C Activity of whole blood culture upon endosome function inhibition ( chloroquine ) and challenge with TLR ligands , heat inactivated ( hi ) , or viable bacteria .

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C Activity of whole blood culture upon endosome function inhibition (chloroquine) and challenge with TLR ligands, heat inactivated (hi), or viable bacteria.


B . Comparisons of MTAP mRNA and protein expression between CL1 - 0 and CL1 - 5 cells detected by RT - qPCR ( top , mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) and Western blot assays using anti - MTAP antibody ( bottom , representative of three independent experiments ) .

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B. Comparisons of MTAP mRNA and protein expression between CL1-0 and CL1-5 cells detected by RT-qPCR (top, mean ± SD, Student t test, n=3, biological replicates, *p < 0.05) and Western blot assays using anti-MTAP antibody (bottom, representative of three independent experiments).


C . Cell invasion abilities of MTAP - overexpressing CL1 - 5 or MTAP - knockout CL1 - 0 and H1650 were determined by Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) . sgCTRL indicates cells transduced with a non - targeting control sgRNA , and sgMTAP indicates cells transduced with a sgRNA targeting MTAP . Bottom : the protein expression levels of V5 - MTAP and endogenous MTAP were detected by Western blots .

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C. Cell invasion abilities of MTAP-overexpressing CL1-5 or MTAP-knockout CL1-0 and H1650 were determined by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). sgCTRL indicates cells transduced with a non-targeting control sgRNA, and sgMTAP indicates cells transduced with a sgRNA targeting MTAP. Bottom: the protein expression levels of V5-MTAP and endogenous MTAP were detected by Western blots.


E . Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays . Top : representative photographs of lungs and H & E staining of the lung sections . The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows . Bottom : quantification of averaged primary tumor sizes , metastatic incidence and nodule number .

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E. Effect of MTAP overexpression on lung cancer metastasis in vivo was demonstrated by orthotopic implantation assays. Top: representative photographs of lungs and H&E staining of the lung sections. The primary tumors are indicated by arrowheads and the metastatic nodules are indicated by arrows. Bottom: quantification of averaged primary tumor sizes, metastatic incidence and nodule number.


F . Kaplan - Meier analyses of overall survival ( top ) and progression - free survival ( bottom ) for 101 patients with lung adenocarcinoma grouped into high - or low - MTAP mRNA expression measured by RT - qPCR . p values were obtained by log - rank test .

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F. Kaplan-Meier analyses of overall survival (top) and progression-free survival (bottom) for 101 patients with lung adenocarcinoma grouped into high- or low-MTAP mRNA expression measured by RT-qPCR. p values were obtained by log-rank test.


A . The necessity of MTAP activity in cell invasion . CL1 - 5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 compared to the vector control group ) . The protein expression levels of exogenous wild - type and mutant V5 - MTAP were detected by Western blots .

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A. The necessity of MTAP activity in cell invasion. CL1-5 and A549 cells were introduced with indicated plasmids and subjected to Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05 compared to the vector control group). The protein expression levels of exogenous wild-type and mutant V5-MTAP were detected by Western blots.


B . Western blot analysis of the symmetric dimethylarginine ( sDMA ) , monomethylarginine ( mMA ) and asymmetric dimethylarginine ( aDMA ) levels in MTAP - overexpressing and MTAP - knockout cells . V5 - MTAP wild - type and D220A mutant were restored in H1650 MTAP - knockout cells . Data shown are representative of three independent experiments .

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B. Western blot analysis of the symmetric dimethylarginine (sDMA), monomethylarginine (mMA) and asymmetric dimethylarginine (aDMA) levels in MTAP-overexpressing and MTAP-knockout cells. V5-MTAP wild-type and D220A mutant were restored in H1650 MTAP-knockout cells. Data shown are representative of three independent experiments.


C . In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti - sDMA antibody . Arrow marks dimethyl - vimentin .

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C. In vitro methyltransferase assays of vimentin by PRMT5 analyzed by Western blots with anti-sDMA antibody. Arrow marks dimethyl-vimentin.


D . Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells . Arrow marks dimethyl - vimentin . Data shown are representative of three independent experiments .

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D. Immunoprecipitation analysis for vimentin dimethylation in response to PRMT5 knockdown in H1650 cells. Arrow marks dimethyl-vimentin. Data shown are representative of three independent experiments.


E - F . Immunoprecipitation analysis for vimentin dimethylation in control and MTAP - knockout cells . Arrows mark vimentin , asterisks mark heavy chains , and arrowheads mark nonspecific bands . Data shown are representative of three independent experiments .

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E-F. Immunoprecipitation analysis for vimentin dimethylation in control and MTAP-knockout cells. Arrows mark vimentin, asterisks mark heavy chains, and arrowheads mark nonspecific bands. Data shown are representative of three independent experiments.


G . Vimentin - knockdown counteracts MTAP - loss - induced effect on cell invasion . MTAP - knockout and control H1650 cells were transfected with vimentin - specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays ( mean ± SD , Student t test , n = 3 , biological replicates , * p < 0 . 05 ) . The silence efficiency of vimentin was examined by Western blots .

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G. Vimentin-knockdown counteracts MTAP-loss-induced effect on cell invasion. MTAP-knockout and control H1650 cells were transfected with vimentin-specific siRNAs for 72 hours and analyzed by Boyden chamber invasion assays (mean ± SD, Student t test, n=3, biological replicates, *p < 0.05). The silence efficiency of vimentin was examined by Western blots.


F . Validation of the PRMT5 - catalyzed methylation sites on V5 - vimentin . HEK293T cells were transfected with indicated constructs and subjected to immunoprecipitation analysis . The intensity of sDMA signals was quantified by ImageJ software , normalized to the intensity of corresponding immunoprecipitated V5 - vimentin , then relative to the V5 - vimentin wild - type group and represented as percentage . Data were the averaged percentage from three independent experiments .

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F. Validation of the PRMT5-catalyzed methylation sites on V5-vimentin. HEK293T cells were transfected with indicated constructs and subjected to immunoprecipitation analysis. The intensity of sDMA signals was quantified by ImageJ software, normalized to the intensity of corresponding immunoprecipitated V5-vimentin, then relative to the V5-vimentin wild-type group and represented as percentage. Data were the averaged percentage from three independent experiments.


B . Reconstitution of vimentin - knockout cells with wild - type , 4RK - and 4RF - mutated V5 - vimentin . Both protein and mRNA levels of vimentin were determined by Western blots and RT - qPCR . Data shown are representative of three independent experiments .

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B. Reconstitution of vimentin-knockout cells with wild-type, 4RK- and 4RF-mutated V5-vimentin. Both protein and mRNA levels of vimentin were determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.


C . Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT - qPCR . Data shown are representative of three independent experiments .

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C. Vimentin protein and mRNA levels in indicated paired cells were measured by Western blots and RT-qPCR. Data shown are representative of three independent experiments.


D . Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT - qPCR . Data shown are representative of three independent experiments .

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D. Effect of MTA on vimentin protein and mRNA levels determined by Western blots and RT-qPCR. Data shown are representative of three independent experiments.


E . Western blot analysis of vimentin protein levels in MTAP - knockout and control cells transfected with PRMT5 siRNAs ( left ) or Flag - PRMT5 ( right ) . Arrows mark the site of Flag - PRMT5 and endogenous PRMT5 . Data shown are representative of three independent experiments .

E. Western blot analysis of vimentin protein levels in MTAP-knockout and control cells transfected with PRMT5 siRNAs (left) or Flag-PRMT5 (right). Arrows mark the site of Flag-PRMT5 and endogenous PRMT5. Data shown are representative of three independent experiments.


F . Effect of PRMT5 on the levels of ectopically expressed wild - type , 4RK - and 4RF - mutated V5 - vimentin in HEK293T cells assessed by Western blots . Data shown are representative of three independent experiments . G . Effects of wild - type and catalytically dead R368A mutant PRMT5 on the level of V5 - vimentin in HEK293T cells detected by Western blot assays . Data shown are representative of three independent experiments .

F. Effect of PRMT5 on the levels of ectopically expressed wild-type, 4RK- and 4RF-mutated V5-vimentin in HEK293T cells assessed by Western blots. Data shown are representative of three independent experiments. G. Effects of wild-type and catalytically dead R368A mutant PRMT5 on the level of V5-vimentin in HEK293T cells detected by Western blot assays. Data shown are representative of three independent experiments.


A . Measurement of V5 - vimentin wild - type , 4RK and 4RF mutant protein levels in CL1 - 5 vimentin - knockout cells treated with 10 μM MG132 by Western blots . Data shown are representative of three independent experiments .

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A. Measurement of V5-vimentin wild-type, 4RK and 4RF mutant protein levels in CL1-5 vimentin-knockout cells treated with 10 μM MG132 by Western blots. Data shown are representative of three independent experiments.


B . Western blot analysis of vimentin protein levels in control and MTAP - knockout cells treated with 10 μM MG132 . Data shown are representative of three independent experiments .

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B. Western blot analysis of vimentin protein levels in control and MTAP-knockout cells treated with 10 μM MG132. Data shown are representative of three independent experiments.


Immunoprecipitation analysis of ubiquitination ( C ) of V5 - vimentin in HEK293T cells transfected with wild - type , 4RK - or 4RF - mutated vimentin in the presence of HA - tagged ubiquitin . Arrows mark the sites of V5 - vimentin . Data shown are representative of three independent experiments .

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Immunoprecipitation analysis of ubiquitination (C) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.


K48 - linked polyubiquitination ( D ) of V5 - vimentin in HEK293T cells transfected with wild - type , 4RK - or 4RF - mutated vimentin in the presence of HA - tagged ubiquitin . Arrows mark the sites of V5 - vimentin . Data shown are representative of three independent experiments .

K48-linked polyubiquitination (D) of V5-vimentin in HEK293T cells transfected with wild-type, 4RK- or 4RF-mutated vimentin in the presence of HA-tagged ubiquitin. Arrows mark the sites of V5-vimentin. Data shown are representative of three independent experiments.


E . Immunoprecipitation analysis for K48 - linked polyubiquitination of vimentin in CL1 - 0 control and MTAP - knockout cells . Arrow marks the site of vimentin . Data shown are representative of three independent experiments .

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E. Immunoprecipitation analysis for K48-linked polyubiquitination of vimentin in CL1-0 control and MTAP-knockout cells. Arrow marks the site of vimentin. Data shown are representative of three independent experiments.


F - G . Western blot analysis of V5 - vimentin wild - type , 4RK and 4RF mutant levels in CL1 - 0 cells ( F ) or endogenous vimentin levels in control and MTAP - knockout cells ( G ) treated with 50 μg / ml cycloheximide ( CHX ) . Bottom : the intensity of V5 - vimentin and vimentin signals was quantified by ImageJ software , normalized to the internal control β - actin , then normalized to zero time point , and plotted against time points ( mean ± SE , n = 3 , biological replicates ) .

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F-G. Western blot analysis of V5-vimentin wild-type, 4RK and 4RF mutant levels in CL1-0 cells (F) or endogenous vimentin levels in control and MTAP-knockout cells (G) treated with 50 μg/ml cycloheximide (CHX). Bottom: the intensity of V5-vimentin and vimentin signals was quantified by ImageJ software, normalized to the internal control β-actin, then normalized to zero time point, and plotted against time points (mean ± SE, n=3, biological replicates).


A - B . The correlation of MTAP and vimentin or PRMT5 and vimentin at protein ( A ) and mRNA ( B ) levels from 89 lung adenocarcinoma patients . r is the Spearman ' s rank correlation coefficient .

A-B. The correlation of MTAP and vimentin or PRMT5 and vimentin at protein (A) and mRNA (B) levels from 89 lung adenocarcinoma patients. r is the Spearman's rank correlation coefficient.


C . Representative images of IHC staining .

C. Representative images of IHC staining.


D . An association between MTAP and vimentin levels in 124 lung cancer patients was examined by Spearman ' s rank correlation .

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D. An association between MTAP and vimentin levels in 124 lung cancer patients was examined by Spearman's rank correlation.


E . Kaplan - Meier analyses of overall survival for lung cancer patients grouped by MTAP and vimentin levels from IHC staining . p value was obtained by log - rank test .

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E. Kaplan-Meier analyses of overall survival for lung cancer patients grouped by MTAP and vimentin levels from IHC staining. p value was obtained by log-rank test.


( B ) Notch signaling effector Hes5 is expressed high in NSCs while Eomes ( Tbr2 ) and Btg2 are expressed high in both NSCs and BPs at the mRNA level . Each dot defines the mean and lines define the Standard Deviation ( SD ) . 3 - 4 biological replicates were collected for each time point .

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(B) Notch signaling effector Hes5 is expressed high in NSCs while Eomes (Tbr2) and Btg2 are expressed high in both NSCs and BPs at the mRNA level. Each dot defines the mean and lines define the Standard Deviation (SD). 3-4 biological replicates were collected for each time point.


( C ) Heatmap validating the known cell - type specific marker gene expression from RNA sequencing data . ( E ) Heatmap illustrating the novel marker genes identified from the RNA sequencing data , as signature genes for NSCs , BPs and NBNs . NSCs - Neural stem cells , BPs - Basal progenitors , NBNs - Newborn neurons , E - Embryonic day , PN - Post natal , VZ - Ventricular zone , SVZ - Subventricular zone , IZ - Intermediate Zone . Expression values on the heatmaps are log2 ( transcripts per million ) .

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(C) Heatmap validating the known cell-type specific marker gene expression from RNA sequencing data. (E) Heatmap illustrating the novel marker genes identified from the RNA sequencing data, as signature genes for NSCs, BPs and NBNs. NSCs- Neural stem cells, BPs-Basal progenitors, NBNs-Newborn neurons, E-Embryonic day, PN-Post natal, VZ-Ventricular zone, SVZ- Subventricular zone, IZ-Intermediate Zone. Expression values on the heatmaps are log2 (transcripts per million).


( D ) Expression profiles of PC1 and PC2 along developmental time ( top ) and expression profiles of the top three genes with highest negative and positive projection on PC1 ( first and second column ) and highest negative and positive projection on PC2 ( third and fourth column ) for NSCs . Data Information : In D , ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

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(D) Expression profiles of PC1 and PC2 along developmental time (top) and expression profiles of the top three genes with highest negative and positive projection on PC1 (first and second column) and highest negative and positive projection on PC2 (third and fourth column) for NSCs. Data Information: In D, (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( H ) Expression profiles of PC1 and PC2 across all samples with time from left to right and NSCs in green , BPs in red , and NBNs in purple ( top ) , and the gene expression profiles of top three genes with highest negative and positive projection on PC1 ( first and second column ) and highest negative and positive projection on PC2 ( third and fourth column ) . Data Information : In H , ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

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(H) Expression profiles of PC1 and PC2 across all samples with time from left to right and NSCs in green, BPs in red, and NBNs in purple (top), and the gene expression profiles of top three genes with highest negative and positive projection on PC1 (first and second column) and highest negative and positive projection on PC2 (third and fourth column). Data Information: In H, (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( L ) As in panel D but now for BPs . Data Information : In ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

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(L) As in panel D but now for BPs. Data Information: In (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( P ) As in panels D and L but now for NBNs . Data Information : In P ( bottom ) , the x - axis is embryonic days , and the y - axis is log2 ( TPM ) .

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(P) As in panels D and L but now for NBNs. Data Information: In P (bottom), the x-axis is embryonic days, and the y-axis is log2(TPM).


( B ) Heatmap illustrating the dynamics of expression of cortical layering markers in NSCs at population level . In B , , heatmaps are based on z - score of log2 ( TPM ) expression values .

(B) Heatmap illustrating the dynamics of expression of cortical layering markers in NSCs at population level. In B, , heatmaps are based on z-score of log2(TPM) expression values.


( C ) Examples of expression dynamics of deep layer markers Tbr1 , Ctip2 and upper layer markers Satb2 , Cux2 in NSCs , BPs and NBNs , profiles at population level ( left ) and single cell level ( right ) . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point .

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(C) Examples of expression dynamics of deep layer markers Tbr1, Ctip2 and upper layer markers Satb2, Cux2 in NSCs, BPs and NBNs, profiles at population level (left) and single cell level (right). Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( D ) Experimental validation of NSCs isolated at E13 . 5 using Hes5 : : GFP transgenic embryos , showing no detectable protein for Tbr1 , Ctip2 and Satb2 . NSCs do express Brn2 ( Pou3f2 ) in vitro and in vivo at protein level . Scale bar = 20μm .

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(D) Experimental validation of NSCs isolated at E13.5 using Hes5::GFP transgenic embryos, showing no detectable protein for Tbr1, Ctip2 and Satb2. NSCs do express Brn2 (Pou3f2) in vitro and in vivo at protein level. Scale bar = 20μm.


( E ) Heatmap illustrating the dynamics of expression of cortical layering markers in BPs at population level . ( G ) Heatmap illustrating the dynamics of expression of cortical layering markers in NBNs at population level . heatmaps are based on z - score of log2 ( TPM ) expression values .

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(E) Heatmap illustrating the dynamics of expression of cortical layering markers in BPs at population level. (G) Heatmap illustrating the dynamics of expression of cortical layering markers in NBNs at population level. heatmaps are based on z-score of log2(TPM) expression values.


( F ) Experimental validation of BPs isolated at E16 . 5 using Tbr2 : : GFP transgenic embryos , showing no detectable protein for Tbr1 , Ctip2 and Satb2 . Scale bar = 20μm . ( H ) Experimental validation of NBNs isolated at E16 . 5 using Tbr2 : : GFP transgenic embryos , showing protein expression for Tbr1 , Ctip2 , Satb2 and Brn2 ( Pou3f2 ) . Scale bar = 20μm .

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(F) Experimental validation of BPs isolated at E16.5 using Tbr2::GFP transgenic embryos, showing no detectable protein for Tbr1, Ctip2 and Satb2. Scale bar = 20μm. (H) Experimental validation of NBNs isolated at E16.5 using Tbr2::GFP transgenic embryos, showing protein expression for Tbr1, Ctip2, Satb2 and Brn2(Pou3f2). Scale bar = 20μm .


( D ) Expression profile of selected target or modulator of key signaling pathways : BMP , Wnt , Shh and Notch signaling . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point .

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(D) Expression profile of selected target or modulator of key signaling pathways: BMP, Wnt, Shh and Notch signaling. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( B ) Expression profile of selected stem cell maintenance markers Hes1 , Hey1 and Id4 . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point . ( C ) Expression profile of selected neurogenic markers Neurog2 , Neurod2 , and Neurod6 . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point . ( D ) Expression profile of selected gliogenic markers Olig1 , Olig2 and Id1 . Each dot defines the mean and lines define the SD . 3 - 4 biological replicates were collected for each time point .

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(B) Expression profile of selected stem cell maintenance markers Hes1, Hey1 and Id4. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point. (C) Expression profile of selected neurogenic markers Neurog2, Neurod2, and Neurod6. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point. (D) Expression profile of selected gliogenic markers Olig1, Olig2 and Id1. Each dot defines the mean and lines define the SD. 3-4 biological replicates were collected for each time point.


( E ) Normalized expression levels of the native core promoters . Their activities spanned over a broad range of three orders of magnitude ( promoter constructs contained block 1 . 11 and block 7 . 11 combination as nucleosomal sequences ) . Each color represents a different class of core promoter architecture . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements . ( including new cell transfection procedures and measurements of promoter activities ) .

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(E) Normalized expression levels of the native core promoters. Their activities spanned over a broad range of three orders of magnitude (promoter constructs contained block 1.11 and block 7.11 combination as nucleosomal sequences). Each color represents a different class of core promoter architecture. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (including new cell transfection procedures and measurements of promoter activities).


( I ) Scatter plot of expression levels obtained in D . mel . S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D . mel . embryos , revealing a high correlation ( Pearson coefficient 0 . 91 ) between the two datasets . Error bars represent standard deviations of 3 - 4 biological replicate measurements .

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(I) Scatter plot of expression levels obtained in D. mel. S2 cells by our Luciferase assay pipeline vs mNeonGreen reporter expression in living D. mel. embryos, revealing a high correlation (Pearson coefficient 0.91) between the two datasets. Error bars represent standard deviations of 3-4 biological replicate measurements.


( C ) Boxplots depicting block 1 effects for tested core promoters . Effects of different block 7s were merged in each column ( within the same block 1 ) : the median SD is 0 . 66 for developmental promoters compared to 1 . 23 for constitutive promoters ( lower right corner ) ; Wilcoxon rank - sum test * * * p = 3 . 1 × 10 - 4 , significant . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements . ( D ) Boxplots depicting block 7 effects for tested core promoters . Effects of different block 1s were merged in each column ( within the same block 7 ) : the median SD is 0 . 54 for developmental promoters compared to 0 . 64 for constitutive promoters ( lower right corner ) ; Wilcoxon rank - sum test p = 0 . 3 , not significant . Block 7 with column " w / o B7 " represents the results obtained from promoters without block 7 sequence . Developmental and constitutive promoters are highlighted in green and red , respectively . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . Individual points represent values over 1 . 5 times the interquartile range . 3 - 4 biological replicate measurements .

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

(C) Boxplots depicting block 1 effects for tested core promoters. Effects of different block 7s were merged in each column (within the same block 1): the median SD is 0.66 for developmental promoters compared to 1.23 for constitutive promoters (lower right corner); Wilcoxon rank-sum test ***p = 3.1×10-4, significant. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements. (D) Boxplots depicting block 7 effects for tested core promoters. Effects of different block 1s were merged in each column (within the same block 7): the median SD is 0.54 for developmental promoters compared to 0.64 for constitutive promoters (lower right corner); Wilcoxon rank-sum test p = 0.3, not significant. Block 7 with column "w/o B7" represents the results obtained from promoters without block 7 sequence. Developmental and constitutive promoters are highlighted in green and red, respectively. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. Individual points represent values over 1.5 times the interquartile range. 3-4 biological replicate measurements.


( B - C ) Comparison of normalized expression levels between wild - type configuration and motif knockouts for two types of core promoters ( developmental : CG8157 ( B ) ; constitutive : RpL5 ( C ) ) . Upper panels : schematic depiction of the wild - type motif compositions ( TTGTT motif in RpL5 is ignored due to its strong overlap with R - INR ) . Two - sample t - test : ns , not significant , * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . The middle hinge represents the median . The Interquartile range the difference between the 75th and 25th percentiles . 3 - 4 biological replicate measurements .

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

(B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements.


( A ) Consensus replacement . Heatmap depicting the mean expression fold changes compared to wild - type expressions after replacing with motif consensus sequences derived by XXmotif . Constitutive and developmental promoters are highlighted in red and green , respectively .

(A) Consensus replacement. Heatmap depicting the mean expression fold changes compared to wild-type expressions after replacing with motif consensus sequences derived by XXmotif. Constitutive and developmental promoters are highlighted in red and green, respectively.


( B ) Expression fold changes ( ecdysone inducibility ) versus measurements without ecdysone and grouped by native core promoter sequences . The colors refer to different core promoter architectures . Three types of line are used to indicate the expression fold change with no increase ( y = x ; solid line ) , 2 - fold increase ( y = x + 1 ; dotted line ) and 4 - fold increase ( y = x + 2 ; dashed line ) . Red vertical dashed line : log2 basal expressions = 2 .

O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression fold changes (ecdysone inducibility) versus measurements without ecdysone and grouped by native core promoter sequences. The colors refer to different core promoter architectures. Three types of line are used to indicate the expression fold change with no increase (y = x; solid line), 2-fold increase (y = x + 1; dotted line) and 4-fold increase (y = x + 2; dashed line). Red vertical dashed line: log2 basal expressions = 2.


( C ) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters . Disrupted INR ( highlighted with the black dotted line rectangle ) had a slightly negative effect on changing the core promoter responsiveness to ecdysone . ( ~ 2 . 3 - fold reduction on average , Wilcoxon rank - sum test p = 2 . 1 × 10 - 5 ) . Constitutive and developmental promoters highlighted in red and green , respectively .

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

(C) Heatmap depicting the ecdysone inducibility fold changes caused by individual knockout of motifs in different core promoters. Disrupted INR (highlighted with the black dotted line rectangle) had a slightly negative effect on changing the core promoter responsiveness to ecdysone. (~ 2.3-fold reduction on average, Wilcoxon rank-sum test p = 2.1×10-5). Constitutive and developmental promoters highlighted in red and green, respectively.


A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21 % O2 vs . 85 % O2 , at post - natal day ( P ) 5 and P14 .

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

A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O2 vs. 85% O2, at post-natal day (P)5 and P14.


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


E Representative mIHC of immune components in CC tumors and adjacent normal tissues . T cells : CD3 ( red ) , B cells : CD20 ( green ) , macrophages : CD68 ( pink ) , NK cells : CD56 ( yellow ) , epithelial cells : Pan - CK ( orange ) . Scale bar = 100 μm .

E Representative mIHC of immune components in CC tumors and adjacent normal tissues. T cells: CD3 (red), B cells: CD20 (green), macrophages: CD68 (pink), NK cells: CD56 (yellow), epithelial cells: Pan-CK (orange). Scale bar =100 μm.


H Heatmap showing the expression of marker genes in each subtype of epithelial cells . SCC , squamous cell carcinoma ; SCJ , squamocolumnar junction ; Imm , immune - related genes ; Prolif , hyperproliferation ; Colu , columnar epithelium ; EMT , epithelial - mesenchymal transition ;

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

H Heatmap showing the expression of marker genes in each subtype of epithelial cells. SCC, squamous cell carcinoma; SCJ, squamocolumnar junction; Imm, immune-related genes; Prolif, hyperproliferation; Colu, columnar epithelium; EMT, epithelial-mesenchymal transition;


J Comparison of Overall survival ( OS ) rates for the high - correlation and low - correlation groups , stratified using the EP0 _ MUC5B ( left panel ) and EP2 _ POSTN ( right panel ) signatures in TCGA . P values are calculated using the log - rank test ( N = 255 ) .

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

J Comparison of Overall survival (OS) rates for the high-correlation and low-correlation groups, stratified using the EP0_MUC5B (left panel) and EP2_POSTN (right panel) signatures in TCGA. P values are calculated using the log-rank test (N=255).


B Boxplots showing the cell - type proportions of T / NK cells for matched tumor and normal samples ( n = 5 ) . The scCODA model and the ALDEx2 model were used to examine the differences in T / NK cells ' composition . Red bars indicate credible and significant results of scCODA . Stars indicate the significance calculated by ALDEx2 model ( * P < 0 . 05 , * * P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots showing the cell-type proportions of T/NK cells for matched tumor and normal samples (n=5). The scCODA model and the ALDEx2 model were used to examine the differences in T/NK cells' composition. Red bars indicate credible and significant results of scCODA. Stars indicate the significance calculated by ALDEx2 model (* P < 0.05, ** P < 0.01).


I Violin plots showing the differentially expressed genes between large and small clone ( color - coded as in Fig 2H ) ;

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I Violin plots showing the differentially expressed genes between large and small clone (color-coded as in Fig 2H);


H Violin plots showing the association of different B - cell signatures with the response ( R , N = 15 ) and no response ( NR , N = 42 ) to Pembrolizumab in the Kim cohort . p values calculated using the student ' s t - test ;

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

H Violin plots showing the association of different B-cell signatures with the response (R, N=15) and no response (NR, N=42) to Pembrolizumab in the Kim cohort. p values calculated using the student's t-test;


I Representative mIHC of plasma cells in CC tumor area . plasma cells : CD38 + Pan - CK - , scale bar = 100 μm ;

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I Representative mIHC of plasma cells in CC tumor area. plasma cells: CD38+Pan-CK-, scale bar = 100 μm;


A , B B cell subsets localization in CC tissues are exhibited by mIHC via differential markers as follow : total B cell : CD20 + , GCB : CD20 + BCL6 - , PC : CD20 - CD38 + ; with scale = 500 μm for A , scale = 200 μm for B ;

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A, B B cell subsets localization in CC tissues are exhibited by mIHC via differential markers as follow: total B cell: CD20+, GCB: CD20+BCL6-, PC: CD20-CD38+; with scale =500 μm for A, scale = 200 μm for B;


C Quantification of B - cell density in Pan - CK + tumor area , adjacent stroma , and TLS . * P < 0 . 05 , two - way ANOVA following Tukey post - hoc test ;

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C Quantification of B-cell density in Pan-CK+ tumor area, adjacent stroma, and TLS. * P < 0.05, two-way ANOVA following Tukey post-hoc test;


E Representative mIHC of CC tumor with CD4 + PD - 1 + CXCL13 + Tfh cells located in TLS ( yellow arrows ) . scale bars = 100 μm for left panel , scale bars = 20 μm for right panel ;

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E Representative mIHC of CC tumor with CD4+PD-1+CXCL13+ Tfh cells located in TLS (yellow arrows). scale bars =100 μm for left panel, scale bars = 20 μm for right panel;


B Boxplots showing the cell - type proportions of myeloid cells for matched tumor and normal samples ( n = 5 ) . The scCODA model and the ALDEx2 model were used to examine the differences in myeloid cells ' composition . Red bars indicate credible and significant results of scCODA . Stars indicate the significance calculated by ALDEx2 model ( * P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots showing the cell-type proportions of myeloid cells for matched tumor and normal samples (n=5). The scCODA model and the ALDEx2 model were used to examine the differences in myeloid cells' composition. Red bars indicate credible and significant results of scCODA. Stars indicate the significance calculated by ALDEx2 model (* P < 0.05).


G Comparison of Overall survival ( OS ) rates for the high - correlation and low - correlation groups , stratified using the Macro _ C1QC ( upper panel ) , and Mast _ CPA3 ( lower panel ) signatures in TCGA ( N = 255 ) . P values are calculated using the log - rank test .

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

G Comparison of Overall survival (OS) rates for the high-correlation and low-correlation groups, stratified using the Macro_C1QC (upper panel), and Mast_CPA3 (lower panel) signatures in TCGA (N=255). P values are calculated using the log-rank test.


B Boxplots showing the cell - type proportions of fibroblast cells for matched tumor and normal samples ( n = 5 ) .

B Boxplots showing the cell-type proportions of fibroblast cells for matched tumor and normal samples (n=5).


J Comparison of Overall survival ( OS ) rates for three clusters identified in I . P values are calculated using the log - rank test ( n = 255 ) .

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

J Comparison of Overall survival (OS) rates for three clusters identified in I. P values are calculated using the log-rank test (n=255).


( A ) Co - immunoprecipitation ( Co - IP ) of Beclin1 with WT or mutant vBcl - 2 . 293T cells were transiently transfected with the indicated constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 and immunoblotting of V5 - tagged Beclin1 .

(A) Co-immunoprecipitation (Co-IP) of Beclin1 with WT or mutant vBcl-2. 293T cells were transiently transfected with the indicated constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of V5-tagged Beclin1.


( B ) Co - IP of WT or mutant vBcl - 2 with endogenous Beclin1 . 293T cells were transfected with the indicated vBcl - 2 constructs , followed by immunoprecipitation of HA - tagged vBcl - 2 and immunoblotting of endogenous Beclin1 . 1 % whole - cell lysates ( WCLs ) was used as the input .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-IP of WT or mutant vBcl-2 with endogenous Beclin1. 293T cells were transfected with the indicated vBcl-2 constructs, followed by immunoprecipitation of HA-tagged vBcl-2 and immunoblotting of endogenous Beclin1. 1% whole-cell lysates (WCLs) was used as the input.


( C ) vBcl - 2 interaction with Bak protein . 293T cells were transfected with WT and mutant forms of vBcl - 2 as indicated . At 48 h posttransfection , WCLs were mixed either with GST - BakΔTM fusion protein ( left panel ) or with GST alone ( right panel ) for an in vitro GST pull - down ( GST PD ) assays . GST fusion proteins used for the pulldown assay are indicated ( bottom panel ) . 1 % WCL was used as the input . Data are representative of at least three experiments yielding similar results .

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(C) vBcl-2 interaction with Bak protein. 293T cells were transfected with WT and mutant forms of vBcl-2 as indicated. At 48 h posttransfection, WCLs were mixed either with GST-BakΔTM fusion protein (left panel) or with GST alone (right panel) for an in vitro GST pull-down (GST PD) assays. GST fusion proteins used for the pulldown assay are indicated (bottom panel). 1% WCL was used as the input. Data are representative of at least three experiments yielding similar results.


( A ) NIH3T3 cells stably expressing the WT and mutant forms of vBcl - 2 were transfected with GFP - LC3 . At 18 h posttransfection , cells were incubated under normal or starvation conditions for 4 h . Autophagy was quantified as mean ± SEM of the combined results from three independent experiments . * * , P < 0 . 0001 .

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(A) NIH3T3 cells stably expressing the WT and mutant forms of vBcl-2 were transfected with GFP-LC3. At 18 h posttransfection, cells were incubated under normal or starvation conditions for 4 h. Autophagy was quantified as mean±SEM of the combined results from three independent experiments. **, P<0.0001.


( B ) NIH3T3 cells expressing the WT or mutant forms of vBcl - 2 as indicated were transfected with GFP - LC3 and treated with 2 µM rapamycin for 6 h . GFP - LC3 was detected using an inverted fluorescence microscope ( top ) . Autophagy was quantified as mean ± SEM of the combined results from three independent experiments . Scale bar , 5 µm ; * * , P < 0 . 01 .

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

(B) NIH3T3 cells expressing the WT or mutant forms of vBcl-2 as indicated were transfected with GFP-LC3 and treated with 2 µM rapamycin for 6 h. GFP-LC3 was detected using an inverted fluorescence microscope (top). Autophagy was quantified as mean±SEM of the combined results from three independent experiments. Scale bar, 5 µm; **, P<0.01.


( C ) NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 , as indicated , were treated with 2 µM rapamycin . LC3 - I and LC3 - II levels were then determined by immunoblotting with an antibody against LC3 ( top ) . Densitometric quantification of the LC3 - II / LC3 - I ratios under normal and rapamycin treatment conditions is shown at the bottom . Similar results were obtained from three independent experiments .

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

(C) NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2, as indicated, were treated with 2 µM rapamycin. LC3-I and LC3-II levels were then determined by immunoblotting with an antibody against LC3 (top). Densitometric quantification of the LC3-II/LC3-I ratios under normal and rapamycin treatment conditions is shown at the bottom. Similar results were obtained from three independent experiments.


( top ) Representative confocal images of GFP - LC3 and HA - tagged vBcl - 2 ( WT and mutants ) in NIH3T3 cells infected with the indicated viruses ( MOI = 5 ) . Nuclei were counterstained with 4 ′ , 6 ′ - diamidino - 2 - phenylindole ( DAPI ) . Scale bar , 5 µm . ( bottom ) Quantification of the percentage of virally infected cells with GFP - LC3 punctate staining . Results shown represent mean ± SEM of combined results from three independent experiments ( 200 cells per experimental condition ) . * , P < 0 . 01 versus HA - WT ; * * , P < 0 . 001 versus HA - WT .

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(top) Representative confocal images of GFP-LC3 and HA-tagged vBcl-2 (WT and mutants) in NIH3T3 cells infected with the indicated viruses (MOI = 5). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI). Scale bar, 5 µm. (bottom) Quantification of the percentage of virally infected cells with GFP-LC3 punctate staining. Results shown represent mean±SEM of combined results from three independent experiments (200 cells per experimental condition). *, P<0.01 versus HA-WT; **, P<0.001 versus HA-WT.


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) ,

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NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A),


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) , or for apoptosis by TUNEL staining ( B )

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

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B)


NIH3T3 cells stably expressing the WT or mutant forms of vBcl - 2 were treated with TNFα and cycloheximide ( CHX ) for 12 h , then assayed for cell viability by trypan blue exclusion assay ( A ) , or for apoptosis by TUNEL staining ( B ) or for the caspase 3 activation using flow cytometry ( C ) . Apoptotic cells in ( B ) were counted under high power magnification ( 60 × magnification ) . Mock , untreated condition . Data represents mean ± SEM of combined results from three independent experiments . Scale bars , 100 µm ( A ) , 5 µm ( B ) . * * , P < 0 . 0001 versus vector cells .

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

NIH3T3 cells stably expressing the WT or mutant forms of vBcl-2 were treated with TNFα and cycloheximide (CHX) for 12 h, then assayed for cell viability by trypan blue exclusion assay (A), or for apoptosis by TUNEL staining (B) or for the caspase 3 activation using flow cytometry (C). Apoptotic cells in (B) were counted under high power magnification (60×magnification). Mock, untreated condition. Data represents mean±SEM of combined results from three independent experiments. Scale bars, 100 µm (A), 5 µm (B). **, P<0.0001 versus vector cells.


( A ) Single - step ( right ) and multiple - step ( left ) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells .

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(A) Single-step (right) and multiple-step (left) growth curves of the WT and recombinant γHV68 viruses in BHK21 cells.


( B ) Acute replication of the WT and mutant vBcl - 2 γHV68 viruses in the lungs of BALB / c mice at 5 dpi ( up ) and 7 dpi ( down ) after intranasal infection determined by viral titers in the lungs of the infected mice ( left ) or by real - time PCR of the viral genomic DNA ( right ) . Mean ± SEM of five mice per group / experiment . Data of 7 dpi is pooled from two separate experiments . The vBcl - 2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [ for Δα1 , P = 0 . 82 ( day 5 ) ; P = 0 . 08 ( day 7 ) ; for ΔBH2 , P = 0 . 49 ( day 5 ) ; P = lungs [ for Δα1 , P = 0 . 25 ( day 5 ) ; P = 0 . 28 ( day 7 ) ; for ΔBH2 , P = 0 . 21 ( day 5 ) ; P = 0 . 37 ( day 7 ) ; unpaired t - tests ] . n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Acute replication of the WT and mutant vBcl-2 γHV68 viruses in the lungs of BALB/c mice at 5 dpi (up) and 7 dpi (down) after intranasal infection determined by viral titers in the lungs of the infected mice (left) or by real-time PCR of the viral genomic DNA (right). Mean±SEM of five mice per group/experiment. Data of 7 dpi is pooled from two separate experiments. The vBcl-2 Δα1 and ΔBH2 mutants did not yield significantly different results when compared to the WT in infectious virus titers [for Δα1, P = 0.82 (day 5); P = 0.08 (day 7); for ΔBH2, P = 0.49 (day 5); P = lungs [for Δα1, P = 0.25 (day 5); P = 0.28 (day 7); for ΔBH2, P = 0.21 (day 5); P = 0.37 (day 7); unpaired t-tests]. n.s., not significant.


Splenic infectious centers measured at 12 dpi ( A , left ) , 14 dpi ( B , left ) , 21 dpi ( C , left ) , 28 dpi ( D , up ) , 35 dpi ( E , up ) , or 42 dpi ( F , up ) and viral genome load measured by real - time PCR at 12 dpi ( A , right ) , 14 dpi ( B , right ) , 21 dpi ( C , right ) , 28 dpi ( D , down ) , 35 dpi ( E , down ) , or 42 dpi ( F , down ) , in the BALB / cmice intranasally infected with the WT or recombinant γHV68 mutants , as indicated ( Mean ± SEM of five mice per group / time point / experiment ) . Data of 14 dpi and 28 dpi is pooled from two and three separate experiments , respectively . Preformed infectious virus was negligible in all spleen samples . No significant difference was detected at 12 dpi ( A ) , 14 dpi ( B ) , and 21 dpi ( C ) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [ for Δα1 , P = 0 . 58 ( day 12 ) ; P = 0 . 75 ( day 14 ) ; P = 0 . 18 ( day 21 ) ; for ΔBH2 , P = 0 . 64 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 81 ( day 21 ) ; unpaired t - tests ] and in viral genome loads [ for Δα1 , P = 0 . 85 ( day 12 ) ; P = 0 . 76 ( day 14 ) ; P = 0 . 96 ( day 21 ) ; for ΔBH2 , P = 0 . 56 ( day 12 ) ; P = 0 . 73 ( day 14 ) ; P = 0 . 25 ( day 21 ) ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased infectious center titers ( top ) of vBcl - 2 mutant viruses , as compared to WT γHV68 , were statistically significant [ vBcl - 2Δα1 versus vBcl - 2AAA ( 42 dpi ) , P = 0 . 46 ; vBcl - 2Δα1 versus WT γHV68 ( 42 dpi ) , P = 0 . 08 ; unpaired t - tests ] . At 28 dpi ( D ) , 35 dpi ( E ) , and 42 dpi ( F ) , the decreased viral DNA loads ( bottom ) of the vBcl - 2Δα1 , vBcl - 2AAA , and vBcl - 2 null mutants , as compared to WT γHV68 and the vBcl - 2ΔBH2 mutant , were statistically significant as follows ( unpaired t - tests ) : at day 28 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 01 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 01 ; vBcl - 2 - null versus WT γHV68 , P < 0 . 01 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2 - null versus vBcl - 2ΔBH2 , P < 0 . 001 ; at day 35 , vBcl - 2Δα1 versus WT γHV68 , P < 0 . 05 ; vBcl - 2AAA versus WT γHV68 , P < 0 . 05 ; vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 01 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 05 ; at day 42 , vBcl - 2Δα1 versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2AAA versus vBcl - 2ΔBH2 , P < 0 . 001 ; vBcl - 2ΔBH2 versus WT γHV68 , P = 0 . 33 . I . C . , infectious center . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

Splenic infectious centers measured at 12 dpi (A, left), 14 dpi (B, left), 21 dpi (C, left), 28 dpi (D, up), 35 dpi (E, up), or 42 dpi (F, up) and viral genome load measured by real-time PCR at 12 dpi (A, right), 14 dpi (B, right), 21 dpi (C, right), 28 dpi (D, down), 35 dpi (E, down), or 42 dpi (F, down), in the BALB/cmice intranasally infected with the WT or recombinant γHV68 mutants, as indicated (Mean±SEM of five mice per group/time point/experiment). Data of 14 dpi and 28 dpi is pooled from two and three separate experiments, respectively. Preformed infectious virus was negligible in all spleen samples. No significant difference was detected at 12 dpi (A), 14 dpi (B), and 21 dpi (C) with the Δα1 and ΔBH2 mutant viruses when compared to the WT in infectious center titers [for Δα1, P = 0.58 (day 12); P = 0.75 (day 14); P = 0.18 (day 21); for ΔBH2, P = 0.64 (day 12); P = 0.73 (day 14); P = 0.81 (day 21); unpaired t-tests] and in viral genome loads [for Δα1, P = 0.85 (day 12); P = 0.76 (day 14); P = 0.96 (day 21); for ΔBH2, P = 0.56 (day 12); P = 0.73 (day 14); P = 0.25 (day 21); unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased infectious center titers (top) of vBcl-2 mutant viruses, as compared to WT γHV68, were statistically significant [vBcl-2Δα1 versus vBcl-2AAA (42 dpi), P = 0.46; vBcl-2Δα1 versus WT γHV68 (42 dpi), P = 0.08; unpaired t-tests]. At 28 dpi (D), 35 dpi (E), and 42 dpi (F), the decreased viral DNA loads (bottom) of the vBcl-2Δα1, vBcl-2AAA, and vBcl-2 null mutants, as compared to WT γHV68 and the vBcl-2ΔBH2 mutant, were statistically significant as follows (unpaired t-tests): at day 28, vBcl-2Δα1 versus WT γHV68, P<0.01; vBcl-2AAA versus WT γHV68, P<0.01; vBcl-2-null versus WT γHV68, P<0.01; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2-null versus vBcl-2ΔBH2, P<0.001; at day 35, vBcl-2Δα1 versus WT γHV68, P<0.05; vBcl-2AAA versus WT γHV68, P<0.05; vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.01; vBcl-2AAA versus vBcl-2ΔBH2, P<0.05; at day 42, vBcl-2Δα1 versus vBcl-2ΔBH2, P<0.001; vBcl-2AAA versus vBcl-2ΔBH2, P<0.001; vBcl-2ΔBH2 versus WT γHV68, P = 0.33. I.C., infectious center. *, P<0.05; **, P<0.01; ***, P<0.001.


A . Retinal whole mount immunostaining for Isolectin - B4 ( red ) and Desmin ( green ) in WT ( top panel ) and Glut1iECKO ( bottom panel ) mice showing pericyte coverage in the vasculature . Left and right panels show representative images with 20x or 63x magnification , respectively . B . Quantification of the pericytes coverage in the retinal vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 9 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

A. Retinal whole mount immunostaining for Isolectin-B4 (red) and Desmin (green) in WT (top panel) and Glut1iECKO (bottom panel) mice showing pericyte coverage in the vasculature. Left and right panels show representative images with 20x or 63x magnification, respectively. B. Quantification of the pericytes coverage in the retinal vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


C . Immunostaining for CD31 ( green ) , CD13 ( white ; pericyte marker ) and αSMA ( cyan ) using paraffin section from WT ( top panel ) and Glut1iECKO ( bottom panel ) mice .

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

C. Immunostaining for CD31 (green), CD13 (white; pericyte marker) and αSMA (cyan) using paraffin section from WT (top panel) and Glut1iECKO (bottom panel) mice.


D . Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 7 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) E . Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 13 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

D. Quantification of the pericytes coverage using CD13 immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=7 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) E. Quantification of the smooth muscle cell coverage using αSMA immunostaining in the brain vasculature of WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


J . Representative confocal images of fibrin ( green ) , CD31 ( red ) and DAPI ( blue ) immunostaining in the brain vasculature of WT ( top panel ) and Glut1iECKO ( bottom panel ) mice K . Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 13 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) L . Quantification of extravascular fibrin deposits in WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 9 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . ) M . Quantification of lactate in the cerebrospinal fluid of WT ( blue ) and Glut1iECKO ( orange ) mice . ( n = 3 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

J. Representative confocal images of fibrin (green), CD31 (red) and DAPI (blue) immunostaining in the brain vasculature of WT (top panel) and Glut1iECKO (bottom panel) mice K. Quantification of the number of cadaverine 555 positive brain parenchyma cells in WT (blue) and Glut1iECKO (orange) mice. (n=13 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) L. Quantification of extravascular fibrin deposits in WT (blue) and Glut1iECKO (orange) mice. (n=9 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.) M. Quantification of lactate in the cerebrospinal fluid of WT (blue) and Glut1iECKO (orange) mice. (n=3 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


A . Quantification of the mTFP / Venus fluorescence ratio of laconic ( lactate sensor ) showing concentration - dependent lactate uptake in HBVPs . ( n = 8 from 3 independent experiments . Error bars indicate the standard error of the mean ( SEM ) ) B . Representative images showing mTFP ( blue ) and Venus ( yellow ) fluorescence in control and lactate ( 20mM ) treated HBVPs .

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

A. Quantification of the mTFP/Venus fluorescence ratio of laconic (lactate sensor) showing concentration-dependent lactate uptake in HBVPs. (n=8 from 3 independent experiments. Error bars indicate the standard error of the mean (SEM)) B. Representative images showing mTFP (blue) and Venus (yellow) fluorescence in control and lactate (20mM) treated HBVPs.


F . Representative images ( high magnification ; 60x ) showing mTFP ( cyan ) , Venus ( yellow ) and VECAD ( purple ) fluorescence from BMECs and laconic - infected HBVPs on a 2 well chamber . The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact . G . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs with endothelial contact ( EC - PC contact ) or HBVPs alone ( PC only ) . ( n = 16 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

F. Representative images (high magnification; 60x) showing mTFP (cyan), Venus (yellow) and VECAD (purple) fluorescence from BMECs and laconic-infected HBVPs on a 2 well chamber. The upper panel shows HBVPs with endothelial contact and the lower panel shows HBVPs without endothelial contact. G. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs with endothelial contact (EC-PC contact) or HBVPs alone (PC only). (n=16 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


H . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or GLUT1 ( lower panel ) siRNA treated BMECs . VECAD immunostaining ( purple ) was used for endothelial staining . I . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or GLUT1 ( siGLUT1 ) siRNA - treated BMECs . ( n = 12 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

H. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or GLUT1 (lower panel) siRNA treated BMECs. VECAD immunostaining (purple) was used for endothelial staining. I. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or GLUT1 (siGLUT1) siRNA-treated BMECs. (n=12 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


E . Western blot analysis for siRNA efficiency of MCT1 .

E. Western blot analysis for siRNA efficiency of MCT1.


G . Western blot analysis for siRNA efficiency of MCT12 .

G. Western blot analysis for siRNA efficiency of MCT12.


I . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or MCT1 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial staining . J . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or MCT1 ( siMCT1 ) siRNA - treated BMECs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

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

I. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT1 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. J. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT1 (siMCT1) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


K . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in HBVPs which have direct contact with control ( upper panel ) or MCT5 ( lower panel ) siRNA - treated BMECs . VECAD immunostaining was used for endothelial staining . L . Quantification of the mTFP / Venus fluorescence ratio of laconic in HBVPs which have direct contact with control ( siCON ) or MCT5 ( siMCT5 ) siRNA - treated BMECs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test . )

K. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in HBVPs which have direct contact with control (upper panel) or MCT5 (lower panel) siRNA-treated BMECs. VECAD immunostaining was used for endothelial staining. L. Quantification of the mTFP/Venus fluorescence ratio of laconic in HBVPs which have direct contact with control (siCON) or MCT5 (siMCT5) siRNA-treated BMECs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.)


D . Western blot analysis showing expression level of MCT12 and β - actin in HBVPs and BMECs . E . Western blot analysis for siRNA efficiency of MCT12 .

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

D. Western blot analysis showing expression level of MCT12 and β-actin in HBVPs and BMECs. E. Western blot analysis for siRNA efficiency of MCT12.


G . Representative images showing mTFP ( cyan ) and Venus ( yellow ) fluorescence in control ( upper panel ) or MCT12 ( lower panel ) siRNA treated HBVPs in a 2 well chamber assay system with BMECs . VECAD ( purple ) immunostaining was used for endothelial staining . H . Quantification of the mTFP / Venus fluorescence ratio of Laconic in control ( blue ) or MCT12 ( orange ) siRNA treated HBVPs . ( n = 8 from independent experiments . Error bars indicate the standard error of the mean ( SEM ) from unpaired Student ' s t test .

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

G. Representative images showing mTFP (cyan) and Venus (yellow) fluorescence in control (upper panel) or MCT12 (lower panel) siRNA treated HBVPs in a 2 well chamber assay system with BMECs. VECAD (purple) immunostaining was used for endothelial staining. H. Quantification of the mTFP/Venus fluorescence ratio of Laconic in control (blue) or MCT12 (orange) siRNA treated HBVPs. (n=8 from independent experiments. Error bars indicate the standard error of the mean (SEM) from unpaired Student's t test.


A . Permeability assay using vibratome section showing the cadaverine 555 dye ( red ) , CD31 ( green ) and CD13 ( purple ) in the brain vasculature of Glut1iECKO mice with ( bottom ) or without ( top ) lactate administration . Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice , but not in lactate supplemented Glut1iECKO mice .

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

A. Permeability assay using vibratome section showing the cadaverine 555 dye (red), CD31 (green) and CD13 (purple) in the brain vasculature of Glut1iECKO mice with (bottom) or without (top) lactate administration. Note the extravascular accumulation of cadaverine dye in control Glut1iECKO mice, but not in lactate supplemented Glut1iECKO mice.