChange ) | > 0 . 2 ) . The interaction row indicates changes beyond simple additive effect ( white = additive effect ) . Each gene was annotated with an interaction class ( int . class ) as explained in ( C ) , using the | log2 ( FoldChange ) | > 0 . 2 ) threshold to assign change direction . E Fractions of genes in each interaction class showed in ( D ) .

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D Changes in expression for genes co-regulated by separate Cebpa/Meis1/Spi1 and Hoxb8 perturbations (FDR <0.1 and |log2(FoldChange)| > 0.2). The interaction row indicates changes beyond simple additive effect (white = additive effect). Each gene was annotated with an interaction class (int. class) as explained in (C), using the |log2(FoldChange)| > 0.2) threshold to assign change direction. E Fractions of genes in each interaction class showed in (D).


A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8 - FL cells .

A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8-FL cells.


B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment . Enrichment over a random value and the p - value ( based on the hypergeometric test ) are shown ( bars corresponding to non - significant enrichment values are semi - transparent ) . Analysis was performed separately for genes up - and down - regulated .

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B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment. Enrichment over a random value and the p-value (based on the hypergeometric test) are shown (bars corresponding to non-significant enrichment values are semi-transparent). Analysis was performed separately for genes up- and down-regulated.


C , D Genomic views of relevant TF binding to Cebpa and Gata3 loci ( supporting direct regulatory interactions identified in Figs 3A , B , C ) . The red arrow indicates a putative enhancer element , which is bound by Ebf1 and flanked by AcK27 - rich regions .

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C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.


C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8 - FL cells . Each quadrant corresponds to a set of target genes with a common regulation by Gata3 / Ebf1 - I - inhibition / inhibition , II - activation / inhibition , III - activation / activation , IV - inhibition / activation . For each group the sums of scaled expression values in mouse LK / LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression . For landscape annotation see Fig 7B andE . . Changes in expression for example genes are provided in Appendix Fig 2G - J . Blue line indicates the linear fit with shaded areas as confidence intervals .

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C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8-FL cells. Each quadrant corresponds to a set of target genes with a common regulation by Gata3/Ebf1 - I - inhibition/inhibition, II - activation/inhibition, III - activation/activation, IV - inhibition/activation. For each group the sums of scaled expression values in mouse LK/LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression. For landscape annotation see Fig 7B andE.. Changes in expression for example genes are provided in Appendix Fig 2G-J. Blue line indicates the linear fit with shaded areas as confidence intervals.


E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8 - FL cells and re - expression of Ebf1 in Ebf1 - / - pre - pro B cells ( Li et al , 2018 ) . Blue line indicates the linear fit with shaded areas as confidence intervals .

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E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8-FL cells and re-expression of Ebf1 in Ebf1-/- pre-pro B cells (Li et al, 2018). Blue line indicates the linear fit with shaded areas as confidence intervals.


F , G Expression of the key marker genes ( F ) and Ebf1 , Pax5 , Gata3 , Cebpa factors ( G ) along pseudotime corresponding to the B - cell differentiation trajectory in human foetal liver cells , data from ( Popescu et al , 2019 ) .

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F, G Expression of the key marker genes (F) and Ebf1, Pax5, Gata3, Cebpa factors (G) along pseudotime corresponding to the B-cell differentiation trajectory in human foetal liver cells, data from (Popescu et al, 2019).


B . Still images of GFP ( syncytia ) and Propidium Iodide ( PI ) ( cell death ) at different time - points . Scale bar : 100 µm .

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B. Still images of GFP (syncytia) and Propidium Iodide (PI) (cell death) at different time-points. Scale bar: 100 µm.


C . Quantification of U2OS - ACE2 fusion and death by time - lapse microscopy . Results are mean ± sd from 3 fields per condition .

C. Quantification of U2OS-ACE2 fusion and death by time-lapse microscopy. Results are mean±sd from 3 fields per condition.


D . S staining of infected U2OS - ACE2 cells analyzed by immunofluorescence . The Hoechst dye stains the nuclei . Scale bar : 40 µm .

D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm.


E . Surface S staining of infected U2OS - ACE2 cells analyzed by flow cytometry . Results are representative of at least three independent experiments .

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E. Surface S staining of infected U2OS-ACE2 cells analyzed by flow cytometry. Results are representative of at least three independent experiments.


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . A . TMPRSS2 increases fusion and cell mortality . Right panel : Areas of GFP + cells and PI +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and PI+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . B . IFITM1 , but not IFITM2 and 3 , inhibits SARS - CoV - 2 - induced syncytia formation . Right panels : Area of GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. B. IFITM1, but not IFITM2 and 3, inhibits SARS-CoV-2-induced syncytia formation. Right panels: Area of GFP+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . C . The fusion of IFITM1 + cells with U2OS - ACE2 syncytia is drastically reduced . Right panel : Area of IFITM + GFP +

Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. C. The fusion of IFITM1+ cells with U2OS-ACE2 syncytia is drastically reduced. Right panel: Area of IFITM+ GFP+ cells


Cells were infected at the indicated multiplicity of infection ( MOI ) and analyzed after 20h . D . IFITM1 decreases the number of infected cells . Right panel : Fraction of cells positive for S , normalized to control cells ( transduced with pQCXIP - empty vector ) .

Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. D. IFITM1 decreases the number of infected cells. Right panel: Fraction of cells positive for S, normalized to control cells (transduced with pQCXIP-empty vector ).


B . Representative images of cell - cell fusion . Scale bar : 100 µm .

B. Representative images of cell-cell fusion. Scale bar: 100 µm.


C . Quantification of GFP + areas . Results are mean ± sd from 5 independent experiments .

C. Quantification of GFP+ areas. Results are mean±sd from 5 independent experiments.


D . TMPRSS2 accelerates fusion . Cells were monitored by video microscopy and the GFP area was quantified over time . Left panel : one representative experiment . Results are mean ± sd from 3 fields per condition . Right panel : Mean ± sd from 7 independent experiments ( at 12h post transfection ) .

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D. TMPRSS2 accelerates fusion. Cells were monitored by video microscopy and the GFP area was quantified over time. Left panel: one representative experiment. Results are mean±sd from 3 fields per condition. Right panel: Mean±sd from 7 independent experiments (at 12h post transfection).


E . Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S - expressing 293T cells . One representative out of three independent experiment is shown .

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E. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells. One representative out of three independent experiment is shown.


A . S - expressing cells ( Donor cells ) , ACE2 - expressing cells ( Acceptor cells ) were co - transfected with TMPRSS2 , IFITM or control plasmids . Cell fusion was quantified by measuring the GFP + area after 18h . The indicated combinations were tested . The impact of IFITMs was measured in absence of TMPRSS2 ( left panel ) , in presence of TMPRSS2 in donor ( middle panel ) or acceptor cells ( right panel ) . Data are mean ± sd of 4 independent experiments . Statistical analysis : One - Way ANOVA , ns : non - significant , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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A. S-expressing cells (Donor cells), ACE2-expressing cells (Acceptor cells) were co-transfected with TMPRSS2, IFITM or control plasmids. Cell fusion was quantified by measuring the GFP+ area after 18h. The indicated combinations were tested. The impact of IFITMs was measured in absence of TMPRSS2 (left panel), in presence of TMPRSS2 in donor (middle panel) or acceptor cells (right panel). Data are mean±sd of 4 independent experiments. Statistical analysis: One-Way ANOVA, ns: non-significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.


Impact of TMPRSS2 on IFITMs levels . B . TMPRSS2 does not decrease IFITM amounts measured by flow cytometry . 293T cells were transfected with the indicated IFITM plasmids , with or without TMPRSS2 , and analyzed 18h later .

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Impact of TMPRSS2 on IFITMs levels. B. TMPRSS2 does not decrease IFITM amounts measured by flow cytometry. 293T cells were transfected with the indicated IFITM plasmids, with or without TMPRSS2, and analyzed 18h later.


C . Impact of TMPRSS2 on S and ACE2 , measured by Western Blot . 293T cells were transfected with or without IFITM1 , TMPRSS2 , S or ACE2 plasmids , and analyzed 18h later .

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C. Impact of TMPRSS2 on S and ACE2, measured by Western Blot. 293T cells were transfected with or without IFITM1, TMPRSS2, S or ACE2 plasmids, and analyzed 18h later.


Impact of TMPRSS2 on S levels . D . TMPRSS2 decreases S surface levels , measured by flow cytometry . 293T cells were transfected with S plasmid , with or without TMPRSS2 , and analyzed 18h later . Murine polyclonal anti - S antibodies , one serum from a convalescent COVID - 19 patient ( sera n ° 111 ) , and two monoclonal antibodies ( anti - S1 and anti - S2 ) were tested . Data are representative of three independent experiments .

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Impact of TMPRSS2 on S levels. D. TMPRSS2 decreases S surface levels, measured by flow cytometry. 293T cells were transfected with S plasmid, with or without TMPRSS2, and analyzed 18h later. Murine polyclonal anti-S antibodies, one serum from a convalescent COVID-19 patient (sera n°111), and two monoclonal antibodies (anti-S1 and anti-S2) were tested. Data are representative of three independent experiments.


A . Identification of FASTKD2 interacting proteins by AP - MS and BioID . 26 high confidence interactors detected by both techniques are listed on the right .

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A. Identification of FASTKD2 interacting proteins by AP-MS and BioID. 26 high confidence interactors detected by both techniques are listed on the right.


B . Prey specificity graph for BioID interactome of TRUB2 protein . Prey specificity was calculated as the relative enrichment of interaction of individual preys and TRUB2 , compared to their interaction with 138 other baits ( 42 mitochondrial baits , 96 baits from other cellular compartments ) .

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B. Prey specificity graph for BioID interactome of TRUB2 protein. Prey specificity was calculated as the relative enrichment of interaction of individual preys and TRUB2, compared to their interaction with 138 other baits (42 mitochondrial baits, 96 baits from other cellular compartments).


A . Immunoblot analysis of indicated proteins in control and siRNA treated cells .

A. Immunoblot analysis of indicated proteins in control and siRNA treated cells.


B . BN - PAGE analysis of siRNA - mediated depletion shows an OXPHOS defect as revealed by subunit - specific antibodies against individual OXPHOS complexes . C . Quantification of the levels of individual OXPHOS complexes normalized to Complex II levels . The graph represents the relative abundance of individual complexes in cells treated with the specified siRNA versus controls . The bars represent the mean ± SEM of 3 - 7 independent experiments . P - values were calculated using paired two - tailed t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B. BN-PAGE analysis of siRNA-mediated depletion shows an OXPHOS defect as revealed by subunit-specific antibodies against individual OXPHOS complexes.C. Quantification of the levels of individual OXPHOS complexes normalized to Complex II levels. The graph represents the relative abundance of individual complexes in cells treated with the specified siRNA versus controls. The bars represent the mean ± SEM of 3-7 independent experiments. P-values were calculated using paired two-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).


D , E . Pulse - labeling mitochondrial translation experiment of the 13 mitochondria - encoded polypeptides ( seven subunits of complex I [ ND ] , three subunits of complex IV [ COX ] , two subunits of complex V [ ATP ] and one subunit of complex III [ cyt b ] ) in control and siRNA treated cells . ( E ) Detail of a mitochondrial pulse - labeling experiment showing a severe decrease in the translation of ATP6 and ATP8 in TRUB2 depleted cells .

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D, E. Pulse-labeling mitochondrial translation experiment of the 13 mitochondria-encoded polypeptides (seven subunits of complex I [ND], three subunits of complex IV [COX], two subunits of complex V [ATP] and one subunit of complex III [cyt b]) in control and siRNA treated cells. (E) Detail of a mitochondrial pulse-labeling experiment showing a severe decrease in the translation of ATP6 and ATP8 in TRUB2 depleted cells.


A , B . Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in control cells ( A ) Individual fractions were separated by SDS - PAGE and immunoblotted with the indicated antibodies . The migration of the mt - SSU ( 28S ) , the mt - LSU ( 39S ) and the mitochondrial monosome ( 55S ) are shown .

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A, B. Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in control cells (A) Individual fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) are shown.


A , B . Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in cells treated with siRNA ( B ) . Individual fractions were separated by SDS - PAGE and immunoblotted with the indicated antibodies . The migration of the mt - SSU ( 28S ) , the mt - LSU ( 39S ) and the mitochondrial monosome ( 55S ) are shown .

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A, B. Identification of mitochondrial ribosomal proteins and pseudouridine synthase interacting proteins by sucrose gradient centrifugation in cells treated with siRNA (B). Individual fractions were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) are shown.


C . Quantification of the levels of the mt - SSU ( 28S ) , the mt - LSU ( 39S ) and the mitochondrial monosome ( 55S ) normalized to the SDHA levels . The graph represents the relative abundance of individual subunits in cells treated with specified siRNA versus controls . The bars represent the mean ± SEM .

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C. Quantification of the levels of the mt-SSU (28S), the mt-LSU (39S) and the mitochondrial monosome (55S) normalized to the SDHA levels. The graph represents the relative abundance of individual subunits in cells treated with specified siRNA versus controls. The bars represent the mean ± SEM.


D . 2D - immunoblot analysis ( BN - PAGE / SDS - PAGE ) of pseudouridine synthase interacting proteins . The migration ( sizes in kDa ) of known protein complexes in the first dimension is indicated on the top of the blot .

D. 2D-immunoblot analysis (BN-PAGE/SDS-PAGE) of pseudouridine synthase interacting proteins. The migration (sizes in kDa) of known protein complexes in the first dimension is indicated on the top of the blot.


B . Quantification of Δ ( the difference between the rate of deletions in CMC treated vs un - treated cells ) for individual ψ sites in control and siRNA treated cells . The bars represent the mean ± SEM of two independent experiments . P - values were calculated using a one - tailed t - test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

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B. Quantification of Δ (the difference between the rate of deletions in CMC treated vs un-treated cells) for individual ψ sites in control and siRNA treated cells. The bars represent the mean ± SEM of two independent experiments. P-values were calculated using a one-tailed t-test (* P<0.05; ** P<0.01; ***P<0.001).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for 4 h ( as described in the Materials and Methods ) , and then incubated for 24 h ( left ) or the indicated periods of time ( right ) . Cells were fixed , stained with DAPI to visualize nuclei ( blue ) , and immunolabeled with an anti - LC3 antibody . Primary antibody was detected using an Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( green ) . Left : representative immunofluorescence images of LC3 punctae ; right : quantification of data ( LC3 - punctated cells were counted manually ) . * * * p < 0 . 001 , vs . Mtb - WT - infected condition . Scale bars , 5 µm . (

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for 4 h (as described in the Materials and Methods), and then incubated for 24 h (left) or the indicated periods of time (right). Cells were fixed, stained with DAPI to visualize nuclei (blue), and immunolabeled with an anti-LC3 antibody. Primary antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). Left: representative immunofluorescence images of LC3 punctae; right: quantification of data (LC3-punctated cells were counted manually). ***p<0.001, vs. Mtb-WT-infected condition. Scale bars, 5 µm. (


( B ) BMDMs were infected with Mtb - Δeis in the absence or presence of 3 - methyladenine ( 3 - MA ; 10 mM ) and subjected to confocal analysis as described in Figure 1A . LC3 - punctated cells were counted manually . Each condition was assayed in triplicate , and at least 250 cells were counted in each well . * * * p < 0 . 001 , vs . SC .

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(B) BMDMs were infected with Mtb-Δeis in the absence or presence of 3-methyladenine (3-MA; 10 mM) and subjected to confocal analysis as described in Figure 1A. LC3-punctated cells were counted manually. Each condition was assayed in triplicate, and at least 250 cells were counted in each well. ***p<0.001, vs. SC.


( C ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . Experimental conditions were identical to those outlined in panel A . Gel images representative of three experiments are shown .

(C) Immunoblot analyses performed using Abs raised to LC3 or β-actin. Experimental conditions were identical to those outlined in panel A. Gel images representative of three experiments are shown.


( D ) Electron micrographs of Mtb - Δeis - infected BMDMs under low ( left ) and high ( right ) magnification show the accumulation of autophagic vesicles ( black arrow , initial autophagic vacuoles ; white arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left ) , 0 . 5 µm ( right ) .

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(D) Electron micrographs of Mtb-Δeis-infected BMDMs under low (left) and high (right) magnification show the accumulation of autophagic vesicles (black arrow, initial autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left), 0.5 µm (right).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs were infected with Mtb - Δeis in the presence or absence of 3 - MA ( 10 mM ) or bafilomycin A1 ( Baf - A1 ; 100 nM ) . Gel images representative of three experiments are shown . The ratio of the intensities of the LC3 - II / LC3 - I and β - actin bands is indicated below each lane ( C and E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % distilled water ( B ) , 0 . 1 % DMSO ( E ) ) .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs were infected with Mtb-Δeis in the presence or absence of 3-MA (10 mM) or bafilomycin A1 (Baf-A1; 100 nM). Gel images representative of three experiments are shown. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane (C and E). UI, uninfected; SC, solvent control (0.1% distilled water (B), 0.1% DMSO (E)).


( A and B ) BMDMs were infected with Mtb - WT , Mtb - Δeis or Mtb - c - eis at different MOIs ( 0 . 1 , 1 and 10 ) for 18 h ( A ) or for the indicated periods of time ( B ; MOI = 10 ) . Supernatants were assessed by ELISA for levels of TNF - α and IL - 6 . Data ( A and B ) are presented as the mean ± SD of five experiments .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis or Mtb-c-eis at different MOIs (0.1, 1 and 10) for 18 h (A) or for the indicated periods of time (B; MOI = 10). Supernatants were assessed by ELISA for levels of TNF-α and IL-6. Data (A and B) are presented as the mean±SD of five experiments.


( C and D ) BMDMs were stimulated with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 30 min . Cells were then incubated with 10 µM DHE or 5 µM DCFH - DA for 15 min , washed thoroughly , and immediately analyzed for superoxide or H2O2 production by flow cytometry ( C , Left ) . Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry ( D , top ) . Quantitative analysis of ROS generation ( C , right ; D , bottom ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(C and D) BMDMs were stimulated with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 30 min. Cells were then incubated with 10 µM DHE or 5 µM DCFH-DA for 15 min, washed thoroughly, and immediately analyzed for superoxide or H2O2 production by flow cytometry (C, Left). Cells were labeled with MitoSOX for 30 min and analyzed for mitochondrial ROS levels by flow cytometry (D, top). Quantitative analysis of ROS generation (C, right; D, bottom). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A - C ) BMDMs were infected with Mtb - Δeis ( MOI = 10 ) for 18 h in the presence or absence of DPI ( 10 µM ) , NAC ( 20 mM ) , catalase ( Cat , 1 mU / mL ) , or tiron ( 5 mM ) . ( A ) Representative immunofluorescence images ( top ) ; percentage of endogenous LC3 - punctated cells ( bottom )

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(A-C) BMDMs were infected with Mtb-Δeis (MOI = 10) for 18 h in the presence or absence of DPI (10 µM), NAC (20 mM), catalase (Cat, 1 mU/mL), or tiron (5 mM). (A) Representative immunofluorescence images (top); percentage of endogenous LC3-punctated cells (bottom)


( B ) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β - actin . Gel images are representative of three experiments . The ratio of the intensities of the LC3 - II / LC3 - I and β - actin bands is indicated below each lane .

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(B) Immunoblot analyses of BMDMs with antibodies raised to LC3 or β-actin. Gel images are representative of three experiments. The ratio of the intensities of the LC3-II/LC3-I and β-actin bands is indicated below each lane.


( C ) Experimental conditions were identical to those outlined in Figure 3A . Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA . Data represent the mean ± SD of five experiments .

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(C) Experimental conditions were identical to those outlined in Figure 3A. Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data represent the mean±SD of five experiments.


( D - F ) BMDMs from WT and NOX2 KO mice were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis for 18 h . ( D ) Numbers of LC3 - punctated cells ( counted manually ) are shown ( at least 250 cells were counted in each well ) .

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(D-F) BMDMs from WT and NOX2 KO mice were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis for 18 h. (D) Numbers of LC3-punctated cells (counted manually) are shown (at least 250 cells were counted in each well).


( E ) Immunoblot analyses performed using Abs raised to LC3 or β - actin . BMDMs from WT and NOX2 KO mice were infected with Mtb - Δeis for 18 h . Gel images representative of three experiments are shown .

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(E) Immunoblot analyses performed using Abs raised to LC3 or β-actin. BMDMs from WT and NOX2 KO mice were infected with Mtb-Δeis for 18 h. Gel images representative of three experiments are shown.


( F ) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA . Data are presented as the mean ± SD of at least three separate experiments , each performed in triplicate . * * * p < 0 . 001 , vs . SC ( A and C ) ; WT mice ( D and F ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(F) Supernatants collected 18 h after infection were assessed for cytokine levels by ELISA. Data are presented as the mean±SD of at least three separate experiments, each performed in triplicate. ***p<0.001, vs. SC (A and C); WT mice (D and F). UI, uninfected; SC, solvent control (0.1% DMSO).


( A ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 10 ) for the indicated periods of time , washed to remove unbound mycobacteria , and then incubated in complete DMEM at 37 ° C in 5 % CO2 . Cell viability was assessed by PI staining and then examined by fluorescence microscopy . ( B and C ) Experimental conditions were identical to those outlined in panel A . BMDMs were infected with the three strains of mycobacteria for 36 h .

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(A) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 10) for the indicated periods of time, washed to remove unbound mycobacteria, and then incubated in complete DMEM at 37°C in 5% CO2. Cell viability was assessed by PI staining and then examined by fluorescence microscopy. (B and C) Experimental conditions were identical to those outlined in panel A. BMDMs were infected with the three strains of mycobacteria for 36 h.


B ) Apoptosis was assessed using a TUNEL / apoptosis detection kit , according to the manufacturer ' s protocol . Cells were then examined under a laser - scanning confocal microscope ( LSM 510 ; Zeiss ) . Percentages of TUNEL - positive , PI - positive , and TUNEL - / PI - double - positive cells were calculated . Data are representative of three separate experiments .

B) Apoptosis was assessed using a TUNEL/apoptosis detection kit, according to the manufacturer's protocol. Cells were then examined under a laser-scanning confocal microscope (LSM 510; Zeiss). Percentages of TUNEL-positive, PI-positive, and TUNEL-/PI-double-positive cells were calculated. Data are representative of three separate experiments.


( C ) Morphological changes in BMDMs infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis at MOIs of 1 and 10 . Representative images are shown . Scale bars : 50 µm ( low magnification ) , 20 µm ( high magnification ) .

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(C) Morphological changes in BMDMs infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis at MOIs of 1 and 10. Representative images are shown. Scale bars: 50 µm (low magnification), 20 µm (high magnification).


( D ) Macrophages were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis in the presence or absence of zVAD - fmk ( 20 µM ) or 3 - MA ( 10 mM ) . Staurosporine ( STS ; 500 nM ) was used as a positive control . After 36 h , cells were stained with PI and then examined by fluorescence microscopy . Data are presented as the mean ± SD of at least three separate experiments , each performed in duplicate . * p < 0 . 05 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( A and B ) ; Mtb - Δeis - infected condition without inhibitors ( D ) . UI , uninfected .

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(D) Macrophages were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis in the presence or absence of zVAD-fmk (20 µM) or 3-MA (10 mM). Staurosporine (STS; 500 nM) was used as a positive control. After 36 h, cells were stained with PI and then examined by fluorescence microscopy. Data are presented as the mean±SD of at least three separate experiments, each performed in duplicate. *p<0.05, ***p<0.001, vs. Mtb-WT-infected condition (A and B); Mtb-Δeis-infected condition without inhibitors (D). UI, uninfected.


( A and B ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis ( MOI = 1 ) for the indicated periods of time , and then subjected to Western blot analysis using Abs raised to p - ERK1 / 2 , p - p38 , p - JNK , and β - actin . Data shown are representative of three independent experiments that all yielded similar results ( A ) . Expression of phospho - MAPK / β - actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically ( B ) .

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(A and B) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis (MOI = 1) for the indicated periods of time, and then subjected to Western blot analysis using Abs raised to p-ERK1/2, p-p38, p-JNK, and β-actin. Data shown are representative of three independent experiments that all yielded similar results (A). Expression of phospho-MAPK/β-actin protein in cytoplasmic extracts of BMDMs was quantified densitometrically (B).


( C and D ) BMDMs were pretreated with U0126 ( 5 , 10 , 20 µM ) , SB203580 ( SB ; 1 , 5 , 10 µM ) , or SP600125 ( SP ; 5 , 10 , 20 µM ) for 45 min , and then infected with Mtb - Δeis for 30 min ( C ) or 36 h ( D ) . ( C ) Cells were then incubated with DHE for 15 min , washed rapidly and thoroughly , and analyzed immediately for superoxide levels by flow cytometry . Quantitative DHE fluorescence data represent the mean ± SD of four experiments . ( D ) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy . (

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(C and D) BMDMs were pretreated with U0126 (5, 10, 20 µM), SB203580 (SB; 1, 5, 10 µM), or SP600125 (SP; 5, 10, 20 µM) for 45 min, and then infected with Mtb-Δeis for 30 min (C) or 36 h (D). (C) Cells were then incubated with DHE for 15 min, washed rapidly and thoroughly, and analyzed immediately for superoxide levels by flow cytometry. Quantitative DHE fluorescence data represent the mean±SD of four experiments. (D) Cell death after 36 h was assessed by PI staining and then examined by fluorescence microscopy. (


( E ) Raw264 . 7 cells were transfected with siRNA specific for JNK1 ( siJNK ) or a non - specific control siRNA ( siNS ) . At 24 h after transfection , cells were infected with Mtb - Δeis for 36 h . Cell death was then assessed by PI staining , and then examined by flow cytometry . Transfection efficiency was assessed by RT - PCR ( inset ) . Data represent the mean ± SD of five random fields and are representative of three independent experiments ( D and E ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition ( B ) ; SC ( C and D ) ; siNS ( E ) . UI , uninfected ; SC , solvent control ( 0 . 1 % DMSO ) .

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(E) Raw264.7 cells were transfected with siRNA specific for JNK1 (siJNK) or a non-specific control siRNA (siNS). At 24 h after transfection, cells were infected with Mtb-Δeis for 36 h. Cell death was then assessed by PI staining, and then examined by flow cytometry. Transfection efficiency was assessed by RT-PCR (inset). Data represent the mean±SD of five random fields and are representative of three independent experiments (D and E). *p<0.05, **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition (B); SC (C and D); siNS (E). UI, uninfected; SC, solvent control (0.1% DMSO).


C57BL / 6 mice were challenged , by aerosol , with 10 - 30 CFU Mtb - WT , Mtb - Δeis , or Mtb - c - eis and sacrificed 4 weeks post - infection . ( A ) High - and low - magnification electron micrographs of lung tissue sections from mice infected with Mtb - Δeis show accumulation of autophagic vesicles ( black arrow , bacteria in autophagic vacuoles ; white arrow , degradative autophagic vacuoles ) . Scale bars : 2 µm ( left upper ) , 0 . 5 µm ( right ) . Numbers of autophagic vacuoles per cell in each TEM section ( left lower ) ( mean ± SD ; n = 50 ) .

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C57BL/6 mice were challenged, by aerosol, with 10-30 CFU Mtb-WT, Mtb-Δeis, or Mtb-c-eis and sacrificed 4 weeks post-infection. (A) High- and low-magnification electron micrographs of lung tissue sections from mice infected with Mtb-Δeis show accumulation of autophagic vesicles (black arrow, bacteria in autophagic vacuoles; white arrow, degradative autophagic vacuoles). Scale bars: 2 µm (left upper), 0.5 µm (right). Numbers of autophagic vacuoles per cell in each TEM section (left lower) (mean±SD; n = 50).


( B ) Quantitative RT - PCR analysis of lung tissue from Mtb - WT - , Mtb - Δeis - , and Mtb - c - eis - infected mice . Total RNA was extracted from paraffin - embedded lung tissue sections , as described in the Materials and Methods .

(B) Quantitative RT-PCR analysis of lung tissue from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice. Total RNA was extracted from paraffin-embedded lung tissue sections, as described in the Materials and Methods.


( C ) To assess in vivo cell death , bronchoalveolar lavage fluid cells from Mtb - WT - , Mtb - Δeis - , and Mtb - c - eis - infected mice were subjected to PI staining , and analyzed by flow cytometry . Data are presented as the mean ± SEM ( n = 4 ) .

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(C) To assess in vivo cell death, bronchoalveolar lavage fluid cells from Mtb-WT-, Mtb-Δeis-, and Mtb-c-eis-infected mice were subjected to PI staining, and analyzed by flow cytometry. Data are presented as the mean±SEM (n = 4).


( D ) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb - WT , Mtb - Δeis , or Mtb - c - eis . Data are presented as log10 CFU ± SEM ( n = 4 ) .

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(D) Numbers of CFUs in lung and spleen 4 weeks after infection with Mtb-WT, Mtb-Δeis, or Mtb-c-eis. Data are presented as log10 CFU±SEM (n = 4).


( E ) BMDMs were infected with Mtb - WT , Mtb - Δeis , or Mtb - c - eis and then analyzed by CFU assay . CFU data represent the mean ± SD of four individual experiments . * * p < 0 . 01 , * * * p < 0 . 001 , vs . Mtb - WT - infected condition . UI , uninfected .

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(E) BMDMs were infected with Mtb-WT, Mtb-Δeis, or Mtb-c-eis and then analyzed by CFU assay. CFU data represent the mean±SD of four individual experiments. **p<0.01, ***p<0.001, vs. Mtb-WT-infected condition. UI, uninfected.


( A ) Intracellular superoxide production was analyzed by flow cytometric analysis . BMDMs were infected with Mtb - Δeis ( MOI = 10 ) in the presence or absence of recombinant Eis protein ( rEis ; 5 , 10 , 20 µg / mL ) or 30 kDa Mtb antigen ( 30 k ; 5 , 10 , 20 µg / mL ) . Upper , representative flow cytometric analysis ; lower , quantitation of superoxide generation .

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(A) Intracellular superoxide production was analyzed by flow cytometric analysis. BMDMs were infected with Mtb-Δeis (MOI = 10) in the presence or absence of recombinant Eis protein (rEis; 5, 10, 20 µg/mL) or 30 kDa Mtb antigen (30 k; 5, 10, 20 µg/mL). Upper, representative flow cytometric analysis; lower, quantitation of superoxide generation.


( B ) THP - 1 cells were transfected with mock , Eis - WT , or Eis - ΔAT constructs , and infected with Mtb - Δeis for 30 min . Cells were stained with DHE ( for superoxide ) or DCFH - DA ( for H2O2 ) and subjected to flow cytometric analysis . Inset , transfection efficiency .

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(B) THP-1 cells were transfected with mock, Eis-WT, or Eis-ΔAT constructs, and infected with Mtb-Δeis for 30 min. Cells were stained with DHE (for superoxide) or DCFH-DA (for H2O2) and subjected to flow cytometric analysis. Inset, transfection efficiency.


( C and D ) Experimental conditions were identical to those outlined in panels A and B , respectively . Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF - α and IL - 6 . Data are presented as the mean ± SD of five experiments .

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(C and D) Experimental conditions were identical to those outlined in panels A and B, respectively. Supernatants were collected 18 h after infection and assessed by ELISA for levels of TNF-α and IL-6. Data are presented as the mean±SD of five experiments.