( D ) The C - terminus of EI24 is required for IRE1 binding . HeLa cells transfected with the indicated EI24 mutants were treated as in ( B ) , after which protein - protein interactions were analyzed by immunoprecipitation ( IP ) . The star represents IgG light chains . ( E ) The C - terminus of EI24 binds to IRE1 in a persistent fashion . HeLa cells transfected with the indicated EI24 domains were treated as in ( B ) . Protein - protein interactions were analyzed by IP . The star represents IgG light chains . (

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(D) The C-terminus of EI24 is required for IRE1 binding. HeLa cells transfected with the indicated EI24 mutants were treated as in (B), after which protein-protein interactions were analyzed by immunoprecipitation (IP). The star represents IgG light chains. (E) The C-terminus of EI24 binds to IRE1 in a persistent fashion. HeLa cells transfected with the indicated EI24 domains were treated as in (B). Protein-protein interactions were analyzed by IP. The star represents IgG light chains. (


( I ) EI24 overexpression cannot prevent the activation of the phospho - mimicking ( 3SE ) IRE1 mutant . Wild - type and IRE1 3SE knock - in HeLa cells were transfected with empty vector or Flag - EI24 . Transfected cells were treated as in ( B ) . Protein expressions were analyzed by immunoblotting .

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(I) EI24 overexpression cannot prevent the activation of the phospho-mimicking (3SE) IRE1 mutant. Wild-type and IRE1 3SE knock-in HeLa cells were transfected with empty vector or Flag-EI24. Transfected cells were treated as in (B). Protein expressions were analyzed by immunoblotting.


( L ) Expression of full - length EI24 or the △ TM34 or △ C mutant in EI24 knockout HeLa cells for comparison of their functions . HeLa cells transfected with the indicated EI24 mutants were treated as in ( B ) . Protein expressions were analyzed by immunoblotting .

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(L) Expression of full-length EI24 or the △TM34 or △C mutant in EI24 knockout HeLa cells for comparison of their functions. HeLa cells transfected with the indicated EI24 mutants were treated as in (B). Protein expressions were analyzed by immunoblotting.


( B , C ) EI24 knockout causes resting - state ER calcium reduction ( B ) and cytosolic calcium elevation ( C ) under ER stress . Control ( sgGFP ) and EI24 knockout ( sgEI24 ) HeLa cells were first perfused in TM - free buffer , and switched to the buffer containing 4 μM TM to induce ER stress where indicated . Resting state calcium levels in the ER ( B ) and cytosol ( C ) with or without TM are displayed in columns . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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(B, C) EI24 knockout causes resting-state ER calcium reduction (B) and cytosolic calcium elevation (C) under ER stress. Control (sgGFP) and EI24 knockout (sgEI24) HeLa cells were first perfused in TM-free buffer, and switched to the buffer containing 4 μM TM to induce ER stress where indicated. Resting state calcium levels in the ER (B) and cytosol (C) with or without TM are displayed in columns. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( G ) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol . The measurements were similarly performed as in ( E ) , and were from the same batch of cells . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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(G) Knockdown of IP3R1 prevents calcium mobilization from the ER to the cytosol. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( I ) Wild - type EI24 , but not the TM34 - or C - terminus - deleted mutant , preserves ER calcium stores in EI24 knockout cells . The measurements were similarly performed as in ( E ) , and were from the same batch of cells . Data information : N = 20 cells for one replicate . Data are mean ± SEM ( n = 3 technical replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test .

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(I) Wild-type EI24, but not the TM34- or C-terminus-deleted mutant, preserves ER calcium stores in EI24 knockout cells. The measurements were similarly performed as in (E), and were from the same batch of cells. Data information: N = 20 cells for one replicate. Data are mean ± SEM (n = 3 technical replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test.


( B ) Knockdown of IP3R1 prevents ER stress - induced apoptosis in EI24 knockout cells . Cells were cultured with or without 4 μM TM for 8 hr . Percentages of early apoptotic cells were measured by flow cytometry . Data information : N ≈ 10000 cells for one biological replicate . Data are mean ± SEM ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t -

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(B) Knockdown of IP3R1 prevents ER stress-induced apoptosis in EI24 knockout cells. Cells were cultured with or without 4 μM TM for 8 hr. Percentages of early apoptotic cells were measured by flow cytometry. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test


( D ) Knockdown of IP3R1 prevents ER stress - induced apoptosis . Cells were treated with 4 μM TM for the indicated times . Protein expressions were analyzed by immunoblotting .

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(D) Knockdown of IP3R1 prevents ER stress-induced apoptosis. Cells were treated with 4 μM TM for the indicated times. Protein expressions were analyzed by immunoblotting.


( F - H ) Reintroduction of full - length EI24 , but not the TM34 - or C - terminus - deleted mutant ( F , G ) or the C - terminus alone ( H ) , inhibited ER stress - induced apoptosis in EI24 knockout cells . Cells treated with or without TM were analyzed for apoptosis by flow cytometry ( F , H ) or by immunoblotting ( G ) . Arrows in ( H ) denote decreased or increased apoptotic levels . Data information : N ≈ 10000 cells for one biological replicate . Data are mean ± SEM ( n = 3 or 4 biologically independent replicates ) . ns , no significant difference ; * * * P < 0 . 001 ; two - tailed Student ' s t - test for F ) , one - tailed Student ' s t - test for ( H ) .

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(F-H) Reintroduction of full-length EI24, but not the TM34- or C-terminus-deleted mutant (F, G) or the C-terminus alone (H), inhibited ER stress-induced apoptosis in EI24 knockout cells. Cells treated with or without TM were analyzed for apoptosis by flow cytometry (F, H) or by immunoblotting (G). Arrows in (H) denote decreased or increased apoptotic levels. Data information: N ≈ 10000 cells for one biological replicate. Data are mean ± SEM (n = 3 or 4 biologically independent replicates). ns, no significant difference; ***P < 0.001; two-tailed Student's t-test for F), one-tailed Student's t-test for (H).


B t - SNE plots showing integrated analysis of Prrx1 - Cre - ( 8 - week - old mice , n = 3 males ) and Lepr - Cre - traced ( 8 - week - old mice , n = 4 males ) cells . Cells were colored by clusters ( left ) or samples ( right ) . Top 20 PCs were chosen for the clustering .

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B t-SNE plots showing integrated analysis of Prrx1-Cre- (8-week-old mice, n=3 males) and Lepr-Cre-traced (8-week-old mice, n=4 males) cells. Cells were colored by clusters (left) or samples (right). Top 20 PCs were chosen for the clustering.


D Violin plots showing the expression of feature genes for each cluster .

D Violin plots showing the expression of feature genes for each cluster.


E In situ hybridization of Col3a1 on femur sections . Col3a1 was expressed in the perichondral ( a ) and periosteal ( b ) regions , but not in the bone marrow . B : Bone ; BM : Bone marrow ; Peri : Periosteum ; Endo : Endosteum . Scale bars were 100 µm . Arrowheads indicated the periosteum .

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E In situ hybridization of Col3a1 on femur sections. Col3a1 was expressed in the perichondral (a) and periosteal (b) regions, but not in the bone marrow. B: Bone; BM: Bone marrow; Peri: Periosteum; Endo: Endosteum. Scale bars were 100 µm. Arrowheads indicated the periosteum.


E Violin plots showing the expression of feature genes for each cluster .

E Violin plots showing the expression of feature genes for each cluster.


F Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Arrowheads indicated tdTomato + periosteal cells . Two representative periosteal regions were magnified ( a and b ) . GP : Growth plate . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm . G Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice 7 days after fracture . Aggrecan ( green ) , tdTomato ( red ) , DAPI ( blue ) . Arrowheads indicated tdTomato + periosteal cells . Two representative regions were magnified ( a and b ) . Scale bars were 100 µm .

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F Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Arrowheads indicated tdTomato+ periosteal cells. Two representative periosteal regions were magnified (a and b). GP: Growth plate. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm. G Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice 7 days after fracture. Aggrecan (green), tdTomato (red), DAPI (blue). Arrowheads indicated tdTomato+ periosteal cells. Two representative regions were magnified (a and b). Scale bars were 100 µm.


B Dot plots showing the gene expression patterns of selected genes in each cluster .

B Dot plots showing the gene expression patterns of selected genes in each cluster.


D Flow cytometry analysis of BrdU incorporation in uncultured Lepr - Cre + Notch3 + / - BMSCs ( n = 4 independent experiments ) . Eight - week - old Lepr - Cre ; tdTomato mice were given a single intraperitoneal injection of BrdU ( 100 mg / kg body mass ) and maintained on 0 . 5 mg / ml of BrdU in the drinking water for 14 days .

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D Flow cytometry analysis of BrdU incorporation in uncultured Lepr-Cre+Notch3+/- BMSCs (n=4 independent experiments). Eight-week-old Lepr-Cre; tdTomato mice were given a single intraperitoneal injection of BrdU (100 mg/kg body mass) and maintained on 0.5 mg/ml of BrdU in the drinking water for 14 days.


E Immunofluorescent images of the distal femur in 8 - week - old Lepr - Cre ; tdTomato mice . Notch3 ( green ) , tdTomato ( red ) , CD31 ( gray ) , DAPI ( blue ) . Sinusoid ( a ) , arteriole ( b ) , artery ( c ) and avascular bone marrow ( BM , d ) regions were shown . Arrowheads indicated Notch3 + tdTomato + adventitial cells closely associated with ECs . Arrows indicated Notch3 - tdTomato + reticular cells in the bone marrow that were not associated with ECs . Scale bars were 10 µm . F Localization quantification of Lepr - Cre + Notch3 + cells relative to bone marrow ECs ( n = 5 biological replicates from three independent experiments ) . Cells within 5 µm diameter of ECs were considered adjacent .

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E Immunofluorescent images of the distal femur in 8-week-old Lepr-Cre; tdTomato mice. Notch3 (green), tdTomato (red), CD31 (gray), DAPI (blue). Sinusoid (a), arteriole (b), artery (c) and avascular bone marrow (BM, d) regions were shown. Arrowheads indicated Notch3+ tdTomato+ adventitial cells closely associated with ECs. Arrows indicated Notch3- tdTomato+ reticular cells in the bone marrow that were not associated with ECs. Scale bars were 10 µm. F Localization quantification of Lepr-Cre+Notch3+ cells relative to bone marrow ECs (n=5 biological replicates from three independent experiments). Cells within 5 µm diameter of ECs were considered adjacent. G


H Volcano plot showing DEGs in the bulk RNA - seq analysis ( n = 3 biological replicates ) . Green dots represent down - regulated genes . Red dots represent up - regulated genes .

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H Volcano plot showing DEGs in the bulk RNA-seq analysis (n=3 biological replicates). Green dots represent down-regulated genes. Red dots represent up-regulated genes.


O Heatmap showing the average expression levels ( column - wide Z score ) of secreted factors with clustering .

O Heatmap showing the average expression levels (column-wide Z score) of secreted factors with clustering.


C Heatmap showing pseudotime - dependent gene expression . Bars on the top were colored as in ( A ) . Representative genes were highlighted on both sides .

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C Heatmap showing pseudotime-dependent gene expression. Bars on the top were colored as in (A). Representative genes were highlighted on both sides.


F Representative images of adipogenic , osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr - Cre + BMSCs . Scale bars were 100 µm .

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F Representative images of adipogenic, osteogenic and chondrogenic differentiation following shRNA knockdown in Lepr-Cre+ BMSCs. Scale bars were 100 µm.


G qPCR analysis of the expression levels of Pparg , Bglap and Acan in control or knockdown group ( n = 3 independent experiments ) . The statistical significance of differences was analyzed by one - way ANOVAs with Tukey ' s multiple comparison tests . Data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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G qPCR analysis of the expression levels of Pparg, Bglap and Acan in control or knockdown group (n=3 independent experiments). The statistical significance of differences was analyzed by one-way ANOVAs with Tukey's multiple comparison tests. Data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


B Violin plots showing the expression of feature genes for each cluster .

B Violin plots showing the expression of feature genes for each cluster.


F Immunostaining of Sca - 1 on femur sections of 8 - week - old Lepr - Cre ; tdTomato mice . Arrows indicated Sca - 1 + tdTomato + cells inside cortical bones of the femur diaphysis . Arrowheads indicated Sca - 1 + tdTomato + cells in the femur periosteum . B : Bone . BM : Bone marrow . Peri : Periosteum . Scale bars were 100 µm .

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F Immunostaining of Sca-1 on femur sections of 8-week-old Lepr-Cre; tdTomato mice. Arrows indicated Sca-1+ tdTomato+ cells inside cortical bones of the femur diaphysis. Arrowheads indicated Sca-1+ tdTomato+ cells in the femur periosteum. B: Bone. BM: Bone marrow. Peri: Periosteum. Scale bars were 100 µm.


G Representative flow cytometry plots of long bone periosteum in 8 - week - old Lepr - Cre ; tdTomato mice . The percentage of DAPI - CD45 - Ter119 - Tie2 - tdTomato + Sca - 1 + / - cells were shown .

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G Representative flow cytometry plots of long bone periosteum in 8-week-old Lepr-Cre; tdTomato mice. The percentage of DAPI- CD45- Ter119- Tie2- tdTomato+ Sca-1+/- cells were shown.


I , J The average number of CFU - F colonies formed ( I ) and the average cell number per colony ( J ) were shown ( n = 4 independent experiments ) . The statistical significance of differences was assessed using one - way ANOVAs with Tukey ' s multiple comparison tests . All data represented Mean ± SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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I, J The average number of CFU-F colonies formed (I) and the average cell number per colony (J) were shown (n=4 independent experiments). The statistical significance of differences was assessed using one-way ANOVAs with Tukey's multiple comparison tests. All data represented Mean ± SD. *p<0.05, **p<0.01, ***p<0.001.


A , B Representative oil red staining after 7 days of adipogenic differentiation in Lepr - Cre + subsets ( A ) and qPCR analysis of Adipoq and Pparg expression ( B ) ( n = 3 independent experiments ) . Scale bars were 100 µm .

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A, B Representative oil red staining after 7 days of adipogenic differentiation in Lepr-Cre+ subsets (A) and qPCR analysis of Adipoq and Pparg expression (B) (n=3 independent experiments). Scale bars were 100 µm.


C , D In vitro chondrogenic differentiation of sorted periosteal cells . Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection ( C ) . Scale bars were 100 µm . Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression ( D ) ( n = 3 independent experiments ) .

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C, D In vitro chondrogenic differentiation of sorted periosteal cells. Representative alcian blue staining after chondrogenic differentiation for 21 days and cryosection (C). Scale bars were 100 µm. Chondrogenic efficiencies were quantified by qPCR analysis of Acan and Col2a1 expression (D) (n=3 independent experiments).


E , F In vitro osteogenic differentiation of sorted periosteal cells . Representative alizarin red staining after osteogenic differentiation for 14 days . Scale bars were 200 µm . Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression ( F ) ( n = 3 independent experiments ) .

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E, F In vitro osteogenic differentiation of sorted periosteal cells. Representative alizarin red staining after osteogenic differentiation for 14 days. Scale bars were 200 µm. Osteogenic efficiencies were quantified by qPCR analysis of Sp7 and Bglap expression (F) (n=3 independent experiments).


D ) SDS - PAGE analysis of purified tubulin from brains of all mouse models used in the current study , stained with Coomassie Brilliant - Blue . Alpha - and β - tubulin are separated using a specific SDS - PAGE

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D) SDS-PAGE analysis of purified tubulin from brains of all mouse models used in the current study, stained with Coomassie Brilliant-Blue. Alpha- and β-tubulin are separated using a specific SDS-PAGE protocol


E ) Immunoblot analysis of all purified tubulin variants . The molecular weight in ( D ) and ( E ) is indicated in kDa .

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E) Immunoblot analysis of all purified tubulin variants. The molecular weight in (D) and (E) is indicated in kDa.


B ) Representative IRM and TIRF microscopy images showing microtubule positions and Tau - meGFP intensities for different PTM variants ( grey : wild type ; blue : Ttll1 - / - ; yellow : Ttll7 - / - ; green : Ttll1 - / - Ttll7 - / - ; pink : Atat1 ‑ / ‑ ) . In the last column , false - coloured TIRF images highlight differences of Tau - meGFP intensities on different types of microtubules ( Movie EV1 ) . C ) Quantification of integrated intensity of Tau - meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples . Single assays ( Fig EV2A ) of one experiment were combined after normalisation to the wild - type microtubule values ( A . U . = arbitrary units ) . Each data point represents the normalised fluorescence intensity value of one microtubule . D ) Summary quantification the three independent experiments shown in ( C ) . Each point is a normalised mean of the triplicates , error bars represent standard deviation .

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B) Representative IRM and TIRF microscopy images showing microtubule positions and Tau-meGFP intensities for different PTM variants (grey: wild type; blue: Ttll1-/-; yellow: Ttll7-/-; green: Ttll1-/-Ttll7-/-; pink: Atat1‑/‑). In the last column, false-coloured TIRF images highlight differences of Tau-meGFP intensities on different types of microtubules (Movie EV1). C) Quantification of integrated intensity of Tau-meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples. Single assays (Fig EV2A) of one experiment were combined after normalisation to the wild-type microtubule values (A.U. = arbitrary units). Each data point represents the normalised fluorescence intensity value of one microtubule. D) Summary quantification the three independent experiments shown in (C). Each point is a normalised mean of the triplicates, error bars represent standard deviation.


E ) Saturation curve of increasing concentration Tau - mCherry binding to GMPCPP wild - type and Ttll1 - / - Ttll7 - / - microtubules . Each data point is shown as mean ± standard deviation of the raw integrated intensity values ( A . U . × 104 ) of three experiments with independently purified tubulin samples ( Fig EV2D ) . Dissociation constants Kd and R2 coefficient from non - linear fits of each microtubule type are indicated below the plot .

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E) Saturation curve of increasing concentration Tau-mCherry binding to GMPCPP wild-type and Ttll1-/-Ttll7-/- microtubules. Each data point is shown as mean ± standard deviation of the raw integrated intensity values (A.U. ×104) of three experiments with independently purified tubulin samples (Fig EV2D). Dissociation constants Kd and R2 coefficient from non-linear fits of each microtubule type are indicated below the plot.


C ) Decay curves of normalised microtubule length plotted against time upon incubation with katanin ( A . U . = arbitrary units ) for single microtubules highlighted by arrow heads with yellow borders in ( B ) . D ) Half - life times ( seconds ) of all microtubules from the assays of which representative images are displayed in ( B ) and representative decay curves are shown in ( C ) . Each data point represents half - life of a single microtubule . Medians with interquartile ranges shown . Mann - Whitney test , p - values displayed .

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C) Decay curves of normalised microtubule length plotted against time upon incubation with katanin (A.U. = arbitrary units) for single microtubules highlighted by arrow heads with yellow borders in (B). D) Half-life times (seconds) of all microtubules from the assays of which representative images are displayed in (B) and representative decay curves are shown in (C). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown. Mann-Whitney test, p-values displayed.


E ) Analysis of normalised microtubule half - lifetimes from 14 independent experiments ( Fig EV3 ) . Each data point represents half - life of a single microtubule . Medians with interquartile ranges shown , Mann - Whitney test , p - values displayed .

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E) Analysis of normalised microtubule half-lifetimes from 14 independent experiments (Fig EV3). Each data point represents half-life of a single microtubule. Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.


B ) Kymographs showing single Kif5B - EGFP motor molecules moving on one single microtubule of known PTM state ( Movie EV4 ) . Horizontal scale bars 2 µm ; vertical scale bars 5 C ) Scatter plots showing the average velocities ( µm / s ) exhibited by single Kif5b - EGFP molecules on the different microtubule types ( each data point represents one Kif5b - EGFP molecule ) . Data pooled from 29 independent experiments ( Appendix Fig S2 ) . Medians with interquartile ranges shown , Mann - Whitney test , p - values displayed .

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B) Kymographs showing single Kif5B-EGFP motor molecules moving on one single microtubule of known PTM state (Movie EV4). Horizontal scale bars 2 µm; vertical scale bars 5 C) Scatter plots showing the average velocities (µm/s) exhibited by single Kif5b-EGFP molecules on the different microtubule types (each data point represents one Kif5b-EGFP molecule). Data pooled from 29 independent experiments (Appendix Fig S2). Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.


D , E ) Survival probability ( Kaplan - Meier plots , taking varying microtubule lengths into account ; see Methods ) of the interaction time ( D ) and run length ( E ) of Kif5b - EGFP molecules on different microtubule types . Data in ( C , D , E ) were pooled from individual experiments ( Appendix Fig S2 ) without normalisation . The 50 % probability values are show in the plots . Statistical analysis displayed in the tables below : hazard ratios with 95 % confidence intervals pairing all data from different microtubule types ( * pairs differing with significance level p < 0 . 05 ) .

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D,E) Survival probability (Kaplan-Meier plots, taking varying microtubule lengths into account; see Methods) of the interaction time (D) and run length (E) of Kif5b-EGFP molecules on different microtubule types. Data in (C,D,E) were pooled from individual experiments (Appendix Fig S2) without normalisation. The 50% probability values are show in the plots. Statistical analysis displayed in the tables below: hazard ratios with 95% confidence intervals pairing all data from different microtubule types (*pairs differing with significance level p<0.05).


( B ) Phenotyping of the same mice , presented in the same order , inoculated i . n . with 0 . 3 × 105 TCID50 of SARS - CoV - 2 at the age of 5 - 7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E - specific qRT - PCR . Red lines indicate the qRT - PCR limits of detection .

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(B) Phenotyping of the same mice, presented in the same order, inoculated i.n. with 0.3 × 105 TCID50 of SARS-CoV-2 at the age of 5-7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection.


( C , D ) Quantification ( C ) and images ( D ) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B , presented in the same order .

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(C, D) Quantification (C) and images (D) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B, presented in the same order.


( B - E ) Examples of lesions observed in the lungs of infected mice at higher magnification . The two top panels depict mild ( B ) ( scale bar : 100 μm ) and mild to moderate ( C ) interstitium thickening accompanied by dense inflammatory infiltrates predominantly localized in the vicinity of bronchioles ( yellow stars ) and also present around blood vessels ( green stars ) ( scale bar : 200 μm ) . ( D ) Alveoli filled with a proteinaceous exudate containing a few cells ( green arrows ) ( scale bar : 50 μm ) . ( E ) Discrete degenerative lesions of the bronchiolar epithelium , such as perinuclear clear spaces ( blue arrows ) , hyper - eosinophilic cells with condensed nuclei ( orange arrow ) and some intraluminal fibrinous and cell debris ( black arrow ) in an overall well - preserved epithelium ( scale bar : 20 μm ) .

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(B-E) Examples of lesions observed in the lungs of infected mice at higher magnification. The two top panels depict mild (B) (scale bar: 100 μm) and mild to moderate (C) interstitium thickening accompanied by dense inflammatory infiltrates predominantly localized in the vicinity of bronchioles (yellow stars) and also present around blood vessels (green stars) (scale bar: 200 μm). (D) Alveoli filled with a proteinaceous exudate containing a few cells (green arrows) (scale bar: 50 μm). (E) Discrete degenerative lesions of the bronchiolar epithelium, such as perinuclear clear spaces (blue arrows), hyper-eosinophilic cells with condensed nuclei (orange arrow) and some intraluminal fibrinous and cell debris (black arrow) in an overall well-preserved epithelium (scale bar: 20 μm).


( G ) Representative NCoV - 2 - specific IHC image of the brain in NI control or SARS - CoV - 2 - infected mice at 3 dpi . Scale bar : 500 μm . ( H ) Closer view of a NCoV - 2 positive area from an infected mouse . Scale bar : 500 μm .

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(G) Representative NCoV-2-specific IHC image of the brain in NI control or SARS-CoV-2-infected mice at 3 dpi. Scale bar: 500 μm. (H) Closer view of a NCoV-2 positive area from an infected mouse. Scale bar: 500 μm.


( A ) Comparative permissiveness of various organs from B6 . K18 - hACE2IP - THV and B6 . K18 - ACE22prlmn / Jax transgenic mice ( n = 5 - 6 / group ) to SARS - CoV - 2 replication , as determined at 3 dpi by conventional E - specific or sub - genomic Esg - specific qRT - PCR . Red lines indicate the qRT - PCR limits of detection . ( B ) Comparative quantitation of hACE - 2 mRNA in the lungs and brain of B6 . K18 - hACE2IP - THV and B6 . K18 - ACE22prlmn / Jax transgenic mice ( n = 5 - 6 / group ) . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , ns = not significant ) .

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(A) Comparative permissiveness of various organs from B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group) to SARS-CoV-2 replication, as determined at 3 dpi by conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection. (B) Comparative quantitation of hACE-2 mRNA in the lungs and brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group). Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ns = not significant).


( C ) Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression in the lungs or brain of B6 . K18 - hACE2IP - THV and B6 . K18 - ACE22prlmn / Jax mice at 3 dpi ( n = 5 - 6 / group ) . Data were normalized versus untreated controls .

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(C) Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression in the lungs or brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax mice at 3 dpi (n = 5-6/group). Data were normalized versus untreated controls.


( C ) Viral RNA content as determined in various organs at 3 dpi ( n = 6 / group ) by use of conventional E - specific or sub - genomic Esg - specific qRT - PCR . Red lines indicate the qRT - PCR detection limits . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(C) Viral RNA content as determined in various organs at 3 dpi (n = 6/group) by use of conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR detection limits. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).


( D ) Percentages of NK cells or neutrophils in the lungs of LV : : S - or sham - vaccinated and SARS - CoV - 2 - challenged B6 . K18 - hACE2IP - THV transgenic mice at 3 dpi ( n = 6 / group ) . Percentages were calculated versus total lung live CD45 + cells . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(D) Percentages of NK cells or neutrophils in the lungs of LV::S- or sham-vaccinated and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Percentages were calculated versus total lung live CD45+ cells. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).


( E ) Relative log2 fold change in cytokine and chemokine mRNA expression in the brain of LV : : S - or sham - immunized and SARS - CoV - 2 - challenged B6 . K18 - hACE2IP - THV transgenic mice at 3 dpi ( n = 6 / group ) . Means ± SD are shown . Data were normalized versus untreated controls . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(E) Relative log2 fold change in cytokine and chemokine mRNA expression in the brain of LV::S- or sham-immunized and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Means ± SD are shown. Data were normalized versus untreated controls. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).


B6 . K18 - hACE2IP - THV mice were primed ( i . m . ) at wk 0 and boosted ( i . n . ) at wk 5 ( n = 5 ) with non - integrative LV : : S . Control mice were injected with an empty LV ( sham ) . ( B , C ) Cytometric strategy to detect lung CD8 + T central memory ( Tcm , CD44 + CD62L + CD69 - ) , T effector memory ( Tem , CD44 + CD62L - CD69 - ) and T resident memory ( Trm , CD44 + CD62L - CD69 + CD103 + ) and ( C ) percentages of these subsets among CD8 + T - cells in LV : : S - vaccinated ( n = 9 ) or sham ( n = 5 ) mice . Data information : Statistical significance of the difference between the two groups was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham). (B, C) Cytometric strategy to detect lung CD8+ T central memory (Tcm, CD44+CD62L+CD69-), T effector memory (Tem, CD44+CD62L-CD69-) and T resident memory (Trm, CD44+CD62L-CD69+CD103+) and (C) percentages of these subsets among CD8+ T-cells in LV::S-vaccinated (n = 9) or sham (n = 5) mice. Data information: Statistical significance of the difference between the two groups was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01).


B6 . K18 - hACE2IP - THV mice were primed ( i . m . ) at wk 0 and boosted ( i . n . ) at wk 5 ( n = 5 ) with non - integrative LV : : S . Control mice were injected with an empty LV ( sham ) . ( D ) Cytometric strategy to detect SCoV - 2 - specific CD8 + T cells by use of the H - 2Db - SCoV - 2 : 538 - 546 dextramer in the lungs of LV : : S or sham - vaccinated mice . Inside CD8 + dextramer + T - cell subset , Tcm , Tem and Trm have been distinguished .

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B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham). (D) Cytometric strategy to detect SCoV-2-specific CD8+ T cells by use of the H-2Db-SCoV-2:538-546 dextramer in the lungs of LV::S or sham-vaccinated mice. Inside CD8+ dextramer+ T-cell subset, Tcm, Tem and Trm have been distinguished.


B6 . K18 - hACE2IP - THV mice were primed ( i . m . ) at wk 0 and boosted ( i . n . ) at wk 5 ( n = 5 ) with non - integrative LV : : S . Control mice were injected with an empty LV ( sham ) . ( F , G ) Anti - SCoV - 2 IgG or IgA titers ( F ) and neutralizing activity ( EC50 ) ( G ) in the sera or lung homogenates at 3 dpi . Samples from individual mice ( n = 4 / group ) were studied . Data information : Statistical significance of the difference between the two groups was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham). (F, G) Anti-SCoV-2 IgG or IgA titers (F) and neutralizing activity (EC50) (G) in the sera or lung homogenates at 3 dpi. Samples from individual mice (n = 4/group) were studied. Data information: Statistical significance of the difference between the two groups was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01).


( A ) B6 . K18 - hACE2IP - THV mice were immunized with LV : : S via i . m . or i . n . at wk 0 and boosted via i . m . or i . n . at wk 5 ( n = 5 / group ) with non - integrative LV : : S . Control mice were injected i . m . i . n . with an empty LV ( sham ) . Viral RNA contents were determined by conventional E - specific RT - PCR at 3 dpi , in the brain , lung and nasal washes . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 , ns = not significant ) .

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(A) B6.K18-hACE2IP-THV mice were immunized with LV::S via i.m. or i.n. at wk 0 and boosted via i.m. or i.n. at wk 5 (n = 5/group) with non-integrative LV::S. Control mice were injected i.m. i.n. with an empty LV (sham). Viral RNA contents were determined by conventional E-specific RT-PCR at 3 dpi, in the brain, lung and nasal washes. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ****= p < 0.0001, ns = not significant).


( B ) Lung histology in B6 . K18 - hACE2IP - THV mice , vaccinated with LV : : S after SARS - CoV - 2 inoculation . H & E ( rows 1 and 3 ) ( scale bar : 500 μm ) and NCoV - 2 - specific IHC ( rows 2 and 4 ) ( scale bar : 50 μm ) staining of whole lung sections ( scale bar : 50 μm ) from the primed ( i . m . ) , boosted ( i . n . ) and challenged B6 . K18 - hACE2IP - THV mice compared with their sham controls . H & E and NCoV - 2 - specific IHC were performed on contiguous sections . The IHC fields correspond to the rectangles in the corresponding H & E images above them . A representative NCoV - 2 - specific IHC on a lung section from a non - infected ( NI ) mouse is also shown .

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(B) Lung histology in B6.K18-hACE2IP-THV mice, vaccinated with LV::S after SARS-CoV-2 inoculation. H&E (rows 1 and 3) (scale bar: 500 μm) and NCoV-2-specific IHC (rows 2 and 4) (scale bar: 50 μm) staining of whole lung sections (scale bar: 50 μm) from the primed (i.m.), boosted (i.n.) and challenged B6.K18-hACE2IP-THV mice compared with their sham controls. H&E and NCoV-2-specific IHC were performed on contiguous sections. The IHC fields correspond to the rectangles in the corresponding H&E images above them. A representative NCoV-2-specific IHC on a lung section from a non-infected (NI) mouse is also shown.


( A ) Example of CD3 - positive cells in an olfactory bulb from an LV : : S i . m . - i . n . vaccinated and protected mice and representative results from this group versus sham - vaccinated and unprotected mice at 3 dpi ( n = 7 - 9 / group ) . Scale bar : 50 μm . Statistical significance was evaluated by Mann - Whitney test ( * * = p < 0 . 01 ) .

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(A) Example of CD3-positive cells in an olfactory bulb from an LV::S i.m.-i.n. vaccinated and protected mice and representative results from this group versus sham-vaccinated and unprotected mice at 3 dpi (n = 7-9/group). Scale bar: 50 μm. Statistical significance was evaluated by Mann-Whitney test (**= p <0.01).


Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups .

Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups.


Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups . ( C , D ) Innate immune cells in the olfactory bulbs ( C ) or brain ( D ) . The CD11b + Ly6C + Ly6G + population in the olfactory bulbs are neutrophils and the CD11b + Ly6C + Ly6G - cells of the brain are inflammatory monocytes .

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Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups. (C, D) Innate immune cells in the olfactory bulbs (C) or brain (D). The CD11b+ Ly6C+ Ly6G+ population in the olfactory bulbs are neutrophils and the CD11b+ Ly6C+ Ly6G- cells of the brain are inflammatory monocytes.


( E ) Brain H & E histology at 3 dpi . The top right and both bottom panels show examples , in two different mice , of leukocyte clusters ( arrows ) alongside the ventricular wall . No such clusters were detected in the LV : : S i . m . - i . n . vaccinated mice ( top left panel ) . Scale bar : 200 μm . The close up view ( bottom right panel ) highlights the thickened , disorganized ependymal lining , compared to the normal ependymal cells and cilia of an LV : : S i . m . - i . n . vaccinated mouse ( top left panel ) . Scale bar : 50 μm .

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(E) Brain H&E histology at 3 dpi. The top right and both bottom panels show examples, in two different mice, of leukocyte clusters (arrows) alongside the ventricular wall. No such clusters were detected in the LV::S i.m.-i.n. vaccinated mice (top left panel). Scale bar: 200 μm. The close up view (bottom right panel) highlights the thickened, disorganized ependymal lining, compared to the normal ependymal cells and cilia of an LV::S i.m.-i.n. vaccinated mouse (top left panel). Scale bar: 50 μm.


( B ) Brain or lung viral RNA contents , determined by conventional E - specific or sub - genomic Esg - specific qRT - PCR at 3 dpi . Two mice out of the 5 sham - vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 ) .

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(B) Brain or lung viral RNA contents, determined by conventional E-specific or sub-genomic Esg-specific qRT-PCR at 3 dpi. Two mice out of the 5 sham-vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).


( E ) Wild type or µMT KO mice ( n = 5 - 9 / group ) were injected by LV : : S or sham following the time line shown in ( A ) , then pretreated with Ad5 : : hACE2 4 days before challenge with the ancestral SARS - CoV - 2 strain . Lung viral RNA contents were determined at 3 dpi . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 ) .

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(E) Wild type or µMT KO mice (n = 5-9/group) were injected by LV::S or sham following the time line shown in (A), then pretreated with Ad5::hACE2 4 days before challenge with the ancestral SARS-CoV-2 strain. Lung viral RNA contents were determined at 3 dpi. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).


D Changes ( Δ g ) in inguinal WAT ( iWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant . E Changes ( Δ g ) in epididymal WAT ( eWAT ) mass of mice in ( C ) . n = 5 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test . N . S . , not significant .

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D Changes (Δ g) in inguinal WAT (iWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant. E Changes (Δ g) in epididymal WAT (eWAT) mass of mice in (C). n=5. *P<0.05, **P<0.01, ***P<0.001 relative to GFP by Student's t-test. N.S., not significant.


F Expression of Pltp mRNA in indicated tissues . Data are shown by relative Pltp mRNA levels normalized by 18s expression . n = 3 for all tissues ( except BAT and WAT n = 5 , Testis n = 2 ) . * P < 0 . 05 Pltp expression in BAT vs other tissues ( except testis and lung ) by Student ' s t - test .

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

F Expression of Pltp mRNA in indicated tissues. Data are shown by relative Pltp mRNA levels normalized by 18s expression. n=3 for all tissues (except BAT and WAT n=5, Testis n=2). *P<0.05 Pltp expression in BAT vs other tissues (except testis and lung) by Student's t-test.


G Plasma PLTP activity from control ( Ppargflox / flox ) and BAT - less mice ( Pparg UCP1 - KO , Ucp1 - Cre x Ppargflox / flox ) . Following 6 - h fasting plasma was obtained from PpargUCP1 - KO mice and littermate control . n = 3 for both groups . * P < 0 . 05 relative to control mice by Student ' s t - test .

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

G Plasma PLTP activity from control (Ppargflox/flox) and BAT-less mice (Pparg UCP1-KO, Ucp1-Cre x Ppargflox/flox). Following 6-h fasting plasma was obtained from PpargUCP1-KO mice and littermate control. n=3 for both groups. *P < 0.05 relative to control mice by Student's t-test.


H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type ( WT ) and aP2 - PRDM16 mice [ 24 ] . Peptide levels was determined by quantitative proteomics . n = 2 for both groups .

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H PLTP protein levels in the culture conditioned medium from primary differentiated inguinal adipocytes of wild type (WT) and aP2-PRDM16 mice [24]. Peptide levels was determined by quantitative proteomics. n=2 for both groups.


I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ . ChIP - seq data for PRDM16 and PPARγ were obtained from [ 31 , 32 ] , respectively .

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I Transcriptional regulation of the Pltp gene by PRDM16 and PPARγ. ChIP-seq data for PRDM16 and PPARγ were obtained from [31,32], respectively.


B Changes in body weight of mice in ( A ) during 10 weeks of HFD . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s 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 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 Changes in body weight of mice in (A) during 10 weeks of HFD. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05, **P<0.01, ***P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


C Changes in food intake of mice in ( A ) during 10 weeks of HFD ( cohort 1 ) . n = 9 for GFP ( cage 1 = 5 mice ; cage 2 = 4 mice ) and n = 8 for PLTP ( cage 1 = 4 mice ; cage 2 = 4 mice )

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C Changes in food intake of mice in (A) during 10 weeks of HFD (cohort 1). n=9 for GFP (cage 1 = 5 mice; cage 2 = 4 mice) and n=8 for PLTP (cage 1 = 4 mice; cage 2 = 4 mice)


D Body composition of mice in ( A ) at 10 weeks of HFD determined by EchoMRI . n = 9 for GFP and n = 8 for PLTP ( cohort 1 ) . * P < 0 . 05 relative to GFP by two - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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D Body composition of mice in (A) at 10 weeks of HFD determined by EchoMRI. n=9 for GFP and n=8 for PLTP (cohort 1). *P<0.05 relative to GFP by two-way ANOVA followed by Tukey's test. N.S., not significant.


E Whole - body energy expenditure of mice at 3 weeks of HFD . Area under the curve ( AUC ) of VO2 is shown in the right graph . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . * P < 0 . 05 relative to GFP by Student ' s t - test .

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E Whole-body energy expenditure of mice at 3 weeks of HFD. Area under the curve (AUC) of VO2 is shown in the right graph. n=7 for GFP and n=8 for PLTP (cohort 2). *P<0.05 relative to GFP by Student's t-test.


F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis . n = 7 for GFP and n = 8 for PLTP ( cohort 2 ) . N . S . , not significant by Student ' s t -

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F Physical movement of mice at 3 weeks of HFD as determined by metabolic cage analysis. n=7 for GFP and n=8 for PLTP (cohort 2). N.S., not significant by Student's t-test


G OCR in the interscapular BAT and iWAT of control and PLTP - treated mice . One week after the treatment , BAT and iWAT were isolated from mice ( 7 weeks old ) that received adenovirus for GFP or PLTP . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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G OCR in the interscapular BAT and iWAT of control and PLTP-treated mice. One week after the treatment, BAT and iWAT were isolated from mice (7 weeks old) that received adenovirus for GFP or PLTP. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


H Gene expression of indicated thermogenic genes in ( A ) from interscapular BAT for GFP ( n = 8 ) and PLTP ( n = 8 ) groups ( cohort 1 ) except n = 7 for Ucp1 of the PLTP group . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test . N . S . , not significant .

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 B-MEASURED_VAR 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

H Gene expression of indicated thermogenic genes in (A) from interscapular BAT for GFP (n=8) and PLTP (n=8) groups (cohort 1) except n=7 for Ucp1 of the PLTP group. Tbp was used as an internal control. *P<0.05, **P<0.01 relative to GFP by Student's t-test. N.S., not significant.


I H & E staining of interscapular BAT from mice in ( A ) at 10 weeks of HFD ( cohort 1 ) . Representative images from n = 3 mice for both groups . Scale Bar , 50µm .

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I H&E staining of interscapular BAT from mice in (A) at 10 weeks of HFD (cohort 1). Representative images from n=3 mice for both groups. Scale Bar, 50µm.


A Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 10 weeks of HFD . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 9 for GFP and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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 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

A Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 10 weeks of HFD. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=9 for GFP and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


B Insulin tolerance test of mice in ( A ) at 10 weeks of HFD . Mice were fasted for four hours and administered insulin ( 0 . 75 U / kg body weight ) . n = 9 for GFP control and n = 7 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s 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 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

B Insulin tolerance test of mice in (A) at 10 weeks of HFD. Mice were fasted for four hours and administered insulin (0.75 U/kg body weight). n=9 for GFP control and n=7 for PLTP. *P<0.05, **P<0.01 relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test.


C Fasting insulin levels of mice in ( A ) at 10 weeks of HFD . Blood samples were harvested after eight hours of fasting . n = 5 for GFP and n = 4 for PLTP . * P < 0 . 05 relative to GFP by Student ' s t - test .

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C Fasting insulin levels of mice in (A) at 10 weeks of HFD. Blood samples were harvested after eight hours of fasting. n=5 for GFP and n=4 for PLTP. *P<0.05 relative to GFP by Student's t-test.


D Glucose tolerance test of mice that received AAV control ( GFP ) or PLTP at 6 weeks of HFD . Note that no difference was found in body weight between the groups by two - way ANOVA repeated measures followed by Bonferroni ' s test . . After eight hours of fasting , 2 g / kg body weight of glucose was administered . n = 7 for GFP control and n = 10 for PLTP . AUC of glucose is shown in the right graph . * P < 0 . 05 by Student ' s t - test .

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 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

D Glucose tolerance test of mice that received AAV control (GFP) or PLTP at 6 weeks of HFD. Note that no difference was found in body weight between the groups by two-way ANOVA repeated measures followed by Bonferroni's test.. After eight hours of fasting, 2 g/kg body weight of glucose was administered. n=7 for GFP control and n=10 for PLTP. AUC of glucose is shown in the right graph. *P<0.05 by Student's t-test.


E 18F - FDG PET / CT scan images of mice that received adenovirus GFP ( control ) or PLTP at 14 weeks of HFD . Mice received CL316 , 243 at dose of 1 mg / kg of body weight 30 min before the PET / CT images . Representative images of n = 5 for both groups . Yellow arrows indicate anatomical references as bladder , heart and interscapular BAT .

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E 18F-FDG PET/CT scan images of mice that received adenovirus GFP (control) or PLTP at 14 weeks of HFD. Mice received CL316,243 at dose of 1 mg/kg of body weight 30 min before the PET/CT images. Representative images of n=5 for both groups. Yellow arrows indicate anatomical references as bladder, heart and interscapular BAT.


F Quantification of 18F - FDG uptake in indicated tissues from mice in ( E ) . After the 18F - FDG PET / CT imaging , indicated tissues were harvested and the radioactivity was measured by scintillation counter . n = 5 for both groups . * P < 0 . 05 relative to GFP by Student ' s t - test . N . S . , not significant .

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

F Quantification of 18F-FDG uptake in indicated tissues from mice in (E). After the 18F-FDG PET/CT imaging, indicated tissues were harvested and the radioactivity was measured by scintillation counter. n=5 for both groups. *P<0.05 relative to GFP by Student's t-test. N.S., not significant.


A Lipidomics - based quantification of indicated lipid species in mice that received tail - vein injection of adenovirus expressing PLTP or GFP ( control ) . Blood and tissues were harvested after 10 weeks of HFD . n = 4 for GFP and n = 5 for PLTP in mice on HFD . LPC = ( lyso ) phosphatidylcholine , LPE = ( lyso ) phosphatidylethalonamine , PA = phosphatidic acid , SM = sphingomyelin , TAG = triglycerides , FFA = free fatty acid . Statistical analysis is demonstrated ( P value ) relative to GFP by Student ' s t - test .

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 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

A Lipidomics-based quantification of indicated lipid species in mice that received tail-vein injection of adenovirus expressing PLTP or GFP (control). Blood and tissues were harvested after 10 weeks of HFD. n=4 for GFP and n=5 for PLTP in mice on HFD. LPC = (lyso)phosphatidylcholine, LPE = (lyso)phosphatidylethalonamine, PA = phosphatidic acid, SM = sphingomyelin, TAG = triglycerides, FFA = free fatty acid. Statistical analysis is demonstrated (P value) relative to GFP by Student's t-test.


B Plasma total cholesterol levels in mice that received adenovirus GFP ( control ) or PLTP . Blood and tissues were harvested seven days after adenovirus infection . n = 8 in mice on RD and n = 7 in mice on HFD for 14 weeks . * * * P < 0 . 001 relative to GFP by Student ' s t - test .

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B Plasma total cholesterol levels in mice that received adenovirus GFP (control) or PLTP. Blood and tissues were harvested seven days after adenovirus infection. n=8 in mice on RD and n=7 in mice on HFD for 14 weeks. ***P<0.001 relative to GFP by Student's t-test.


C Relative mRNA levels of indicated genes in the liver of mice on HFD . Tbp was used as an internal control . n = 4 for GFP and n = 6 for PLTP . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test .

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C Relative mRNA levels of indicated genes in the liver of mice on HFD. Tbp was used as an internal control. n=4 for GFP and n=6 for PLTP. *P<0.05, **P<0.01 relative to GFP by Student's t-test.


D Bile acid levels in the liver of mice in ( B ) . n = 8 in mice on RD and n = 6 in mice on HFD . * P < 0 . 05 , * * P < 0 . 01 relative to GFP by Student ' s t - test .

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D Bile acid levels in the liver of mice in (B). n=8 in mice on RD and n=6 in mice on HFD. *P<0.05, **P<0.01 relative to GFP by Student's t-test.


E Bile acid levels in the plasma of mice in ( B ) . n = 8 in mice on RD and n = 7 in mice on HFD . * * * P < 0 . 001 relative to GFP by Student ' s t - test . F Bile acid levels in the feces of mice in ( B ) . Feces were collected from mice on RD and HFD after seven days of treatment . n = 7 in mice on RD and HFD . * * P < 0 . 01 , * * * P < 0 . 001 relative to GFP by Student ' s t - test .

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E Bile acid levels in the plasma of mice in (B). n=8 in mice on RD and n=7 in mice on HFD. ***P<0.001 relative to GFP by Student's t-test. F Bile acid levels in the feces of mice in (B). Feces were collected from mice on RD and HFD after seven days of treatment. n=7 in mice on RD and HFD. **P<0.01, ***P<0.001 relative to GFP by Student's t-test.


H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid ( CA ) . Cells were treated with CA at indicated concentrations for 24 h . 0 . 1 % ETOH was used as vehicle control ( Ctr ) . n = 3 . Ucp1 mRNA expression was normalized by Tbp as an internal control . * * P < 0 . 01 relative to Ctr by one - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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 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

H Relative expression of Ucp1 in differentiated brown adipocytes treated with cholic acid (CA). Cells were treated with CA at indicated concentrations for 24 h. 0.1% ETOH was used as vehicle control (Ctr). n=3. Ucp1 mRNA expression was normalized by Tbp as an internal control. **P<0.01 relative to Ctr by one-way ANOVA followed by Tukey's test. N.S., not significant.


B Relative expression of Pltp in liver of mice that received adenovirus GFP ( control ) or PLTP . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * P < 0 . 01 relative to GFP by ANOVA one - way followed by Tukey ' s test . N . S . , not significant .

O O O O B-MEASURED_VAR 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 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

B Relative expression of Pltp in liver of mice that received adenovirus GFP (control) or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. **P<0.01 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.


C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * * P < 0 . 001 relative to GFP by ANOVA one - way followed by Tukey ' s test . N . S . , not significant .

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

C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.


D Plasma PLTP activity in mice that received adenovirus GFP or PLTP . n = 4 for GFP , n = 6 for PLTP and n = 6 for PLTPM159E . * * * P < 0 . 001 PLTP relative to GFP and PLTPM159E ; * P < 0 . 05 PLTPM159E relative to GFP by ANOVA one - way followed by Tukey ' s test .

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

D Plasma PLTP activity in mice that received adenovirus GFP or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 PLTP relative to GFP and PLTPM159E; *P<0.05 PLTPM159E relative to GFP by ANOVA one-way followed by Tukey's test.


E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD . n = 8 for GFP , n = 10 for PLTP and n = 10 for PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 relative to PLTPM159E by two - way ANOVA repeated measures followed by Bonferroni ' s test .

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E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD. n=8 for GFP, n=10 for PLTP and n=10 for PLTPM159E. *P<0.05, **P<0.01 relative to PLTPM159E by two-way ANOVA repeated measures followed by Bonferroni's test.


F Relative Ucp1 expression in BAT of mice that received AAV GFP ( control ) or PLTP at 10 weeks of HFD . n = 4 for GFP , n = 5 for PLTP and n = 4 for PLTPM159E . Tbp was used as an internal control . * P < 0 . 05 , * * P < 0 . 01 by ANOVA one - way followed by Tukey ' s test .

O O B-MEASURED_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 B-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 O O O O O O

F Relative Ucp1 expression in BAT of mice that received AAV GFP (control) or PLTP at 10 weeks of HFD. n=4 for GFP, n=5 for PLTP and n=4 for PLTPM159E. Tbp was used as an internal control. *P<0.05, **P<0.01 by ANOVA one-way followed by Tukey's test.


G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD . n = 5 for GFP and n = 6 for both PLTP and PLTPM159E . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 PLTP relative to GFP by two - way ANOVA repeated measures followed by Bonferroni ' s test . AUC of glucose , shown in the right graph , was analyzed by one - way ANOVA followed by Tukey ' s test . N . S . , not significant .

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 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-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

G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD. n=5 for GFP and n=6 for both PLTP and PLTPM159E. *P<0.05, **P<0.01, ***P<0.001 PLTP relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. AUC of glucose, shown in the right graph, was analyzed by one-way ANOVA followed by Tukey's test. N.S., not significant.


H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD . n = 7 for all groups . * P = 0 . 05 by ANOVA one - way followed by Fisher ' s LSD test . N . S . , not significant .

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H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD. n=7 for all groups. *P=0.05 by ANOVA one-way followed by Fisher's LSD test. N.S., not significant.


I Plasma bile acids in ( H ) . n = 7 for all groups . * * * P < 0 . 001 PLTP relative to GFP and PLTPM159E by ANOVA one - way followed by Tukey ' s test .

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I Plasma bile acids in (H). n=7 for all groups. ***P<0.001 PLTP relative to GFP and PLTPM159E by ANOVA one-way followed by Tukey's test.


J Plasma phospholipid in ( H ) . n = 7 for all groups . * * * P < 0 . 001 PLTP and PLTPM159E relative to GFP ; * P < 0 . 05 PLTP relative to PLTPM159E by ANOVA one - way followed by Fisher ' s LSD test .

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J Plasma phospholipid in (H). n=7 for all groups. ***P<0.001 PLTP and PLTPM159E relative to GFP; *P<0.05 PLTP relative to PLTPM159E by ANOVA one-way followed by Fisher's LSD test.


Testicular maturation arrest in the patient ( WHT3759 ) . Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin / eosin . Seminiferous tubules contained meiotic germ cells such as zygotene ( ZS , arrowheads ) and pachytene spermatocytes ( PS , arrows ) , but no post - meiotic germ cells such as round spermatids were detectable . Scale bar , 25 μm .

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Testicular maturation arrest in the patient (WHT3759). Testicular tissue obtained by biopsy was sectioned and stained with hematoxylin/eosin. Seminiferous tubules contained meiotic germ cells such as zygotene (ZS, arrowheads) and pachytene spermatocytes (PS, arrows), but no post-meiotic germ cells such as round spermatids were detectable. Scale bar, 25 μm.


Western blot analysis of TEX11 in the testes of 3 - month - old mice with different Tex11 gene dosages . ACTB serves as a loading control .

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Western blot analysis of TEX11 in the testes of 3-month-old mice with different Tex11 gene dosages. ACTB serves as a loading control.


Testis weight of 3 - month - old males .

Testis weight of 3-month-old males.


Sperm count of 3-month-old males.


Histological analysis of testes from adult wild - type , Tex11 KI / KO , and Tex11 − / Y males . RS , round spermatids ; ES , elongating spermatids ; Pa , pachytene spermatocytes . Scale bar , 50 μm .

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Histological analysis of testes from adult wild-type, Tex11 KI/KO, and Tex11−/Y males. RS, round spermatids; ES, elongating spermatids; Pa, pachytene spermatocytes. Scale bar, 50 μm.


The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11 − / Y spermatocytes in 3 - month - old males . Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male ; for each genotype , three males were analyzed . Abbreviations for Tex11 genotypes : − / Y , Tex11 knockout ( Yang et al , 2008 ) ; KI ; − / Y , Tex11 knockin and knockout ; + / Y , wild type ; KI ; + / Y , Tex11 knockin plus wild - type Tex11 .

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The Tex11 knockin allele rescues chromosomal synapsis defects in Tex11−/Y spermatocytes in 3-month-old males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed. Abbreviations for Tex11 genotypes: −/Y, Tex11 knockout (Yang et al, 2008); KI;−/Y, Tex11 knockin and knockout; +/Y, wild type; KI;+/Y, Tex11 knockin plus wild-type Tex11.


Reduced testis weight in juvenile but not adult Tex11 KI ; − / Y males . The time frames of the first wave and adult waves of spermatogenesis are indicated .

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Reduced testis weight in juvenile but not adult Tex11 KI; −/Y males. The time frames of the first wave and adult waves of spermatogenesis are indicated.


Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25 - day - old Tex11 KI / KO males . Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male ; for each genotype , three males were analyzed .

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Increased chromosomal asynapsis in spermatocytes and reduced levels of TEX11 protein in the testes of 25-day-old Tex11 KI/KO males. Chromosomal synapsis defects were assessed by SYCP1 and SYCP2 immunostaining of spread nuclei from 100 pachytene spermatocytes per male; for each genotype, three males were analyzed.


Pachytene spermatocytes from 25 - day - old Tex11 KI / KO males contain significantly fewer MLH1 foci compared to wild - type spermatocytes . Values are shown as average ± standard deviation . Error bar is standard deviation .

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Pachytene spermatocytes from 25-day-old Tex11 KI/KO males contain significantly fewer MLH1 foci compared to wild-type spermatocytes. Values are shown as average ± standard deviation. Error bar is standard deviation.


The number of MLH1 foci in pachytene spermatocytes from 3 - month - old males positively correlates with increasing Tex11 gene dosage .

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The number of MLH1 foci in pachytene spermatocytes from 3-month-old males positively correlates with increasing Tex11 gene dosage.


Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11 − / − , Tex11 + / − , and wild - type embryonic day - 17 . 5 ( E17 . 5 ) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci .

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Quantification of MLH1 foci in pachytene oocytes from the ovaries of Tex11−/−, Tex11+/−, and wild-type embryonic day-17.5 (E17.5) fetuses reveals a positive correlation of Tex11 gene dosage and the number of MLH1 foci.


A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild - type and point mutant KI / KO males . ACTB serves as a control .

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A Western blot analysis reveals comparable levels of TEX11 protein in the testes of wild-type and point mutant KI/KO males. ACTB serves as a control.


B - D Body weight ( B ) , testisweight ( C ) in Tex11 KI ( V749A ) / KO males .

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B-D Body weight (B), testisweight (C) in Tex11 KI(V749A)/KO males.


B - D Significantly reduced sperm count ( per pair of epididymides ) in Tex11 KI ( V749A ) / KO males ( D ) .

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B-D Significantly reduced sperm count (per pair of epididymides) in Tex11 KI(V749A)/KO males (D).


E Mating test of 3 - to 5 - month - old males .

E Mating test of 3- to 5-month-old males.


F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI ( V749A ) / KO males . 100 pachytene spermatocytes per mouse were examined by surface spread analysis .

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F Dramatically increased chromosomal asynapsis in spermatocytes from Tex11 KI(V749A)/KO males. 100 pachytene spermatocytes per mouse were examined by surface spread analysis.


G Number of MLH1 foci in pachytene spermatocytes . Note a significantly higher number of MLH1 foci in spermatocytes from KI ( Q173R ) / KO males .

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G Number of MLH1 foci in pachytene spermatocytes. Note a significantly higher number of MLH1 foci in spermatocytes from KI(Q173R)/KO males.


Reverse Transcriptase PCR showing that bd1971 , and control gene dnaK , are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus . RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E . coli cells . Primers were designed to anneal specifically to the gene of interest . L = 100bp DNA ladder , AP = Attack Phase cells , 15 - 45 = 15 - 45 minutes respectively since infection , 1h - 4h = hours respectively since infection . Ec = E . coli strain S17 - 1 RNA ( negative control : no Bdellovibrio RNA ) ; NT = control with no template RNA ; gen = B . bacteriovorus HD100 genomic DNA ( positive control ) . The cartoon above represents each stage in the predatory cycle .

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Reverse Transcriptase PCR showing that bd1971, and control gene dnaK, are expressed throughout the predatory cycle of Bdellovibrio bacteriovorus. RNA was isolated at the time points indicated across the top of the gel during one round of synchronous Bdellovibrio infection of E. coli cells. Primers were designed to anneal specifically to the gene of interest. L = 100bp DNA ladder, AP = Attack Phase cells, 15-45 = 15-45 minutes respectively since infection, 1h-4h = hours respectively since infection. Ec = E. coli strain S17-1 RNA (negative control: no Bdellovibrio RNA); NT = control with no template RNA; gen = B. bacteriovorus HD100 genomic DNA (positive control). The cartoon above represents each stage in the predatory cycle.


Epifluorescent images of E . coli invaded by B . bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection . Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images ( see methods ) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation . Fluorescence images and merges with phase contrast images are shown . Scale bars are 2 µm . Images are representative of three independent experiments .

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Epifluorescent images of E. coli invaded by B. bacteriovorus HD100 with mCherry tagged Bd1971 at the times indicated throughout the invasive process during one round of synchronous infection. Fluorescence was acquired with a two second exposure and maximum sensitivity gain but different balance and contrast levels in the fluorescence channel were used to produce these images (see methods) as the fluorescence was fainter at later timepoints but we wished to show whether or not the cellular localization within the Bdellovibrio altered during predation. Fluorescence images and merges with phase contrast images are shown. Scale bars are 2 µm. Images are representative of three independent experiments.


Relative levels of c - di - GMP in matched cell biomass host - independent ( HI ) cultures of bd1971 : : kan compared with fliC1 merodiploid KnR HI control strain ( Wild - Type for bd1971 ) . The bd1971 mutant strain contains significantly more c - di - GMP than the control . Data are from 3 independent experiments .

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Relative levels of c-di-GMP in matched cell biomass host-independent (HI) cultures of bd1971::kan compared with fliC1 merodiploid KnR HI control strain (Wild-Type for bd1971). The bd1971 mutant strain contains significantly more c-di-GMP than the control. Data are from 3 independent experiments.


Relative levels of c - di - GMP in matched cell biomass wild - type predatory cultures of HD100 strain , silent Bd1971 deletion strain ( Δ1971 ) , Bd1971D306307A strain ( bd1971DD ) and Bd1971R67D strain ( bd1971R ) . All mutant strains had higher levels of c - di - GMP compared to the wild type strain . Error bars show SEM . Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants .

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Relative levels of c-di-GMP in matched cell biomass wild-type predatory cultures of HD100 strain, silent Bd1971 deletion strain (Δ1971), Bd1971D306307A strain (bd1971DD) and Bd1971R67D strain (bd1971R). All mutant strains had higher levels of c-di-GMP compared to the wild type strain. Error bars show SEM. Data are from six independent experiments for HD100 and ΔBd1971 and from four independent experiments for Bd1971DD and Bd1971R mutants.


Apo - Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations ( 2 . 5 mM ) of cAMP , cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP ( 5 mM ) was measured spectrophotometrically . Values are the average of three repeats , error bars represent standard deviation .

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Apo-Bd1971 was supplemented with 10 mM MgCl2 or CaCl2 in conjunction with saturating concentrations (2.5 mM) of cAMP, cGMP or no nucleotide and phosphodiesterase activity with respect to the generic substrate PNPP (5 mM) was measured spectrophotometrically. Values are the average of three repeats, error bars represent standard deviation.


HPLC traces of reaction products generated from 100 µM c - di - GMP incubated with apo - Bd1971 ( Control ) , or protein supplemented with either 100 µM cAMP or cGMP . Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure .

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HPLC traces of reaction products generated from 100 µM c-di-GMP incubated with apo-Bd1971 (Control), or protein supplemented with either 100 µM cAMP or cGMP. Peaks were identified by reference to standards also resolved by HPLC and are labeled in the figure.


ITC titration curves and relative heats of binding for ( A ) cAMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) and heat changes measured . The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment .

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ITC titration curves and relative heats of binding for (A) cAMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL . Successive injections of 6 µl of cNMP ( 0 . 5 - 1 . 6 mM ) were made into 1 . 4 ml of apo - Bd1971ΔEAL ( 50 - 150 µM ) and heat changes measured . The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment .

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ITC titration curves and relative heats of binding for cGMP binding to Bd1971ΔEAL. Successive injections of 6 µl of cNMP (0.5 - 1.6 mM) were made into 1.4 ml of apo-Bd1971ΔEAL (50 - 150 µM) and heat changes measured. The top panels show the ITC titrations and the bottom panels show the binding isotherm associated with each ITC experiment.


Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity . Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars . Results are representative of at least three independent experiments .

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Data were fitted with an allosteric sigmoidal model in GraphPad Prism software and used to determine the kinetic parameters associated with cNMP dependent phosphodiesterase activity.Data points for both sets of results are the average of three experimental replicates with the standard deviation shown by intersecting bars. Results are representative of at least three independent experiments.


Spot tests of co - transformants of either Bd0367 ( DgcA ; GGDEF protein ) response regulator domain or Bd3125 ( CdgA ; a degenerate GGDEF protein likely involved in c - di - GMP binding and signalling ) with full length Bd1971 , or the N - terminal ( 1 - 145 ) , or C - terminal ( 145 - 400 ) portions of Bd1971 in either pUT18c or pKT25 as indicated . Positive control was co - transformants with pUT18c - zip and pKT25 - zip , negative control was the empty vectors . The co - transformants were plated on LB - X - Gal medium with positive interaction resulting in blue colouration , which can be seen for all of the interactions in both plasmid combinations , but not for the negative control . Images are representative of at least 4 independent experiments .

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Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.


Confirmation of BTH interactions by β - Galactosidase activity assays . The interaction is significantly stronger between Bd3125 and the N - terminal domain of Bd1971 than the interaction with the C - terminus of Bd1971 . The response regulator domain of Bd0367 showed significant interaction with Bd1971 , but the full length Bd0367 did not . * p > 0 . 05 by Student ' s t - test N / S - not significant . Data are from at least three independent experiments , error bars are the standard error of the

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Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean


A RNA - seq . of freshly isolated , proliferating and differentiating satellite cells reveals an increased expression of linc - MYH in proliferating and differentiating MuSCs , but no expression is found in in freshly isolated MuSCs .

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A RNA-seq. of freshly isolated, proliferating and differentiating satellite cells reveals an increased expression of linc-MYH in proliferating and differentiating MuSCs, but no expression is found in in freshly isolated MuSCs.


Expression of linc - MYH is confined to skeletal muscle in mouse ( B ) tissues .

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Expression of linc-MYH is confined to skeletal muscle in mouse (B) tissues.


Expression of linc - MYH is confined to skeletal muscle in human ( C ) tissues .

Expression of linc-MYH is confined to skeletal muscle in human (C) tissues.


D Expression of linc - MYH is not detected in limb buds at E10 . 5 ( n = 3 ) , E13 . 5 ( n = 3 ) and E15 . 5 ( n = 3 ) . Expression of linc - MYH in hindlimb muscle of E18 . 5 , newborn and 7 day old animals ( n = 3 / 3 / 3 ) . Expression of linc - MYH in m . soleus ( soleus ) , m . tibialis anterior ( TA ) and and m . extensor digitorum longus ( EDL ) muscle at 3 weeks ( n = 3 ) and 8 weeks of age ( n = 5 ) . Biological replicates were used for all PCR experiments , data are mean ± S . E . M .

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D Expression of linc-MYH is not detected in limb buds at E10.5 (n = 3), E13.5 (n = 3) and E15.5 (n = 3). Expression of linc-MYH in hindlimb muscle of E18.5, newborn and 7 day old animals (n = 3/3/3). Expression of linc-MYH in m. soleus (soleus), m. tibialis anterior (TA) and and m. extensor digitorum longus (EDL) muscle at 3 weeks (n = 3) and 8 weeks of age (n = 5). Biological replicates were used for all PCR experiments, data are mean ± S.E.M.


E Murine linc - Myh is localized in nuclear extracts of C2C12 cells . Xist ( nuclear ) and Gapdh ( mainly cytoplasmic ) were used as controls ( n = 2 nuclear / 3 cytoplasmic biological replicates . ) Data are mean ± S . E . M .

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E Murine linc-Myh is localized in nuclear extracts of C2C12 cells. Xist (nuclear) and Gapdh (mainly cytoplasmic) were used as controls (n = 2 nuclear / 3 cytoplasmic biological replicates.) Data are mean ± S.E.M.