Figure 5 ( A ) Gel pictures of the saturation phase of RT - qPCR analysis for miR - 204 in SEZ primary culture cells , ChP co - culture and ChP explants 7 day post preparation . ( B ) Gel electrophoresis of RT - qPCR analysis loaded at the saturation phase for miR - 204 in SEZ culture medium and SEZ medium from the ChP co - culture . ( C ) Dot plot showing the proportion of neural stem cells ( GFAP + ) out of DAPI + cells in primary SEZ / ChP co - culture with or without treatment with GW4869 inhibitor or AntimiR204 . ( D - G ) Micrographs depicting the cellular compositions of SEZ cultures after co - culturing with control treated , GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating . Boxed areas correspond to the higher magnification images in adjacent panels ( D ' , E ' , F ' , G ' ) .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Gel pictures of the saturation phase of RT-qPCR analysis for miR-204 in SEZ primary culture cells, ChP co-culture and ChP explants 7 day post preparation.(B) Gel electrophoresis of RT-qPCR analysis loaded at the saturation phase for miR-204 in SEZ culture medium and SEZ medium from the ChP co-culture.(C) Dot plot showing the proportion of neural stem cells (GFAP+) out of DAPI+ cells in primary SEZ/ChP co-culture with or without treatment with GW4869 inhibitor or AntimiR204.(D-G) Micrographs depicting the cellular compositions of SEZ cultures after co-culturing with control treated, GW4869 inhibitor treated or AntimiR204 treated ChP 7 days after plating. Boxed areas correspond to the higher magnification images in adjacent panels (D', E', F', G').


Figure 6 ( B , C ) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle . ( B ' and C ' ) are magnifications of the SEZ and ( B ' ' and C ' ' ) of the ChP . ( D ) Dot plot depicting expression of miR - 204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR - 204 - specific TuD compared to scrambled control . Micrographs depicting immunoreactivity for MEIS2 ( E , F ) priming factors in the LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition and scrambled control White arrows point out the primed LRCs , the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR - 204 interference . Micrographs depicting immunoreactivity for MCM6 ( H , I ) priming factors in the LRCs ( BrdU + only ) 7 days after ChP - specific miR - 204 inhibition and scrambled control White arrows point out the primed LRCs , the arrowheads label the MCM6 + LRCs . Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 ( J ) after ChP specific miR - 204 interference . ( K , L ) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control ( K ) and miR - 204 specific TuD ( L ) . Note the reduction of primed LRCs ( BrdU + only , white arrows in K and L ) upon TuD - 204 injection . ( M ) Dot plot depicting the number of LRCs ( BrdU + only ) in the SEZ 7 days after miR - 204 inhibition .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B, C) Micrographs depicting specific expression of AAV5 encoded GFP in the ChP 7 days after viral delivery in the lateral ventricle. (B' and C') are magnifications of the SEZ and (B'' and C'') of the ChP. (D) Dot plot depicting expression of miR-204 in ChP 7 days after second ventricular injection of AAV5 encoding for miR-204-specific TuD compared to scrambled control. Micrographs depicting immunoreactivity for MEIS2 (E, F) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MEIS2 Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MEIS2 after ChP specific miR-204 interference.Micrographs depicting immunoreactivity for MCM6 (H, I) priming factors in the LRCs (BrdU+ only) 7 days after ChP-specific miR-204 inhibition and scrambled control White arrows point out the primed LRCs, the arrowheads label the MCM6+ LRCs. Dot plots depicting the proportion of LRCs immunoreactive for the priming proteins MCM6 (J) after ChP specific miR-204 interference.(K, L) Micrographs showing the cellular composition in the SEZ 7 days after injection of AVV5 encoding for scrambled control (K) and miR-204 specific TuD (L). Note the reduction of primed LRCs (BrdU+ only, white arrows in K and L) upon TuD-204 injection. (M) Dot plot depicting the number of LRCs (BrdU+ only) in the SEZ 7 days after miR-204 inhibition.


Figure 1 ( A ) TNF concentrations in supernatants of WT and Tnf - / - BMDMs unstimulated or stimulated with 10 ng / ml Pam3CSK4 , quantified by ELISA . ( Left ) 0 h , 4 h and 8 h time points . ( Right ) Close - up of 0 h time point . Dashed line indicates mean of blank measurements . Data are represented as mean ± SD of 3 ( 4 h , 8 h ) or 5 ( 0 h ) biological replicates using cells from 2 ( 4 h , 8 h ) or 3 ( 0 h ) mice . Statistical significance was determined by two - tailed paired - sample t - test . ( E - J ) Nuclear localization of mVenus - RelA upon stimulation with 10 ng / ml TNF ( E - G ) or 10 ng / ml Pam3CSK4 ( H - J ) , measured over time in WT and Tnf - / - BMDMs by fluorescence microscopy and quantified by automated image analysis . ( E , H ) Each row of the heatmap represents the NFκB signaling trajectory of one cell . Trajectories are sorted by maximum amplitude . Example trajectories are shown below . Data from a single representative experiment is depicted . ( F , I ) Indicated dynamic features were calculated from signaling trajectories . Violin plots depict pooled data from 8 ( F ) or 6 ( I ) biological replicates ( total # of cells : 5769 / 5238 ( F ) , 3843 / 3899 ( I ) ) . Marker indicates median of distribution . Statistical significance was determined by Wilcoxon rank sum test . Number indicates log10 ( p - value ) . ( G , J ) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment . Data are represented as mean ± SD of 8 ( G ) or 6 ( J ) biological replicates . Statistical significance was determined by two - tailed paired - sample t - test . ( F , G ) Some replicates for Tnf - / - BMDMs overlap with

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) TNF concentrations in supernatants of WT and Tnf-/- BMDMs unstimulated or stimulated with 10 ng/ml Pam3CSK4, quantified by ELISA. (Left) 0 h, 4 h and 8 h time points. (Right) Close-up of 0 h time point. Dashed line indicates mean of blank measurements. Data are represented as mean ± SD of 3 (4 h, 8 h) or 5 (0 h) biological replicates using cells from 2 (4 h, 8 h) or 3 (0 h) mice. Statistical significance was determined by two-tailed paired-sample t-test.(E - J) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF (E - G) or 10 ng/ml Pam3CSK4 (H - J), measured over time in WT and Tnf-/- BMDMs by fluorescence microscopy and quantified by automated image analysis. (E, H) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment is depicted. (F, I) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 8 (F) or 6 (I) biological replicates (total # of cells: 5769/5238 (F), 3843/3899 (I)). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (G, J) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 8 (G) or 6 (J) biological replicates. Statistical significance was determined by two-tailed paired-sample t-test. (F, G) Some replicates for Tnf-/- BMDMs overlap with data


Figure 2 ( B ) IκBα mRNA expression in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by RT - qPCR . IκBα expression was normalized to GAPDH expression and then to maximum WT value in each time course replicate . Data are represented as mean ± SD of 5 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Some replicates for Tnf - / - BMDMs overlap with dataPhosphorylation of IKK , JNK , ERK , and p38 and total IKK levels in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by SDS - PAGE followed by Western Blotting . ( C ) One of three representative experiments is shown . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Phosphorylation of IKK levels in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h For quantification of p - IKK ( D ) levels , basal levels ( 0 min ) were deducted per genotype before normalizing to maximum WT value in each experiment . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Phosphorylation of JNK levels in WT and Tnf - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , p - JNK ( E ) levels , basal levels ( 0 min ) were deducted per genotype before normalizing to maximum WT value in each experiment . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) IκBα mRNA expression in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by RT-qPCR. IκBα expression was normalized to GAPDH expression and then to maximum WT value in each time course replicate. Data are represented as mean ± SD of 5 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Some replicates for Tnf-/- BMDMs overlap with dataPhosphorylation of IKK, JNK, ERK, and p38 and total IKK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by SDS-PAGE followed by Western Blotting. (C) One of three representative experiments is shown. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.Phosphorylation of IKK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h For quantification of p-IKK (D) levels, basal levels (0 min) were deducted per genotype before normalizing to maximum WT value in each experiment. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.Phosphorylation of JNK levels in WT and Tnf-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, p-JNK (E) levels, basal levels (0 min) were deducted per genotype before normalizing to maximum WT value in each experiment. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point.


Figure 3 ( E ) TNF receptor 1 ( TNFR1 , left ) , TNF receptor 2 ( TNFR2 , center ) , and CD11b ( right ) cell surface expression in Tnf - / - compared to WT BMDMs unstimulated or stimulated for 30 min with 10 ng / ml TNF , measured by flow cytometry . Per experiment , median fluorescence intensity average of 3 culture replicates in Tnf - / - cells is expressed as percent of expression in WT cells . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed one - sample t - test using a hypothesis mean of 100 . ( F ) ( Left ) TNF receptor 1 ( TNFR1 ) cell surface expression at baseline in BMDMs pre - treated with TNFR1 siRNA , control ( ctrl ) siRNA , or untreated ( - ) . Median fluorescence intensity is expressed as percent of expression in untreated WT cells . ( Right ) Percentage of cells with oscillatory NFκB activity ( measured by fluorescence microscopy ) among all responder cells upon indicated siRNA pre - treatment and stimulation with 10 ng / ml TNF . Per condition , 1032 - 1484 responding cells were analyzed . ( Left , right ) Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by one - tailed paired - sample t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(E) TNF receptor 1 (TNFR1, left), TNF receptor 2 (TNFR2, center), and CD11b (right) cell surface expression in Tnf-/- compared to WT BMDMs unstimulated or stimulated for 30 min with 10 ng/ml TNF, measured by flow cytometry. Per experiment, median fluorescence intensity average of 3 culture replicates in Tnf-/- cells is expressed as percent of expression in WT cells. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed one-sample t-test using a hypothesis mean of 100.(F) (Left) TNF receptor 1 (TNFR1) cell surface expression at baseline in BMDMs pre-treated with TNFR1 siRNA, control (ctrl) siRNA, or untreated (-). Median fluorescence intensity is expressed as percent of expression in untreated WT cells. (Right) Percentage of cells with oscillatory NFκB activity (measured by fluorescence microscopy) among all responder cells upon indicated siRNA pre-treatment and stimulation with 10 ng/ml TNF. Per condition, 1032 - 1484 responding cells were analyzed. (Left, right) Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by one-tailed paired-sample t-test.


Figure 4 ( D ) IκBα mRNA expression in Tnf - / - and Tnf - / - p100 - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by RT - qPCR . IκBα expression was normalized to GAPDH expression and then to maximum WT value also obtained with each replicate . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Data for Tnf - / - BMDMs is also included ( E ) Levels of nuclear NFκB DNA binding activity in Tnf - / - and Tnf - / - p100 - / - BMDMs stimulated with 10 ng / ml TNF over 2 h , determined by EMSA . Quantification of the NFκB - DNA complex was normalized to the quantified NFY - DNA complex , and then to maximum WT value also obtained with each replicate . Data are represented as mean ± SD of 3 biological replicates . Statistical significance was determined by two - tailed paired - sample t - test for each time point . Data for Tnf - / - BMDMs is also shown ( F - H ) Nuclear localization of mVenus - RelA upon stimulation with 10 ng / ml TNF was measured over time by fluorescence microscopy in Tnf - / - and Tnf - / - p100 - / - BMDMs and quantified by automated image analysis . ( F ) Each row of the heatmap represents the NFκB signaling trajectory of one cell . Trajectories are sorted by maximum amplitude . Example trajectories are shown below . Data from a single representative experiment are depicted . ( G ) Indicated dynamic features were calculated from signaling trajectories . Violin plots depict pooled data from 6 biological replicates ( total # of cells : 4030 / 4133 ) . Marker indicates median of distribution . Statistical significance was determined by Wilcoxon rank sum test . Number indicates log10 ( p - value ) . ( H ) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment . Data are represented as mean ± SD of 6 biological replicates . Statistical significance was determined by one - tailed paired - sample t - test . ( G , H ) Data for Tnf - / - BMDMs is also

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(D) IκBα mRNA expression in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by RT-qPCR. IκBα expression was normalized to GAPDH expression and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also included(E) Levels of nuclear NFκB DNA binding activity in Tnf-/- and Tnf-/-p100-/- BMDMs stimulated with 10 ng/ml TNF over 2 h, determined by EMSA. Quantification of the NFκB-DNA complex was normalized to the quantified NFY-DNA complex, and then to maximum WT value also obtained with each replicate. Data are represented as mean ± SD of 3 biological replicates. Statistical significance was determined by two-tailed paired-sample t-test for each time point. Data for Tnf-/- BMDMs is also shown(F - H) Nuclear localization of mVenus-RelA upon stimulation with 10 ng/ml TNF was measured over time by fluorescence microscopy in Tnf-/- and Tnf-/-p100-/- BMDMs and quantified by automated image analysis. (F) Each row of the heatmap represents the NFκB signaling trajectory of one cell. Trajectories are sorted by maximum amplitude. Example trajectories are shown below. Data from a single representative experiment are depicted. (G) Indicated dynamic features were calculated from signaling trajectories. Violin plots depict pooled data from 6 biological replicates (total # of cells: 4030/4133). Marker indicates median of distribution. Statistical significance was determined by Wilcoxon rank sum test. Number indicates log10(p-value). (H) Percentage of cells with NFκB trajectories categorized as oscillatory based on a frequency threshold among all responder cells in each experiment. Data are represented as mean ± SD of 6 biological replicates. Statistical significance was determined by one-tailed paired-sample t-test. (G, H) Data for Tnf-/- BMDMs is also included


Figure 5 ( E ) Volcano plot of single - cell gene expression in Tnf - / - vs . WT BMDMs upon TNF stimulation . Dashed lines indicate p - value threshold of 0 . 01 and absolute log2 ( FC ) threshold of 0 . 5 . Only genes induced by P3C4 or TNF stimulation over mock in WT cells are included ( p - value threshold of 0 . 05 and log2 ( FC ) threshold of 0 . 25 ) . ( F ) Violin plots of single - cell gene expression in WT and Tnf - / - BMDMs upon mock , TNF , or Pam3CSK4 stimulation . The 10 genes shown are significantly upregulated in Tnf - / - vs . WT cells upon TNF stimulation ( see ( E ) ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(E) Volcano plot of single-cell gene expression in Tnf-/- vs. WT BMDMs upon TNF stimulation. Dashed lines indicate p-value threshold of 0.01 and absolute log2(FC) threshold of 0.5. Only genes induced by P3C4 or TNF stimulation over mock in WT cells are included (p-value threshold of 0.05 and log2(FC) threshold of 0.25).(F) Violin plots of single-cell gene expression in WT and Tnf-/- BMDMs upon mock, TNF, or Pam3CSK4 stimulation. The 10 genes shown are significantly upregulated in Tnf-/- vs. WT cells upon TNF stimulation (see (E)).


Figure 6 ( D ) Violin plot of log 2 ( FC ) of normalized H3K4me1 ChIP - seq signal in TNF - or Pam3CSK4 - stimulated over unstimulated WT or Tnf - / - BMDMs within Cluster 1 regions ( 533 regions ) . Statistical significance was determined by Kolmogorov - Smirnov test . Data are represented as mean of two biological replicates for each genomic region . In the boxplot , central band indicates the median , boxes the interquartile range ( IQR ) , whiskers values within 1 . 5 IQR from the median . ( E ) Examples of genomic regions with differential H3K4me1 signal in TNF stimulation in Cluster 1 . Tracks for WT and Tnf - / - BMDMs unstimulated or stimulated with TNF or Pam3CSK4 in two biological replicates ( R1 , R2 ) are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(D) Violin plot of log2(FC) of normalized H3K4me1 ChIP-seq signal in TNF- or Pam3CSK4-stimulated over unstimulated WT or Tnf-/- BMDMs within Cluster 1 regions (533 regions). Statistical significance was determined by Kolmogorov-Smirnov test. Data are represented as mean of two biological replicates for each genomic region. In the boxplot, central band indicates the median, boxes the interquartile range (IQR), whiskers values within 1.5 IQR from the median.(E) Examples of genomic regions with differential H3K4me1 signal in TNF stimulation in Cluster 1. Tracks for WT and Tnf-/- BMDMs unstimulated or stimulated with TNF or Pam3CSK4 in two biological replicates (R1, R2) are shown.


Figure 1 ( B ) Violin plots showing the expression of prostaglandin ( PG ) receptors in pre - adipocytes and mature adipocytes from human deep - neck BAT ( E - MTAB - 8564 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Violin plots showing the expression of prostaglandin (PG) receptors in pre-adipocytes and mature adipocytes from human deep-neck BAT(E-MTAB-8564).


Figure 2 ( A ) Oil Red O staining of mouse BAs differentiated from normal or EP3 - deficient brown pre - adipocytes . Scale bar : 50 μm . ( B ) Mean lipid droplet area of differentiated BAs from normal or EP3 - deficient brown pre - adipocytes . * P < 0 . 05 vs WT ( n = 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U tests ( B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ( D ) Representative image of hematoxylin and eosin ( H & E ) staining for iBAT from EP3F / F and EP3F / FMyf5Cre mice . Scale bar : 50 μm . ( E ) Mean adipocyte area of BAs in iBAT from EP3F / F and EP3F / FMyf5Cre mice ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann - Whitney U tests E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( K ) Representative image of H & E staining of iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD . Scale bar : 50 μm . ( L ) Mean adipocyte area of BAs in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are pooled from two independent experiments with biological replicates L , Statistical significance was evaluated by Mann - Whitney U tests L , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Oil Red O staining of mouse BAs differentiated from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm. (B) Mean lipid droplet area of differentiated BAs from normal or EP3-deficient brown pre-adipocytes. *P < 0.05 vs WT (n = 6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests (B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05(D) Representative image of hematoxylin and eosin (H&E) staining for iBAT from EP3F/F and EP3F/FMyf5Cre mice. Scale bar: 50 μm. (E) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(K) Representative image of H&E staining of iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD. Scale bar: 50 μm. (L) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates L, Statistical significance was evaluated by Mann-Whitney U tests L, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


Figure 3 ( A ) N6 - methyladenosine ( m6A ) modification levels of mRNAs in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction , detected by m6A RNA Methylation Quantification Kit ( Colorimetric ) ( n = 4 - 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test ( A * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( B ) qRT - PCR analysis of the relative mRNA levels of m6A writers ( METTL3 , METTL4 , METTL14 and WTAP ) and erasers ( FTO and ALKBH5 ) in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction ( n = 6 - 9 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test B , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( C ) Western blotting analysis of m6A writers and erasers in normal and EP3 - deficient brown pre - adipocytes with or without brown adipogenesis induction . ( D ) Quantification of protein levels in C ( n = 4 - 6 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates , D , Statistical significance was evaluated by unpaired two - tailed student ' s t test D , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( E ) Western blotting analysis of WTAP in iBAT from EP3F / F and EP3F / FMyf5Cre mice fed HFD for 1 week . ( F ) Western blotting analysis of WTAP in BAs differentiated from EP3γ - expressed brown pre - adipocytes . ( G ) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ - expressed brown pre - adipocytes detected by RT - qPCR ( n = 6 - 7 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates G , Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons ( G , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( H ) qRT - PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . ( I ) qRT - PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two - tailed student ' s t test H ) , two - way ANOVA followed by Tukey ' s test for post - hoc comparisons I , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( J ) Representative picture of oil red O - stained BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes with WTAP siRNA treatment . Scale bar : 50 μm . ( K - Q ) qRT - PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ - expressed EP3 - / - brown pre - adipocytes after transfection with WTAP siRNA ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for post - hoc comparisons K - Q ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) N6-methyladenosine (m6A) modification levels of mRNAs in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction, detected by m6A RNA Methylation Quantification Kit (Colorimetric) (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test (A *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(B) qRT-PCR analysis of the relative mRNA levels of m6A writers (METTL3, METTL4, METTL14 and WTAP) and erasers (FTO and ALKBH5) in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction (n = 6-9). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(C) Western blotting analysis of m6A writers and erasers in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction.(D) Quantification of protein levels in C (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates , D, Statistical significance was evaluated by unpaired two-tailed student's t test D, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(E) Western blotting analysis of WTAP in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD for 1 week.(F) Western blotting analysis of WTAP in BAs differentiated from EP3γ-expressed brown pre-adipocytes.(G) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed brown pre-adipocytes detected by RT-qPCR (n = 6-7). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates G, Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons (G, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(H) qRT-PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). (I) qRT-PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test H), two-way ANOVA followed by Tukey's test for post-hoc comparisons I, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(J) Representative picture of oil red O-stained BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes with WTAP siRNA treatment. Scale bar: 50 μm.(K-Q) qRT-PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons K-Q). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


Figure 6 ( A ) Western blotting analysis of ubiquitination of WTAP in the control and EP3 - deficient brown pre - adipocytes with or without sulprostone treatment . Data information : Data are representative of two independent experiments with biological replicates ( B ) Western blotting analysis of ERK1 / 2 phosphorylation in the control and EP3 - deficient brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological replicates ( C ) Effect of ERK1 / 2 inhibitor , ravoxertinib , on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological replicates ( D ) Effect of wortmannin ( Wor ) , pertussis toxin ( PTX ) , U73122 , barbadin , and melittin on ubiquitin - dependent degradation of WTAP in sulprostone - treated brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological replicates ( E ) Analysis of PKA activity in the control and EP3 - deficient brown pre - adipocytes ( n = 4 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates ( F ) Effect of PKA inhibitor , H89 , on ubiquitin - dependent degradation of WTAP in EP3 - / - brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological replicates ( G ) Effect of ERK1 / 2 phosphorylation site mutation ( HA - WTAP - 2A ) on EP3 - mediated ubiquitin - dependent degradation of WTAP in brown pre - adipocytes . Data information : Data are representative of two independent experiments with biological replicates ( H ) Representative picture of oil red O - stained BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological replicates ( I ) Western blotting assay of ERK1 / 2 , WTAP , ZFP410 , PRDM16 , and UCP1 in BAs differentiated from sulprostone - treated brown pre - adipocyte with treatment of PTX , dbcAMP , or ravoxertinib . Data information : Data are representative of two independent experiments with biological

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Western blotting analysis of ubiquitination of WTAP in the control and EP3-deficient brown pre-adipocytes with or without sulprostone treatment. Data information: Data are representative of two independent experiments with biological replicates(B) Western blotting analysis of ERK1/2 phosphorylation in the control and EP3-deficient brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(C) Effect of ERK1/2 inhibitor, ravoxertinib, on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(D) Effect of wortmannin (Wor), pertussis toxin (PTX), U73122, barbadin, and melittin on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(E) Analysis of PKA activity in the control and EP3-deficient brown pre-adipocytes (n = 4). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates(F) Effect of PKA inhibitor, H89, on ubiquitin-dependent degradation of WTAP in EP3-/- brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(G) Effect of ERK1/2 phosphorylation site mutation (HA-WTAP-2A) on EP3-mediated ubiquitin-dependent degradation of WTAP in brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(H) Representative picture of oil red O-stained BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates(I) Western blotting assay of ERK1/2, WTAP, ZFP410, PRDM16, and UCP1 in BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Data information: Data are representative of two independent experiments with biological replicates


Figure 7 ( B ) Representative pictures of nile red - stained human BAs differentiated from hESC - derived brown progenitors . Scale bar : 50 μm . ( C ) Time course analysis of the relative mRNA levels of EP3 receptors in hESC - derived BAs during BA differentiation detected by RT - qPCR ( n = 7 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates ( C , Statistical significance was evaluated by Welch ANOVA test followed by Tamhane ' s T2 test for post - hoc comparisons ( C ) * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( D ) Representative pictures of hESC - derived BAs treated with L798106 alone or combined with H89 . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological replicates ( E ) Effect of H89 on the relative mRNA level of brown marker gene in hESC - derived BAs treated with L798106 detected by RT - qPCR ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for multiple comparisons ( E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( F ) Effect of H89 on WTAP mRNA expression in hESC - derived BAs treated with L798106 ( n = 6 - 7 ) . ( G ) Effect of H89 on ZNF410 mRNA expression in hESC - derived BAs treated with L798106 ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two - way ANOVA followed by Tukey ' s test for multiple comparisons F , G ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) . ( H ) Western blotting assay of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , and UCP1 in hESC - derived BAs treated with L798106 alone or combined with H89 .

O O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B)Representative pictures of nile red-stained human BAs differentiated from hESC-derived brown progenitors. Scale bar: 50 μm.(C) Time course analysis of the relative mRNA levels of EP3 receptors in hESC-derived BAs during BA differentiation detected by RT-qPCR (n = 7-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates (C, Statistical significance was evaluated by Welch ANOVA test followed by Tamhane's T2 test for post-hoc comparisons (C) *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(D) Representative pictures of hESC-derived BAs treated with L798106 alone or combined with H89. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates(E) Effect of H89 on the relative mRNA level of brown marker gene in hESC-derived BAs treated with L798106 detected by RT-qPCR (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons (E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(F) Effect of H89 on WTAP mRNA expression in hESC-derived BAs treated with L798106 (n = 6-7). (G) Effect of H89 on ZNF410 mRNA expression in hESC-derived BAs treated with L798106 (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two-way ANOVA followed by Tukey's test for multiple comparisons F, G). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(H) Western blotting assay of ERK1/2, WTAP, ZNF410, PRDM16, and UCP1 in hESC-derived BAs treated with L798106 alone or combined with H89.


Figure 1A C57BL / 6 WT or Rag2 − / − mice were immunosuppressed with hydrocortisone + / − FK506 and infected with 105 AF CEA10 conidia intranasally . Mortality was assessed using a 20 % weight loss endpoint . Mortality was significantly increased in FK506 immunosuppressed mice reaching 100 % by day 10 p . i . ( P = 0 . 003 for HC versus HC + FK506 in Rag2 − / − using log - rank test , n = 10 per group ) . B , C PAS - stained lung sections of Rag2 − / − mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ . FK506 - treated animals had increased inflammation at day 2 p . i . compared to control . P = 0 . 0126 ( using Student ' s t - test ) , n = 3 - 5 per group . ( C ) Close - up of an infectious focus showing increased inflammation and fungal growth in the FK506 - treated group . Scale bar : 400 μm . D , E Murine macrophages ( J774A . 1 ) were pre - treated with hydrocortisone , and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy . The nuclei are indicated in red , and the GR is indicated in green . Scale bar : 50 μm . FK506 did not cause increased translocation of the glucocorticoid receptor . Data represent mean + SEM calculated from 10 fields of vision . One out of 2 experiments is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group).B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm.D, E Murine macrophages (J774A.1) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.


Figure 2Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . A Weight was monitored daily for 7 days p . i . Immunosuppressed mice showed increased weight loss through days 1 - 3 p . i . Data points represent mean ± SEM . Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . B BALs were performed over a 3 - day period p . i . and fungal killing assessed by enumerating CFUs . Fungal killing was significantly impaired in immunosuppressed mice . Error bars indicate SEM . Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . C , D Flow cytometry analysis of the CD45 + leucocyte populations in BAL . Representative dot plots for CD45 + cells ( C ) and percentages of Ly - 6G - positive neutrophils infiltrating the infected lungs over a 48 - h p . i . time course ( D ) . The bars indicate the mean percentage ( ± SEM ) of neutrophils of total CD45 + leucocytes ( n = 5 mice per group ) . Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p . i . Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . E PAS - stained lung sections of immunocompetent and FK506 - treated animals at 6 h p . i . Scale bar : 50 μm . IC animals showed inflammatory cell infiltrates around conidia ( black arrows ) , whereas in FK506 - treated animals , only uptake of conidia by the resident macrophage population could be seen . Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ . Bars represent mean percentages ( ± SEM ) , n = 3 . Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . G Luminex multiplex cytokine analysis of BAL SNs at 6 h p . i . Bars represent mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1-3 p.i. Data points represent mean ± SEM.Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM.Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n. C, D Flow cytometry analysis of the CD45+leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i.Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen.Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3.Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.


Figure 3At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . A , B Absolute neutrophil numbers in treated and untreated mpx : GFPlarvae were quantified by direct counting of fluorescent neutrophils in non - infected whole fish ( n = 11 - 13 per group ) . There were no significant differences . At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . C For survival analysis , lyz : dsRedlarvae were microinjected with ˜ 10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan - Meier analysis for survival over 6 days post - infection ( n = 16 per group ) . Immunosuppression led to a significant increase in mortality . At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . D TNF - α mRNA levels were measured in whole non - infected or infected larvae 48 h p . i . Immunosuppression led to significant and almost complete inhibition of TNF - α expression during infection . At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFP larvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . E - G Lyz : dsRedlarvae were infected with ˜ 50 eGFP - expressing conidia of A . fumigatus and were imaged daily from 24 h p . i . ( E ) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ . A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown ( 60 h p . i . ) . The arrows indicate infectious foci . ( F ) Neutrophil recruitment was minimal throughout the first 60 h p . i . in immunosuppressed larvae ( n = 8 per group ) . ( G ) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements ( n = 8 per group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.A, B Absolute neutrophil numbers in treated and untreated mpx:GFPlarvae were quantified by direct counting of fluorescent neutrophils in non-infected whole fish (n = 11-13 per group). There were no significant differences.At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.C For survival analysis, lyz:dsRedlarvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan-Meier analysis for survival over 6 days post-infection (n = 16 per group). Immunosuppression led to a significant increase in mortality.At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. D TNF-α mRNA levels were measured in whole non-infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF-α expression during infection.At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. E-G Lyz:dsRedlarvae were infected with ˜50 eGFP-expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group).


Figure 4A J774A . 1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . FK506 significantly reduced TNF - α levels at 5 and 10 ng / ml for AF . A significant reduction in TNF - α was also seen for zymosan , but not LPS . B J774A . 1 cells were treated with FK506 ( 10 ng / ml ) and either left non - stimulated ( NS ) or stimulated with AF SC ( MOI = 1 ) for 6 h . TNF - α mRNA levels were quantified from whole - cell RNA extracts by RT - PCR . FK506 significantly reduced TNF - α expression . C J774A . 1 macrophages were treated with control ( Ctrl . ) or calcineurin A ( CnA ) - targeting siRNA ( 50 nM ) and stimulated with either AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . CnA siRNA led to significant reductions in both AF - and zymosan - dependent TNF - α levels , but had no significant effect on LPS responses .

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Figure 4A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS.B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT-PCR. FK506 significantly reduced TNF-α expression.C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.


Figure 5A J774A . 1macrophages were stimulated with live AF SC ( MOI = 5 ) , and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2 . NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p . i . B J774A . 1 macrophages were stimulated with live or fixed swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFAT translocation was assessed by confocal microscopy . NFAT is indicated in green , the nuclei are indicated in red , and AF conidia can be seen in pink . Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan . Data represent mean ± SEM . Scale bar : 50 μm . Arrows indicate A . fumigatus or zymosan - positive cells . C J774A . 1 macrophages were stimulated with zymosan after FK506 ( 10 ng / ml ) or control treatment , and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF - κB translocation . NFATc2 and NF - κB nuclear translocation was observed , which was maximal at 1 h p . i . , in stimulated macrophages . FK506 specifically inhibited NFATc2 translocation , but had no effect on NF - κB . D J774A . 1 macrophages were pre - treated with FK506 ( 10 ng / ml ) or vehicle and stimulated with live swollen conidia or zymosan . NFATc2 translocation was evaluated 30 min after stimulation . Box and whisker plot shows median ± maximum / minimum . Scale bar : 50 μm . FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan . Statistical analysis was carried out by Student ' s t - test . NS , non - stimulated . Arrows indicate A . fumigatus or zymosan - positive cells .

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Figure 5A J774A.1macrophages were stimulated with live AF SC (MOI=5), and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2. NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p.i.B J774A.1 macrophages were stimulated with live or fixed swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFAT translocation was assessed by confocal microscopy. NFAT is indicated in green, the nuclei are indicated in red, and AF conidia can be seen in pink. Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan. Data represent mean ± SEM. Scale bar: 50 μm. Arrows indicate A. fumigatus or zymosan-positive cells.C J774A.1 macrophages were stimulated with zymosan after FK506 (10 ng/ml) or control treatment, and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF-κB translocation. NFATc2 and NF-κB nuclear translocation was observed, which was maximal at 1 h p.i., in stimulated macrophages. FK506 specifically inhibited NFATc2 translocation, but had no effect on NF-κB.D J774A.1 macrophages were pre-treated with FK506 (10 ng/ml) or vehicle and stimulated with live swollen conidia or zymosan. NFATc2 translocation was evaluated 30 min after stimulation. Box and whisker plot shows median ± maximum/minimum. Scale bar: 50 μm. FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan. Statistical analysis was carried out by Student's t-test. NS, non-stimulated. Arrows indicate A. fumigatus or zymosan-positive cells.


Figure 6A BMDMs from WT , Dectin - 1 − / − or MyD88 − / − mice were infected with swollen conidia ( MOI = 1 ) , and TNF - α was measured in the SN after overnight incubation . TNF - α levels were significantly reduced in Dectin1 − / − and Myd88 − / − BMDMs . B BMDMs were stimulated with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation . Whilst almost complete abrogation of NFAT translocation was seen in Dectin1 − / − macrophages stimulated with zymosan , NFAT responses to AF were unaffected . C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( C ) TNF - α was measured in the SN after overnight incubation . Syk inhibition significantly reduced swollen conidia - dependent or zymosan - dependent TNF - α levels . C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( D ) NFATc2 translocation was quantified by confocal microscopy . Syk inhibition impaired zymosan - dependent NFAT translocation , but did not affect AF - dependent NFAT translocation . C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( E ) Whole - cell lysates of Syk or Ctrl . siRNA - treated cells were separated by SDS - PAGE , followed by Western blotting , and membranes were probed with anti - Syk and anti - β - actin antibodies . F J774A . 1 macrophages were pre - treated with piceatannol and infected with swollen conidia ( MOI = 5 ) . Phagocytosis was quantified by FACS 30 min after stimulation . Syk inhibition led to impaired phagocytosis of zymosan , but had no effect on phagocytosis of SC . G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( G ) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy . G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( H ) For quantification of NF - κB translocation , nuclear extracts were separated by SDS - PAGE , followed by Western blotting . Membranes were probed with anti - NF - κB p65 and anti - HDAC1 antibodies . G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( I ) After overnight stimulation , TNF - α was measured in the SN by ELISA . Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF , but not ionomycin ( G ) . In addition , inhibition of phagocytosis lead to increased translocation of NF - κB ( H ) and increased levels of TNF - α in response to either zymosan or AF .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A BMDMs from WT, Dectin-1−/− or MyD88−/− mice were infected with swollen conidia (MOI = 1), and TNF-α was measured in the SN after overnight incubation. TNF-α levels were significantly reduced in Dectin1−/− and Myd88−/− BMDMs.B BMDMs were stimulated with swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation. Whilst almost complete abrogation of NFAT translocation was seen in Dectin1−/−macrophages stimulated with zymosan, NFAT responses to AF were unaffected.C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (C) TNF-α was measured in the SN after overnight incubation. Syk inhibition significantly reduced swollen conidia-dependent or zymosan-dependent TNF-α levels.C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (D) NFATc2 translocation was quantified by confocal microscopy. Syk inhibition impaired zymosan-dependent NFAT translocation, but did not affect AF-dependent NFAT translocation.C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (E) Whole-cell lysates of Syk or Ctrl. siRNA-treated cells were separated by SDS-PAGE, followed by Western blotting, and membranes were probed with anti-Syk and anti-β-actin antibodies.F J774A.1 macrophages were pre-treated with piceatannol and infected with swollen conidia (MOI = 5). Phagocytosis was quantified by FACS 30 min after stimulation. Syk inhibition led to impaired phagocytosis of zymosan, but had no effect on phagocytosis of SC.G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (G) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy.G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (H) For quantification of NF-κB translocation, nuclear extracts were separated by SDS-PAGE, followed by Western blotting. Membranes were probed with anti-NF-κB p65 and anti-HDAC1 antibodies.G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (I) After overnight stimulation, TNF-α was measured in the SN by ELISA. Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF, but not ionomycin (G). In addition, inhibition of phagocytosis lead to increased translocation of NF-κB (H) and increased levels of TNF-α in response to either zymosan or AF.


Figure 7A J774A . 1 macrophages were pre - treated with bafilomycin A1 ( 100 nM ) and stimulated with swollen conidia ( MOI = 3 ) for 30 min . NFATc2 translocation was assessed by confocal microscopy . Acidification was required for NFATc2 translocation . B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP - expressing swollen conidia by confocal microscopy . Scale bar : 10 μm . The arrow indicates a TLR9 - positive AF - containing phagosome . C BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 1 ) , and after overnight stimulation , TNF - α was measured in the SN by ELISA . TLR9 − / − BMDMs had significantly lower TNF - α levels . D , E BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 5 ) for 30 min , and NFATc2 translocation was quantified by ( D ) confocal microscopy . TLR9 − / − BMDMs exhibited reduced NFATc2 translocation by both approaches . G BMDMs from WT or MyD88 − / − mice were pre - treated with ODN2088 ( 10 μM ) and infected with swollen conidia ( MOI = 5 ) for 30 min . NFATc2 translocation was quantified by confocal microscopy . In both , WT and MyD88 − / − BMDMs , blocking TLR9 significantly impaired NFATc2 translocation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A J774A.1 macrophages were pre-treated with bafilomycin A1 (100 nM) and stimulated with swollen conidia (MOI = 3) for 30 min. NFATc2 translocation was assessed by confocal microscopy. Acidification was required for NFATc2 translocation.B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP-expressing swollen conidia by confocal microscopy. Scale bar: 10 μm. The arrow indicates a TLR9-positive AF-containing phagosome.C BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 1), and after overnight stimulation, TNF-α was measured in the SN by ELISA. TLR9−/− BMDMs had significantly lower TNF-α levels.D, E BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 5) for 30 min, and NFATc2 translocation was quantified by (D) confocal microscopy. TLR9−/− BMDMs exhibited reduced NFATc2 translocation by both approaches.G BMDMs from WT or MyD88−/−mice were pre-treated with ODN2088 (10 μM) and infected with swollen conidia (MOI = 5) for 30 min. NFATc2 translocation was quantified by confocal microscopy. In both, WT and MyD88−/− BMDMs, blocking TLR9 significantly impaired NFATc2 translocation.


Figure 8A , B J774A . 1 macrophages were pre - treated with the BTK inhibitor LFM - A13 ( 12 . 5 μM ) and infected with swollen conidia . ( A ) After overnight stimulation , TNF - α was measured in the SN by ELISA . A , B J774A . 1 macrophages were pre - treated with the BTK inhibitor LFM - A13 ( 12 . 5 μM ) and infected with swollen conidia . ( B ) After 30 min of stimulation , NFATc2 translocation was quantified by confocal microscopy . Both TNF - α levels and NFATc2 translocation were significantly reduced by BTK blockade . C J774A . 1 macrophages were treated with a control or BTK - targeting siRNA ( 25 nM ) , and NFAT translocation was quantified by confocal microscopy . BTK knock - down led to a significant reduction in NFATc2 translocation in response to AF . D , E J774A . 1 macrophages were treated with a control , BTK ( 25 nM ) - or TLR9 ( 75 nM ) - targeting siRNA and additionally pre - treated with either ODN2088 ( 10 μM ) or the BTK inhibitor LFM - A13 ( 12 . 5 μM ) . The cells were infected with swollen conidia ( MOI = 5 ) , and NFATc2 translocation was measured by confocal microscopy . Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA.A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade.C J774A.1 macrophages were treated with a control or BTK-targeting siRNA (25 nM), and NFAT translocation was quantified by confocal microscopy. BTK knock-down led to a significant reduction in NFATc2 translocation in response to AF.D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.


Figure 9Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . Cells were pre - treated for 1 h with FK506 ( 10 ng / ml ) , the NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in the cell culture supernatant by ELISA . A Representative FACS - phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples . Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . Cells were pre - treated for 1 h with FK506 ( 10 ng / ml ) , the NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in the cell culture supernatant by ELISA . B Inhibition of alveolar macrophage responses to AF with FK506 , MCV1 or SC514 led to a significant reduction in TNF - α levels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples.Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.


Figure 1A . Representative FACS plot ( left ) showing a mixed - lineage clone with a T - lymphoid ( CD25 + / CD11b - Gr1 - ) , myeloid ( CD11b + Gr1 + / CD25 - ) , and biphenotypic ( CD11b + Gr1 + / CD25 + ) fraction derived from a single Myc / Bcl2 - transformed DN2cell grown on 1 : 1 OP9 : OP9 - Dll1 co - culture with lineage - promiscuous cytokines IL - 2 , IL - 3 , IL - 6 , IL - 7 , SCF , GM - CSF , Flt3 . Morphology of the indicated flow - sorted cell fractions was analyzed by Giemsa staining ( right ) . B . Phenotype frequencies of clones from Myc / Bcl2 - transformed single cells ( grown and phenotyped as mentioned in A ) . Analyzed were 31 clones from LSKs , 107 from GMPs and 28 from DN2 cells . C . Cumulative survival of 31 ± 2 and 27 ± 6 days of mice that received 5104 to 5105 T - lymphoid cells or myeloid cells , respectively , that were flow - sorted from mixed Myc / Bcl2 + DN2 - derived clones . The summarized data are from 3 mice injected with the T - lymphoid and 4 mice injected with the myeloid fraction of two different clones . D . Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc / Bcl2 - transformed LSK or GMPcells on OP9 : OP9 - Dll1 co - cultures . E . ( top panels ) Serial replating of Myc - Bcl2 + DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro ( gel : left two lanes ) . Upon transplantation , the same clones induced myeloid leukemia that retained the initial rearrangement in vivo ( gel : right two lanes ) as assessed by nested PCR . GL = germline . ( bottom panels ) Methylcellulose - based replating of Myc - Bcl2 + GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration . F . Cumulative survival of mice that received 106cells of TCRβ - rearranged myeloid DN2 - derived clones leading to death within 33 ± 4 days after transplantation . Two DN2 clones were transplanted into 5 recipient mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Representative FACS plot (left) showing a mixed-lineage clone with a T-lymphoid (CD25+/CD11b-Gr1-), myeloid (CD11b+Gr1+/CD25-), and biphenotypic (CD11b+Gr1+/CD25+) fraction derived from a single Myc/Bcl2-transformed DN2cell grown on 1:1 OP9:OP9-Dll1 co-culture with lineage-promiscuous cytokines IL-2, IL-3, IL-6, IL-7, SCF,GM-CSF, Flt3. Morphology of the indicated flow-sorted cell fractions was analyzed by Giemsa staining (right).B. Phenotype frequencies of clones from Myc/Bcl2-transformed single cells (grown and phenotyped as mentioned in A). Analyzed were 31 clones from LSKs, 107 from GMPs and 28 from DN2 cells.C. Cumulative survival of 31±2 and 27±6 days of mice that received 5104 to 5105 T-lymphoid cells or myeloid cells, respectively, that were flow-sorted from mixed Myc/Bcl2+ DN2-derived clones. The summarized data are from 3 mice injected with the T-lymphoid and 4 mice injected with the myeloid fraction of two different clones.D. Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc/Bcl2-transformed LSK or GMPcells on OP9:OP9-Dll1 co-cultures.E. (top panels) Serial replating of Myc-Bcl2+DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro (gel: left two lanes). Upon transplantation, the same clones induced myeloid leukemia that retained the initial rearrangement in vivo (gel: right two lanes) as assessed by nested PCR. GL = germline. (bottom panels) Methylcellulose-based replating of Myc-Bcl2+GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration.F. Cumulative survival of mice that received 106cells of TCRβ-rearranged myeloid DN2-derived clones leading to death within 33±4 days after transplantation. Two DN2 clones were transplanted into 5 recipient mice.


Figure 2A . Representative FACS plots of spleens from leukemic recipient mice that had received LSK , GMP or DN2 cell grafts immediately after retroviral Myc / Bcl2 transduction . Numbers indicate frequencies ( % ) of CD45 . 2 donor cells within the indicated gates summarizing the mean ± SEM of at least 8 recipients per group from a total of 10 independent experiments . B . Cumulative survival of mice that received 2104 to 2105 freshly Myc / Bcl2 - transduced LSK , GMP or DN2 cells ; Graph shows n = 11 Myc - Bcl2 + LSK - , n = 14 DN2 - , and n = 8 GMP - transplanted animals . C . Histological spleen sections of Myc - Bcl2 + LSK - , GMP - or DN2 - transplanted mice displaying myeloperoxidasestaining . Leukemic cell engraftment in spleens ranged from 75 . 1 % - 95 . 5 % . D . Multiplex quantitative RT - PCR demonstrating strong expression of PU . 1 in the myeloid ( M3L : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) and biphenotypic T / myeloid ( BL : CD11b + Gr1 + / CD3 + CD4 + CD8 + ) fractions but not in the T - lymphoid ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) fraction that were flow - sorted from diseased Myc - Bcl2 + DN2 - transplanted mice . Myeloid progeny of Myc / Bcl2 + GMP - transplanted or T - lymphoid progeny of Myc / Bcl2 + DN3 - transplanted animals as well as healthy thymocytes ( Thy ) and BM cells from WT mice served as controls . Results are shown as fold over actin transcripts . E . D - J rearrangement PCR of flow - sorted T - lymphoid ( TL ) , biphenotypic ( BL ) and myeloid ( ML ) fractions of donor splenocytes of a diseased Myc / Bcl2 + DN2 - transplanted mouse . All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus ( Dβ1Jβ1 . 5 ) demonstrating clonality in DN2 leukemia . thy = thymus . F . FACS plot of diseased intrathymic grafts ( CD45 . 2 - gated ) , demonstrating that Myc / Bcl2 + DN2 cells could produce multi - lineage leukemia within a T - cell supporting environment ; Myc / Bcl2 + GMPs remained myeloid restricted under these conditions . Numbers indicate cell frequencies within the gates ( mean ± SEM ) representing six independent experiments . G . Intrathymic transplantation of mock - infected non - leukemic DN2 cells produced predominantly T - lymphoid but also myeloid progeny 23 days after injection . This differentiation outcome resembles that of freshly isolated DN2 cells ( Bell and Bhandoola , 2008 ; Richie Ehrlich et al . , 2011 ) , and indicates that the short - term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells .

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Figure 2A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments.B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+LSK-, n=14 DN2-, and n=8 GMP-transplanted animals.C. Histological spleen sections of Myc-Bcl2+LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidasestaining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%.D. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3-CD4-CD8-) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b-Gr1-/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts.E. D-J rearrangement PCR of flow-sorted T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus.F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments.G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.


Figure 3A . GSEA demonstrates that Myc / Bcl2 + DN2 - derived myeloid blasts were significantly ( FDR > 0 . 05 ) enriched for expression of a pan - dendritic cell gene set . Gene sets were published in ( Schonheit et al . , 2013 ) . B . Heat map of the top 100 differentially expressed genes ( adjusted p - value of ≤ 0 . 05 ) in each myeloid fraction ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from GMP - , LSK - and DN2 - derived leukemias . D . GSEA demonstrates significant enrichment of a normal DN2 - specific gene set ( see methods section ) in the Myc / Bcl2 + DN2 - derived myeloid blasts .

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Figure 3A. GSEA demonstrates that Myc/Bcl2+DN2-derived myeloid blasts were significantly (FDR >0.05) enriched for expression of a pan-dendritic cell gene set. Gene sets were published in (Schonheit et al., 2013).B. Heat map of the top 100 differentially expressed genes (adjusted p-value of ≤ 0.05) in each myeloid fraction (ML: CD11b+Gr1+/CD3-CD4-CD8-) from GMP-, LSK- and DN2-derived leukemias.D. GSEA demonstrates significant enrichment of a normal DN2-specific gene set (see methods section) in the Myc/Bcl2+DN2-derived myeloid blasts.