Figure 4 ( A ) Sections of mouse skin tumors were stained with antibodies to Gata6 and Krt14 . A papilloma tumor found in WT mice treated with DMBA and TPA ( left panel ) , is compared with sebaceous tumors found in K14ΔNLef1 mice ( right panels ) . Data information : Scale bars : 100 μm . ( B ) Tumor burden and tumor incidence in DMBA - treated K14ΔNLef1 ( n = 11 ) and K14ΔNLef1 : cKO ( n = 8 ) mice . ( * * * ) p - value < 0 . 001 ; Wilcoxon matched - pairs signed rank test . Data information : Data are means ± SEM . ( C ) Representative H & E - stained tumors of K14ΔNLef1 and K14ΔNLef1 : cKO mice with quantification of each tumor type relative to the total number of tumors in each group ( middle panel ) and as the average absolute number of tumors per mice ( right panel ) . A total of 268 tumors were analyzed . 143 tumors were found in 7 K14ΔNLef1 mice ( 103 sebaceous tumors and 40 papillomas ) , while 125 were found in 5 K14ΔNLef1 : cKO mice ( 55 sebaceous tumors and 70 papillomas ) . ( * * ) p - value < 0 . 005 , Student ' s t - test . Data information : ( C ) Scale bar : 1 mm . ( D ) Representative images of mouse skin tumor sections labelled with antibodies to Plet1 and Atp6v1c2 . Data information : D ) Scale bars : 100 μm .

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Figure 4(A) Sections of mouse skin tumors were stained with antibodies to Gata6 and Krt14. A papilloma tumor found in WT mice treated with DMBA and TPA (left panel), is compared with sebaceous tumors found in K14ΔNLef1 mice (right panels). Data information: Scale bars: 100 μm.(B) Tumor burden and tumor incidence in DMBA-treated K14ΔNLef1 (n=11) and K14ΔNLef1:cKO (n=8) mice. (***) p-value < 0.001; Wilcoxon matched-pairs signed rank test. Data information: Data are means ± SEM.(C) Representative H&E-stained tumors of K14ΔNLef1 and K14ΔNLef1:cKO mice with quantification of each tumor type relative to the total number of tumors in each group (middle panel) and as the average absolute number of tumors per mice (right panel). A total of 268 tumors were analyzed. 143 tumors were found in 7 K14ΔNLef1 mice (103 sebaceous tumors and 40 papillomas), while 125 were found in 5 K14ΔNLef1:cKO mice (55 sebaceous tumors and 70 papillomas). (**) p-value < 0.005, Student's t-test. Data information: (C) Scale bar: 1 mm.(D) Representative images of mouse skin tumor sections labelled with antibodies to Plet1 and Atp6v1c2. Data information: D) Scale bars: 100 μm.


Figure 5 ( A ) Heat - map highlighting differentially expressed genes ( DEG ) between WT and Gata6 cKO keratinocytes Gata6 direct transcriptional targets from Chip - Seq data are shown in dark grey . ( C ) MSI analysis performed on skin tumors from DMBA - treated K14ΔNLef1 and K14ΔNLef1 : cKO mice ( 5 mice in each group ) . Representative microsatellite profiles of K14ΔNLef1 and K14ΔNLef1 : cKO tumors . Peak heights were normalized to the highest peak in each microsatellite profile to obtain the relative frequency of each allele for 5 different markers . " 0 " indicates the position of the highest peak in bp . ( * ) p - value < 0 . 05 ; ( * * ) p - value < 0 . 001 ; paired t - test . ( D ) Representative immunohistochemistry stainings against Mlh1 performed on skin tumors from DMBA - treated K14ΔNLef1 and K14ΔNLef1 : cKO mice . 4 mice were included in each group . A technical control is displayed ( without primary antibody incubation ) . Semi - quantitative analysis of Mlh1 staining and statistical analysis ( χ2 test ) were performed . Data information : Scale bar : 250 μm . ( E ) Representative immunohistochemistry stainings against Msh2 and semi - quantitative analysis Data information : Scale bar : 250 μm .

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Figure 5(A) Heat-map highlighting differentially expressed genes (DEG) between WT and Gata6 cKO keratinocytes Gata6 direct transcriptional targets from Chip-Seq data are shown in dark grey.(C) MSI analysis performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice (5 mice in each group). Representative microsatellite profiles of K14ΔNLef1 and K14ΔNLef1:cKO tumors. Peak heights were normalized to the highest peak in each microsatellite profile to obtain the relative frequency of each allele for 5 different markers. "0" indicates the position of the highest peak in bp. (*) p-value < 0.05; (**) p-value < 0.001; paired t-test.(D) Representative immunohistochemistry stainings against Mlh1 performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice. 4 mice were included in each group. A technical control is displayed (without primary antibody incubation). Semi-quantitative analysis of Mlh1 staining and statistical analysis (χ2 test) were performed. Data information: Scale bar: 250 μm.(E) Representative immunohistochemistry stainings against Msh2 and semi-quantitative analysis Data information: Scale bar: 250 μm.


Figure 6 ( A ) Normal human scalp skin sections stained with Gata6 and PanKeratin antibodies . Endogenous Gata6 expression is found in the JZ , upper SG and SD . As in mouse skin , Gata6 is not expressed in sweat glands ( SwG ) . ( B ) Representative sections of human skin tumors stained with Gata6 and PanKeratin antibodies . Quantification of the percentage of Gata6 - positive tumors within each histopathological group ( middle left panel ) and quantification of the percentage of Gata6 - positive cells within each positive sample ( middle right panel ) . Note that the least positive sample showed about 3 % of Gata6 - positive cells . The analysis was performed on 73 different tumor samples . SSC : squamous cell carcinoma , SCAP : syringocystadenoma papilliferum , EMPD : extramammary Paget ' s disease , nod . / superf . / scler . BCC : nodular / superficial / sclerodermiform basal cell carcinoma , Steatocystoma M . : steatocystoma multiplex , FSCH : folliculosebaceous cystic hamartoma . Heat - map displaying the - log ( p - value ) of χ2 test computed between the different tumor types ( bottom left panel ) . ( C ) Gata6 expression ( measured in Transcripts Per Million , TPM ) in human SCC ( n = 6 ) , BCC ( n = 6 ) and sebaceous carcinoma ( n = 14 ) samples from ( North et al , 2018 ) . Data are means ± SD . ( * * ) p - value ≤ 0 . 005 , Student ' s t - test . Data information : ( A and B ) Scale bars : 100 μm .

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Figure 6(A) Normal human scalp skin sections stained with Gata6 and PanKeratin antibodies. Endogenous Gata6 expression is found in the JZ, upper SG and SD. As in mouse skin, Gata6 is not expressed in sweat glands (SwG). (B) Representative sections of human skin tumors stained with Gata6 and PanKeratin antibodies. Quantification of the percentage of Gata6-positive tumors within each histopathological group (middle left panel) and quantification of the percentage of Gata6-positive cells within each positive sample (middle right panel). Note that the least positive sample showed about 3% of Gata6-positive cells. The analysis was performed on 73 different tumor samples. SSC : squamous cell carcinoma, SCAP: syringocystadenoma papilliferum, EMPD: extramammary Paget's disease, nod./superf./scler. BCC: nodular/ superficial/ sclerodermiform basal cell carcinoma, Steatocystoma M.: steatocystoma multiplex, FSCH: folliculosebaceous cystic hamartoma. Heat-map displaying the -log(p-value) of χ2 test computed between the different tumor types (bottom left panel). (C) Gata6 expression (measured in Transcripts Per Million, TPM) in human SCC (n=6), BCC (n=6) and sebaceous carcinoma (n=14) samples from (North et al, 2018). Data are means ± SD. (**) p-value ≤ 0.005, Student's t-test. Data information: (A and B) Scale bars: 100 μm.


Figure 7 ( A ) Differentiation status ( poor , moderate or well - differentiated ) , circumscription ( not infiltrative , focally infiltrative or infiltrative ) , log10 total number of mutations and Gata6 mutations in 32 human sebaceous carcinomas ( SebC ) separated in 3 subgroups : pauci - mutational , MSI - related and UV - related For Gata6 mutations , the most deleterious mutation is displayed . n . a . : not assessed . ( C ) Gata6 expression ( measured in Transcripts Per Million , TPM ) in human pauci - mutational ( pauci - mut ) SebC ( n = 4 ) , MSI - related SebC ( n = 4 ) , UV - related SebC ( n = 5 ) samples . Data are means ± SD . ( * ) p - value ≤ 0 . 05 , Student ' s t - test . ( D - F ) Graphs showing the number of mutations per megabase ( Mutations / Mb ) ( D ) , the number of Somatic Single - Nucleotide Variants ( SSNV ) ( E ) and indel ( F ) of Gata6mut ( n = 3 ) or Gata6wt ( n = 6 ) MSI - related SebC ( left panels ) and of Gata6mut ( n = 3 ) or Gata6wt ( n = 7 ) UV - related SebC ( right panels ) . Data are means ± SD . ( G ) Heat - map displaying the mean expression level of MMR genes ( measured in TPM ) in Gata6wt ( n = 10 ) and Gata6mut ( n = 3 ) SebC .

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Figure 7(A) Differentiation status (poor, moderate or well-differentiated), circumscription (not infiltrative, focally infiltrative or infiltrative), log10 total number of mutations and Gata6 mutations in 32 human sebaceous carcinomas (SebC) separated in 3 subgroups: pauci-mutational, MSI-related and UV-related For Gata6 mutations, the most deleterious mutation is displayed. n.a.: not assessed.(C) Gata6 expression (measured in Transcripts Per Million, TPM) in human pauci-mutational (pauci-mut) SebC (n=4), MSI-related SebC (n=4), UV-related SebC (n=5) samples. Data are means ± SD. (*) p-value ≤ 0.05, Student's t-test.(D-F) Graphs showing the number of mutations per megabase (Mutations/Mb) (D), the number of Somatic Single-Nucleotide Variants (SSNV) (E) and indel (F) of Gata6mut (n=3) or Gata6wt (n=6) MSI-related SebC (left panels) and of Gata6mut (n=3) or Gata6wt (n=7) UV-related SebC (right panels). Data are means ± SD.(G) Heat-map displaying the mean expression level of MMR genes (measured in TPM) in Gata6wt (n=10) and Gata6mut (n=3) SebC.


Figure 1 . Co - immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL - 1L using total cell extracts of HEK293T cells transiently expressing Myc - HOIP wildtype ( WT ) , or Myc - HOIP - K784R with HOIL - 1L - HA and Flag - SHARPIN . Anti - Vinculin antibody used to monitor protein loading . Representative data shown from three independent experiments .

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Figure 1. Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using total cell extracts of HEK293T cells transiently expressing Myc-HOIP wildtype (WT), or Myc-HOIP-K784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody used to monitor protein loading. Representative data shown from three independent experiments.


Figure 2 . Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag - NEMO , GFP - SHARPIN , and HOIL - 1L - HA with Myc - HOIP wildtype ( WT ) , Myc - HOIP K784R or Myc - HOIP C885A . Total cell lysates in denaturing conditions subjected to SDS - PAGE followed up by immunoblotting using antibodies as indicated . Anti - Vinculin antibody to monitor loading . Representative data from three independent experiments .

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Figure 2. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag-NEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wildtype (WT), Myc-HOIP K784R or Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS-PAGE followed up by immunoblotting using antibodies as indicated. Anti-Vinculin antibody to monitor loading. Representative data from three independent experiments.


Figure 3Immunoblotting to detect TNF - induced degradation and phosphorylation of IκB - α , or phosphorylation of IKK in immortalized Hoip + / + and HoipK778R / K778R MEFs treated with human TNF ( 20ng / ml ) for the indicated times . Immunoblots of anti - Vinculin antibody and anti - α - Tubulin antibody shown for monitoring loading amount . Representative data from three independent experimentsTNF - dependent induction of Caspase 3 activation in immortalized Hoip + / + or HoipK778R / K778R MEFs measured by using DEVD - AFC . MEFs treated with hTNF ( 100ng / ml ) and CHX ( 1µg / ml ) for 4 hours subjected to the Caspase 3 activity assays . TNF - induced Caspase 8 activity in Hoip + / + or HoipK778R / K778R immortalized MEFs treated with hTNF ( 100ng / ml ) with or without Cycloheximide ( CHX ) ( 1µg / ml ) or z - VAD ( 20µM ) . Representative data from three independent experiments , n = 4 .

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Figure 3Immunoblotting to detect TNF-induced degradation and phosphorylation of IκB-α, or phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF (20ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti-α-Tubulin antibody shown for monitoring loading amount. Representative data from three independent experimentsTNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays.TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data from three independent experiments, n=4.


Figure 4H & E staining , Keratin 14 ( KRT14 ) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age . Scale bars : 50 µm .

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Figure 4H&E staining, Keratin 14 (KRT14) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age. Scale bars: 50 µm.


Figure 5Immunostaining ( H & E , Keratin 14 ( KRT14 ) , Ly6G , F4 / 80 and Cleaved Caspase 3 ) of mouse dorsal skin sections of the indicated genotypes . Scale bars : 50 µm . C Immunoblotting to examine TNF - induced degradation and phosphorylation of IκB - α in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF ( 20ng / ml ) for the indicated times . Representative of three independent experiments . G Immunoblotting of TNF - induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF ( 100ng / ml ) with or without CHX ( 1µg / ml ) for indicated times . Representative of three independent experiments . H TNF - dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD - AFC . MEFs treated with hTNF ( 100ng / ml ) alone , or with CHX ( 1µg / ml ) for 4 hours ( H ) or 2 hours ( I ) subjected to the Caspase 3 activity assays . A representative data of two independent experiments , n = 4 . TNF - induced Caspase 8 activity in immortalized MEFs of the indicated genotype . Luminol - dependent activity of Caspase 8 in immortalized MEFs treated with hTNF ( 100ng / ml ) with or without CHX ( 1µg / ml ) . A representative data of three independent experiments , n = 4 .

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Figure 5Immunostaining (H&E, Keratin 14 (KRT14), Ly6G, F4/80 and Cleaved Caspase 3) of mouse dorsal skin sections of the indicated genotypes. Scale bars: 50 µm. C Immunoblotting to examine TNF-induced degradation and phosphorylation of IκB-α in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (20ng/ml) for the indicated times. Representative of three independent experiments. G Immunoblotting of TNF-induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) for indicated times. Representative of three independent experiments. H TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4 hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two independent experiments, n=4.TNF-induced Caspase 8 activity in immortalized MEFs of the indicated genotype. Luminol-dependent activity of Caspase 8 in immortalized MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) . A representative data of three independent experiments, n=4.


Figure 1NoV levels in the vaccinated mice hindlimb muscle tissue at 2 , 3 or 4 days post infection ( dpi ) with WT NoV were determined by Western blotting to detect the viral coat protein ( CP ) and B2 Staining of β - actin was used as a loading control ( B ) .

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Figure 1NoV levels in the vaccinated mice hindlimb muscle tissue at 2, 3 or 4 days post infection (dpi) with WT NoV were determined by Western blotting to detect the viral coat protein (CP) and B2 Staining of β-actin was used as a loading control (B).


Figure 2D , Western blotting of HSP70 , tsg 101 , CD81 , CD63 , and CD9 in EVs , whole blood , and blood clot derived from suckling mice infected with DMEM or NoVΔB2 . Staining of β - actin was used as a loading control .

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Figure 2D, Western blotting of HSP70, tsg 101, CD81, CD63, and CD9 in EVs, whole blood, and blood clot derived from suckling mice infected with DMEM or NoVΔB2. Staining of β-actin was used as a loading control.


Figure 4J , Total proteins were extracted from the hindlimb muscle tissue of NoVWT - infected BALB / c suckling mice to measure the viral coat protein ( CP ) and B2 protein by Western blotting . Staining of β - actin was used as a loading control .

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Figure 4J, Total proteins were extracted from the hindlimb muscle tissue of NoVWT-infected BALB/c suckling mice to measure the viral coat protein (CP) and B2 protein by Western blotting. Staining of β-actin was used as a loading control.


Figure 1Left panel : Representative blots showing protein levels of YAP and phosphorylated YAP ( Ser127 ) in control ( si - Ctrl ) and YAP knockdown ( si - YAP ) hOSEs . Protein levels were detected by Western blotting . β - Actin was used as a protein loading control . Right panel : Cell proliferation ( cell number ) in control ( si - Ctrl ) and YAP knockdown ( si - YAP ) hOSE cells . Left panel : Representative blots showing protein levels of YAP and phosphorylated YAP ( Ser127 ) in control ( si - Ctrl ) , YAP knockdown ( si - YAP ) , TAZ knockdown ( si - TAZ ) and YAP / TAZ double knockdown ( si - YAP / TAZ ) hOSE Cells . β - Actin was used as a protein loading control . Right panel : Cell proliferation ( cell number ) of control ( si - Ctrl ) , YAP knockdown ( si - YAP ) , TAZ knockdown ( si - TAZ ) and YAP / TAZ double knockdown ( si - YAP & si - TAZ ) hOSE cells . Representative blots showing protein levels of YAP and phosphorylated YAP ( Ser127 ) in control hOSEs ( MXIV ) and hOSEs expressing wild type YAP ( YAP ) or constitutively active YAP ( YAPS127A ) . Cells were collected at passage 4 . β - Actin was used as a protein loading control . Growth of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 4 . Each bar represents mean ± SEM of four independent samples . Growth curves of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . Representative images showing the morphologic changes of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . Scale bar : 50 µm . Representative images showing expression and location of YAP in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . YAP was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI . Scale bar : 50 µm .

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Figure 1Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSEs. Protein levels were detected by Western blotting. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) in control (si-Ctrl) and YAP knockdown (si-YAP) hOSE cells.Left panel: Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP/TAZ) hOSE Cells. β-Actin was used as a protein loading control. Right panel: Cell proliferation (cell number) of control (si-Ctrl), YAP knockdown (si-YAP), TAZ knockdown (si-TAZ) and YAP/TAZ double knockdown (si-YAP&si-TAZ) hOSE cells.Representative blots showing protein levels of YAP and phosphorylated YAP (Ser127) in control hOSEs (MXIV) and hOSEs expressing wild type YAP (YAP) or constitutively active YAP (YAPS127A). Cells were collected at passage 4. β-Actin was used as a protein loading control.Growth of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 4. Each bar represents mean ± SEM of four independent samples. Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7.Representative images showing the morphologic changes of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Scale bar: 50 µm.Representative images showing expression and location of YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. YAP was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI. Scale bar: 50 µm.


Figure 2Representative images showing SA - β - gal staining in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at passage 7 . Scale bar : 50 µm . Quantitative data showing the ratio of SA - β - gal positive cells in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells . Representative blots showing expression of key proteins associated with cell cycle arrest in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells analyzed using Western blotting at the 7th passage . β - Actin was used as a protein loading control . Representative images showing expression and location of phosphorylated pRB ( 807 / 811 ) in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at the 7th passage . Phosphorylated pRB ( 807 / 811 ) was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI ( blue ) .

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Figure 2Representative images showing SA-β-gal staining in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at passage 7. Scale bar: 50 µm.Quantitative data showing the ratio of SA-β-gal positive cells in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.Representative blots showing expression of key proteins associated with cell cycle arrest in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells analyzed using Western blotting at the 7th passage. β-Actin was used as a protein loading control.Representative images showing expression and location of phosphorylated pRB (807/811) in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the 7th passage. Phosphorylated pRB (807/811) was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).


Figure 3Representative blots showing expression of TP53 and RB1 expression in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence or absence of HPV E6 / E7 oncoproteins at the 7th passage . β - Actin was used as a protein loading control . Growth curves of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence of HPV E6 / E7 oncoproteins at the 9th passage . Each point represents mean ± SEM of four independent samples . Representative images showing SA - β - gal staining in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence or absence of HPV E6 / E7 oncoproteins at the 7th passage . Quantitative data showing the ratio of SA - β - gal positive cells in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells in the presence or absence of HPV E6 / E7 oncoproteins . Representative images showed SA - β - gal staining in cultured primary hOSE cells in the presence or absence of HPV E6 / E7 oncoproteins . Cells were collected and stained at the 13th passage . Scale bar : 25 µm . Right graph showing the ratio of SA - β - gal positive cells in P13 hOSE cells transfected with empty vectors ( CTRL - P13 ) or vectors expressing E6 / E7 oncoproteins ( E6 / E7 - P13 ) .

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Figure 3Representative blots showing expression of TP53 and RB1 expression in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins at the 7th passage. β-Actin was used as a protein loading control.Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence of HPV E6/E7 oncoproteins at the 9th passage. Each point represents mean ± SEM of four independent samples.Representative images showing SA-β-gal staining in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins at the 7th passage.Quantitative data showing the ratio of SA-β-gal positive cells in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells in the presence or absence of HPV E6/E7 oncoproteins.Representative images showed SA-β-gal staining in cultured primary hOSE cells in the presence or absence of HPV E6/E7 oncoproteins. Cells were collected and stained at the 13th passage. Scale bar: 25 µm. Right graph showing the ratio of SA-β-gal positive cells in P13 hOSE cells transfected with empty vectors (CTRL-P13) or vectors expressing E6/E7 oncoproteins (E6/E7-P13).


Figure 4Representative blots showing the expression of YAP and RB1 in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without pRB knockdown . Cells were collected at the 7th passage and protein levels were analyzed using Western blotting . β - Actin was used as a protein loading control . Growth curves of hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells after RB1 knockdown . Representative images showing SA - β - gal staining in the hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without RB1 or TP53 knockdown . Cells were collected at the 7th passage .

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Figure 4Representative blots showing the expression of YAP and RB1 in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown. Cells were collected at the 7th passage and protein levels were analyzed using Western blotting. β-Actin was used as a protein loading control.Growth curves of hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells after RB1 knockdown.Representative images showing SA-β-gal staining in the hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without RB1 or TP53 knockdown. Cells were collected at the 7th passage.


Figure 5Representative blots showing LATS and YAP protein levels in hOSEs at the fourth , ninth and thirteenth passage . β - Actin was used as a protein loading control . Representative gel photographs showing mRNA levels of LATS1 and LATS2 in hOSEs with or without LATS2 knockout . CRISPR / Cas - 9 system was used to knock out LATS2 in hOSEs . LATS1 / 2 mRNA levels were analyzed using qRT - PCR at the fourth , ninth and thirteenth passage . Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells . Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry . YAP protein was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI . Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout . YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI ( blue ) . Representative images showing morphological change ( E ) in the 13th passage ( P13 ) hOSE cells with ( LATS2 KO ) or without ( CTRL ) LATS2 knockout . Representative images showing SA - β - gal staining ( F ) in the 13th passage ( P13 ) hOSE cells with ( LATS2 KO ) or without ( CTRL ) LATS2 knockout . Scale bar : 50 µm . Quantitative data showing the ratio of SA - β - gal positive cells in the control ( CTRL ) and LATS2 - knockout ( LATS2KO ) P13 hOSE cells .

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Figure 5Representative blots showing LATS and YAP protein levels in hOSEs at the fourth, ninth and thirteenth passage. β-Actin was used as a protein loading control.Representative gel photographs showing mRNA levels of LATS1 and LATS2 in hOSEs with or without LATS2 knockout. CRISPR/Cas-9 system was used to knock out LATS2 in hOSEs. LATS1/2 mRNA levels were analyzed using qRT-PCR at the fourth, ninth and thirteenth passage.Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells. Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry. YAP protein was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI.Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout. YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).Representative images showing morphological change (E) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout.Representative images showing SA-β-gal staining (F) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout. Scale bar: 50 µm.Quantitative data showing the ratio of SA-β-gal positive cells in the control (CTRL) and LATS2-knockout (LATS2KO) P13 hOSE cells.


Figure 6Representative blots showing expression levels of LATS1 / 2 , CCNE1 , CCND1 and YAP in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells . Cells were collected at passage four ( P4 ) and Passage seven ( P7 ) . Actin was used as internal control . mRNA levels of LATS1 , LATS2 and YAP in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells . Cells were collected at passage seven and protein levels were analyzed using quantitative real - time PCR . Representative gel photographs showing mRNA levels of LATS1 , LATS2 , and YAP in the LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells examined using semi - quantitative RT - PCR at passage seven . Representative images showing the morphologic changes and SA - β - gal staining in control and LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells . Quantitative data showing the ratio of SA - β - gal positive cells in the control and LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells at the seventh passage . Growth curves of the control and LATS2 - knockout hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells .

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Figure 6Representative blots showing expression levels of LATS1/2, CCNE1, CCND1 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage four (P4) and Passage seven (P7). Actin was used as internal control.mRNA levels of LATS1, LATS2 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage seven and protein levels were analyzed using quantitative real-time PCR.Representative gel photographs showing mRNA levels of LATS1, LATS2, and YAP in the LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells examined using semi-quantitative RT-PCR at passage seven.Representative images showing the morphologic changes and SA-β-gal staining in control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.Quantitative data showing the ratio of SA-β-gal positive cells in the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the seventh passage.Growth curves of the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.


Figure 7Representative images showing cells with ectopic expression of LATS2 , which is labelled with GFP ( the control is empty vectors ) . Relative mRNA levels of LATS1 , LATS2 , and YAP in the control and LATS2 - expressing hOSEs at the 4th passage . Relative mRNA levels were determined with RT - PCR . Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2 . Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage . Growth curves of control and LATS2 - overexpressing hOSE cells . Quantitative data showing the ratio of SA - β - gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2 . Representative images showing the effect of LATS2 on the morphology and β - galactosidase activities in cultured primary hOSE cells at the 9th passage . Quantitative data showing the ratio of SA - β - gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2 . mRNA levels of major components of the DREAM complex ( E2F4 , E2F5 , RBBP4 , TFDP2 ) in the control and LATS2 - overexpressing hOSEs at the 9th passage . Relative mRNA levels were examined by real - time PCR . Growth curves of the hOSE - shRB1 cells and hOSE - shRB1 - LATS2 cells at their 16th passages . Representative images showing cellular morphology and β - galactosidase activities in control and LATS2 - overexpressing hOSEs ( K ) . Scale Bar : 50 µm . Right bar graph ( L ) showing quantitative results of SA - β - gal positive cells in the hOSE - shRB1 cells and hOSE - shRB1 - LATS2 cells .

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Figure 7Representative images showing cells with ectopic expression of LATS2, which is labelled with GFP (the control is empty vectors).Relative mRNA levels of LATS1, LATS2, and YAP in the control and LATS2-expressing hOSEs at the 4th passage. Relative mRNA levels were determined with RT-PCR.Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2.Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage.Growth curves of control and LATS2-overexpressing hOSE cells.Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2.Representative images showing the effect of LATS2 on the morphology and β-galactosidase activities in cultured primary hOSE cells at the 9th passage.Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2.mRNA levels of major components of the DREAM complex (E2F4, E2F5, RBBP4, TFDP2) in the control and LATS2-overexpressing hOSEs at the 9th passage. Relative mRNA levels were examined by real-time PCR.Growth curves of the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells at their 16th passages.Representative images showing cellular morphology and β-galactosidase activities in control and LATS2-overexpressing hOSEs (K). Scale Bar: 50 µm. Right bar graph (L) showing quantitative results of SA-β-gal positive cells in the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells.


Figure 8Representative images showing colony formation in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without pRB knockdown or LATS2 knockout . hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging . Quantitative data showing colony numbers in hOSE - MXIV , hOSE - YAP and hOSE - YAPS127A cells with or without pRB knockdown or LATS2 deletion . hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope . Representative images showing tumorigenesis ( red circle area ) of hOSE - YAPS127A cells with or without pRB knockdown or LATS2 deletion . Tumors formed by hOSE - YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area . Right panel showing representative tumors formed by hOSE - YAPS127A - LATS2KO and hOSE - YAPS127A - shRB1 cells . Please note that no tumor xenografts formed in hOSE - YAPS127A alone group . Representative images showing expression of YAP , Ki67 and LATS2 in hOSE - LATS2KO - YAPS127A cell - derived tumors examined by immunohistochemistry .

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Figure 8Representative images showing colony formation in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 knockout. hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging.Quantitative data showing colony numbers in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope.Representative images showing tumorigenesis (red circle area) of hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. Tumors formed by hOSE-YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area. Right panel showing representative tumors formed by hOSE-YAPS127A-LATS2KO and hOSE-YAPS127A-shRB1 cells. Please note that no tumor xenografts formed in hOSE-YAPS127A alone group.Representative images showing expression of YAP, Ki67 and LATS2 in hOSE-LATS2KO-YAPS127A cell-derived tumors examined by immunohistochemistry.


Figure 1 ( A ) Proliferation analysis of hematopoietic CD34 + precursors for up to 6 days ( section i ) . ( A ) Neutrophil morphologic differentiation were assessed at day 6 ( section ii ) . Controls were without treatment . White arrows indicate neutrophilnuclear segmentation . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve . ( A ) Neutrophil bacterial killing were assessed at day 6 ( section iii ) . Controls were without treatment . . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve . ( A ) Neutrophil ROS production were assessed at day 6 ( iv ) . Controls were without treatment . A + G , Am80 - GCSF combination ; CL , chemiluminescence ; RLU , relative light units ; AUC , area under the curve . ( B ) qRT - PCR assessed RA - target gene expression after culturing CD34 + cells for 1 , 2 , and 6 days . ( C ) Flow cytometry analysis of CD66 - CD18 co - expression after culturing CD34 + cells for 6 days , using anti - human CD66 - PE and CD18 - FITC antibodies ( section i ) . Isotypes were used for controls ( section i , top panel ) . CD66 - CD18 co - expression was quantified in section ii . ( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear cells were evaluated by bacterial killing assays ( section iii ) . ( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bactericidal activities of neutrophils induced by Am80 - GCSF from PB mononuclear cells were evaluated by ROS production ( section ii ) . ( D ) Fresh peripheral blood ( PB ) collected from normal human donor . Bacterial killing was assessed in the presence or absence of PBneutrophils ( section i ) . ( E ) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies . Controls were with or without IgG . Normal IgG , φ ; Ab , antibodies ; Comb Ab , combined anti - CD18 and - CD11b antibodies . ( F ) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days . Bacterial killing ( sections i ) and neutralization of bacterial killing in the presence of IgG or anti - CD18 antibody were assessed at day 3 ( section ii ) .

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Figure 1(A) Proliferation analysis of hematopoietic CD34+ precursors for up to 6 days (section i).(A) Neutrophil morphologic differentiation were assessed at day 6 (section ii). Controls were without treatment. White arrows indicate neutrophilnuclear segmentation. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil bacterial killing were assessed at day 6 (section iii). Controls were without treatment. . A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(A) Neutrophil ROS production were assessed at day 6 (iv). Controls were without treatment. A+G, Am80-GCSF combination; CL, chemiluminescence; RLU, relative light units; AUC, area under the curve.(B) qRT-PCR assessed RA-target gene expression after culturing CD34+ cells for 1, 2, and 6 days.(C) Flow cytometry analysis of CD66-CD18 co-expression after culturing CD34+ cells for 6 days, using anti-human CD66-PE and CD18-FITC antibodies (section i). Isotypes were used for controls (section i, top panel). CD66-CD18 co-expression was quantified in section ii.(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by bacterial killing assays (section iii).(D) Fresh peripheral blood (PB) collected from normal human donor. Bactericidal activities of neutrophils induced by Am80-GCSF from PB mononuclear cells were evaluated by ROS production (section ii).(D) Fresh peripheral blood (PB) collected from normal human donor. Bacterial killing was assessed in the presence or absence of PBneutrophils (section i).(E) Fresh PBneutrophils collected from normal human donor were tested for ROS production in the presence or absence of specific antibodies. Controls were with or without IgG. Normal IgG, φ; Ab, antibodies; Comb Ab, combined anti-CD18 and -CD11b antibodies.(F) Fresh PB mononuclear cells collected from normal human donors were treated for 3 days. Bacterial killing (sections i) and neutralization of bacterial killing in the presence of IgG or anti-CD18 antibody were assessed at day 3 (section ii).


Figure 2 ( A ) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia ( AML ) patient . ROS production ( section ii ) were analyzed after cells were treated for 4 days . ( A ) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia ( AML ) patient . Proliferation ( section i ) were analyzed after cells were treated for 4 days . ( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . Proliferation was assessed at day 3 in parallel ( section ii ) . ( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . ROS production were assessed at day 3 in parallel ( section iii ) . ( B ) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days . Transcription of RA - target genes was analyzed with qRT - PCR at day 2 , 3 , and 6 ( sections i ) .

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Figure 2(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. ROS production (section ii) were analyzed after cells were treated for 4 days.(A) Fresh bone marrow mononuclear cells were collected from acute myeloid leukemia (AML) patient. Proliferation (section i) were analyzed after cells were treated for 4 days.(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Proliferation was assessed at day 3 in parallel (section ii).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. ROS production were assessed at day 3 in parallel (section iii).(B) Fresh BM mononuclear cells collected from AML patient were treated for up to 6 days. Transcription of RA-target genes was analyzed with qRT-PCR at day 2, 3, and 6 (sections i).


Figure 3 ( A ) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF ( 25 ng / ml ) for up to 9 days . Dynamic changes in proliferation ( left sections ) and live cells on day 9 ( right sections ) were illustrated for each of specimens . Controls were without treatment . ( B ) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . Dynamic changes in proliferation ( left sections ) and live cells on day 12 ( right sections ) were illustrated for each of specimens . ( C ) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF . Proliferation ( left sections ) and bacterial killing ( right sections ) were analyzed on day 12 for each of samples . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Morphologic differentiation was assessed at day 12 ( section iv ) . White arrows indicate neutrophil nuclear segmentation . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Bacterial killing was assessed at day 12 ( sections iii ) . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . RA - target gene expression was assessed at day 12 ( section v ) . ( B ) Fresh BM mononuclear cells collected from AML patients were treated with 20 , 100 , or 150 nM Am80 alone or combined with GCSF for up to 12 days . ROS production was assessed at day 12 ( section ii ) . ( D ) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF . Proliferation was assessed for up to 12 days ( section i ) .

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Figure 3(A) Fresh BM mononuclear cells collected from AML patients were treated with 20 or 50 nM Am80 alone or combined with medium dose of GCSF (25 ng/ml) for up to 9 days. Dynamic changes in proliferation (left sections) and live cells on day 9 (right sections) were illustrated for each of specimens. Controls were without treatment.(B) Fresh PB or BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. Dynamic changes in proliferation (left sections) and live cells on day 12 (right sections) were illustrated for each of specimens.(C) Fresh BM mononuclear cells collected from AML patients were treated with 150 nM Am80 alone or combined with GCSF. Proliferation (left sections) and bacterial killing (right sections) were analyzed on day 12 for each of samples.(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Morphologic differentiation was assessed at day 12 (section iv). White arrows indicate neutrophil nuclear segmentation.(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Bacterial killing was assessed at day 12 (sections iii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. RA-target gene expression was assessed at day 12 (section v).(B) Fresh BM mononuclear cells collected from AML patients were treated with 20, 100, or 150 nM Am80 alone or combined with GCSF for up to 12 days. ROS production was assessed at day 12 (section ii).(D) Fresh BM mononuclear cells collected from AML patient were treated with 150 nM Am80 alone or combined with GCSF. Proliferation was assessed for up to 12 days (section i).


Figure 4 ( A ) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide ( CPA ) consists of both neutrophil decrease and neutrophil recovery stages . ANC , absolute neutrophil count . ( D ) Vetscan counting PBleukocytes . ( E ) Neutrophils induced by low dose treatment in PB ( section i ) and BM ( section ii ) . ( F ) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post - infection and in spleen ( sections i - iii ) , using blood agar analysis of total extracellular viable bacteria . ( H ) Vetscan counting PBleukocytes . ( I ) Neutrophils induced by medium dose treatment in PB ( section i ) and BM ( section ii ) . ( J ) Similar to panel F , bacterial killing by PBneutrophils was evaluated 3 and 16 hr post - infection and in heart .

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Figure 4(A) Mouse CCIN induced by cancer chemotherapy drug cyclophosphamide (CPA) consists of both neutrophil decrease and neutrophil recovery stages. ANC, absolute neutrophil count.(D) Vetscan counting PBleukocytes.(E) Neutrophils induced by low dose treatment in PB (section i) and BM (section ii).(F) Bacterial killing by PBneutrophils was assessed at 3 and 16 hr post-infection and in spleen (sections i-iii), using blood agar analysis of total extracellular viable bacteria.(H) Vetscan counting PBleukocytes.(I) Neutrophils induced by medium dose treatment in PB (section i) and BM (section ii).(J) Similar to panel F, bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection and in heart.


Figure 5 ( B ) Vetscan counting PBleukocytes . ( C ) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post - infection ( sections i , ii ) , using blood agar analysis of extracellular viable bacteria . ( D E ) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and / or GCSF in BM ( panel D ) and PB ( panel E ) . White arrows indicate neutrophilnuclear segmentation .

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Figure 5(B) Vetscan counting PBleukocytes.(C) Bacterial killing by PBneutrophils was evaluated 3 and 16 hr post-infection (sections i, ii), using blood agar analysis of extracellular viable bacteria.(D E) Neutrophil recovery and morphologic differentiation induced by low doses of Am80 and/or GCSF in BM (panel D) and PB (panel E). White arrows indicate neutrophilnuclear segmentation.


Figure 6 ( B ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 3 independent experiments with similar results . ( C ) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9 . White arrows indicate phagocytosed S . aureus in neutrophils . ( E ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) . Data represent 2 independent experiments with similar results . ( G ) Survival of CCIN mice was calculated with both Kaplan - Meier plots and log - rank test on day 9 since CPA injection ( section i ) as well as assessed 6 days post - infection ( section ii ) .

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Figure 6(B) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 3 independent experiments with similar results.(C) Gram and Giemsa stains analyzed neutrophil phagocytosis and bacterial clearance in mice surviving on day 9. White arrows indicate phagocytosed S. aureus in neutrophils.(E) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii). Data represent 2 independent experiments with similar results.(G) Survival of CCIN mice was calculated with both Kaplan-Meier plots and log-rank test on day 9 since CPA injection (section i) as well as assessed 6 days post-infection (section ii).


Figure 7 ( A B ) Neutrophil recovery and morphologic differentiation were assessed in BM ( panel A ) and PB ( panel B ) of CCIN mice surviving on day 9 . White arrows indicate neutrophil nuclear segmentation . ( C ) PBneutrophil production and nuclear segmentation were assessed in control and Am80 - GCSF mice surviving on day 13 ( section i ) . ( C ) The increased spleen size on day 9 in Am80 - GCSF mice dropped to the levels similar to those in control mice on day 13 ( section iii ) . ( C ) The increased spleen weight on day 9 in Am80 - GCSF mice dropped to the levels similar to those in control mice on day 13 ( sections iii , iv ) . ( C ) PBneutrophil production and nuclear segmentation were assessed in control and Am80 - GCSF mice surviving on day 13 ( section ii ) . ( D ) Significant loss of body weight occurred in GCSFmice on day 9 . ( E ) Ultrastructure analysis of neutrophils collected from mice surviving on day 11 . Neutrophil from GCSFmice showed less granules ( section i , images 1 vs . 2 or 3 ) , more intracytoplasmic vacuoles ( sections i , ii ) , and inner nuclear membrane dilation ( sections i , iii ) . Pg : primary granules ; Sg : secondary granules .

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Figure 7(A B) Neutrophil recovery and morphologic differentiation were assessed in BM (panel A) and PB (panel B) of CCIN mice surviving on day 9. White arrows indicate neutrophil nuclear segmentation.(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section i).(C) The increased spleen size on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (section iii).(C) The increased spleen weight on day 9 in Am80-GCSF mice dropped to the levels similar to those in control mice on day 13 (sections iii, iv).(C) PBneutrophil production and nuclear segmentation were assessed in control and Am80-GCSF mice surviving on day 13 (section ii).(D) Significant loss of body weight occurred in GCSFmice on day 9.(E) Ultrastructure analysis of neutrophils collected from mice surviving on day 11. Neutrophil from GCSFmice showed less granules (section i, images 1 vs. 2 or 3), more intracytoplasmic vacuoles (sections i, ii), and inner nuclear membrane dilation (sections i, iii). Pg: primary granules; Sg: secondary granules.


Figure 8A . Vps10yEGFP localization . Yeast cells carrying Vps10yEGFP and expressing the indicated vps35 alleles as the sole source of Vps35 were logarithmically grown in SC medium . Their vacuoles were labelled with FM4 - 64 . Cells were harvested by brief centrifugation and immediately imaged by confocal microscopy . Single confocal planes are shown . A brightfield image was used to outline the cell boundaries ( shown in the merged images ) . B . Co - localization of Vps10yEGFP and FM4 - 64 in cells from a was measured using Pearson ' s coefficient . 15 Confocal planes with 20 to 30 cells each from 3 independent experiments were analyzed .

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Figure 8A. Vps10yEGFP localization. Yeast cells carrying Vps10yEGFP and expressing the indicated vps35 alleles as the sole source of Vps35 were logarithmically grown in SC medium. Their vacuoles were labelled with FM4-64. Cells were harvested by brief centrifugation and immediately imaged by confocal microscopy. Single confocal planes are shown. A brightfield image was used to outline the cell boundaries (shown in the merged images). B. Co-localization of Vps10yEGFP and FM4-64 in cells from a was measured using Pearson's coefficient. 15 Confocal planes with 20 to 30 cells each from 3 independent experiments were analyzed. Means


Figure 9B . Influence of VPS35 variants on GLUT1 . HK2 cells silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP - VPS35 . GLUT1 was detected by fixation and immunofluorescence staining as in a . Maximum projections of image stacks ( step size in z of 300 nm ) are shown . C . Quantification of GLUT1 immunofluorescence in cells from B . Regions of interest ( ROIs ) corresponding to cells expressing the indicated VPS35 variants , and some regions outside the cells ( background ) , were manually defined using ImageJ software . Total cell fluorescence was integrated and corrected for background fluorescence . 105 cells per condition stemming from 3 independent experiments were analyzed . The analysis was performed with 99 % confidence : * * * p < 0 . 001 . D . Expression of Vps35 variants in cells from B was analyzed by SDS - PAGE and Western blot against Vps35 . Tubulin served as loading control .

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Figure 9B. Influence of VPS35 variants on GLUT1. HK2 cells silenced for VPS35 were transfected with a plasmid carrying siRNA resistant wildtype or mutant forms of GFP-VPS35. GLUT1 was detected by fixation and immunofluorescence staining as in a. Maximum projections of image stacks (step size in z of 300 nm) are shown.C. Quantification of GLUT1 immunofluorescence in cells from B. Regions of interest (ROIs) corresponding to cells expressing the indicated VPS35 variants, and some regions outside the cells (background), were manually defined using ImageJ software. Total cell fluorescence was integrated and corrected for background fluorescence. 105 cells per condition stemming from 3 independent experiments were analyzed. The analysis was performed with 99% confidence: *** p < 0.001.D. Expression of Vps35 variants in cells from B was analyzed by SDS-PAGE and Western blot against Vps35. Tubulin served as loading control.


Figure 1A ) Neutrophils and lymphocytes count . B ) Levels of creatine kinase ( CK ) and creatinine . C ) Levels of procalcitonin ( PCT ) and C - reactive protein ( CRP ) . D ) Levels of erythrocyte sedimentation rate ( ESR ) and ferritin . E ) Levels of D - dimer and lactate dehydrogenase ( LDH ) . F ) Proportion of total CD3 + and CD4 + T cells . G ) Proportion of CD8 + T cells and CD4 + / CD8 + T cell ratio . H ) Proportion of B and natural killer ( NK ) cells . cytokine profile of COVID - 19 patients for IL - 2 , IL - 4 ( I ) cytokine profile of COVID - 19 patients for , IL - 6 , IL - 10 ( J ) and TNF - α and IFN - γ ( K ) .

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Figure 1A) Neutrophils and lymphocytes count.B) Levels of creatine kinase (CK) and creatinine.C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).D) Levels of erythrocyte sedimentation rate (ESR) and ferritin.E) Levels of D-dimer and lactate dehydrogenase (LDH).F) Proportion of total CD3+ and CD4+ T cells.G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio.H) Proportion of B and natural killer (NK) cells.cytokine profile of COVID-19 patients for IL-2, IL-4 (I)cytokine profile of COVID-19 patients for , IL-6, IL-10 (J) and TNF-α and IFN-γ (K).


Figure 2Time from symptom onset to initial diagnosis ( TFSD ) . Time from symptom onset to development of pneumonia ( TFSP ) . Time from symptom onset to treatment ( TFST ) . Time from development of pneumonia to recovery ( TFPR ) .

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Figure 2Time from symptom onset to initial diagnosis (TFSD).   Time from symptom onset to development of pneumonia (TFSP).   Time from symptom onset to treatment (TFST).Time from development of pneumonia to recovery (TFPR).


Figure 3Correlation between IL - 6 and maximal body temperature during hospitalization . Correlation between IL - 6 and C - reactive protein ( CRP ) . Correlation between IL - 6 and lactate dehydrogenase ( LDH ) . Correlation between IL - 6 and D - dimer . Correlation between IL - 6 and ferritin . Levels of baseline IL - 6 in patients who received glucocorticoids , human immunoglobulin , high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not . Error bars , SEM .

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Figure 3Correlation between IL-6 and maximal body temperature during hospitalization.Correlation between IL-6 and C-reactive protein (CRP).Correlation between IL-6 and lactate dehydrogenase (LDH).Correlation between IL-6 and D-dimer.Correlation between IL-6 and ferritin.Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.


Figure 4IL - 6 levels before and after treatment in three patient groups classified according to baseline IL - 6 levels of ≥ 40 pg / mL ( n = 11 ) , ≥ 20 pg / mL ( n = 6 ) or < 20 pg / mL ( n = 9 ) ( left ) . Two representative chest computed tomography ( CT ) scans before and after treatment from each group ( right ) . IL - 6 levels at diagnosis and after disease progression in three exacerbated patients ( left ) . Progressed radiological findings were recorded in two patients ( right ) , while radiological assessment after treatment was not performed in the third patient due to poor general condition . The baseline IL - 6 level was 197 . 39 pg / mL in a 69 - year - old female patient who showed high fever and dyspnea . IL - 6 decreased to 9 . 47 pg / mL after treatment ( day 8 ) , while the symptoms were not relieved . The C - reactive protein ( CRP ) rebounded and procalcitonin ( PCT ) increased together with disease exacerbation . Follow - up chest computed tomography ( CT ) assessment was not performed due to poor general condition , whereas chest X - ray showed aggravated pneumonia . Follow - up sputum culture confirmed the exacerbation was caused by bacterial infection .

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Figure 4IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right).IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition.The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.


Figure 5A ) The chest computed tomography ( CT ) was normal five days after symptom onset . Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities . Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset . B ) Procalcitonin ( PCT ) levels stayed within normal range throughout the course of disease . C ) Lymphocyte count was normal five days after symptom onset , while firstly decreasing then returning to normal range during the course of disease . Both abnormal C - reactive protein ( CRP ) ( D ) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal . In association with changes in chest CT scans , CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset . IL - 6 ( E ) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal . In association with changes in chest CT scans , IL - 6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset . F ) The body temperature was elevated when CRP and IL - 6 levels were rising , but returned to normal when CRP and IL - 6 . Levels were declining .

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Figure 5A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset.B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease.Both abnormal C-reactive protein (CRP) (D) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.


Figure 1B ) Body weights of E18 . 5 wild - type , TKO and QKO foetuses . Data for individual animals are presented as circles with mean ± SEM . Data were analysed by one - way ANOVA followed by multiple comparison with Benjamini and Hochberg correction . C ) Percentages of E18 . 5 QKO foetuses exhibiting the indicated externally visible developmental defects ( numbers above bars represent the number of animals examined ) . Age - matched TKO animals are included for comparison . An example of a QKO E18 . 5 pup presenting with an omphalocele ( asterisk ) , exencephaly ( arrow ) , and spina bifida ( arrowhead ) is shown alongside an age - matched WT control . Scale bar = 5 mm .

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Figure 1B) Body weights of E18.5 wild-type, TKO and QKO foetuses. Data for individual animals are presented as circles with mean ± SEM. Data were analysed by one-way ANOVA followed by multiple comparison with Benjamini and Hochberg correction.C) Percentages of E18.5 QKO foetuses exhibiting the indicated externally visible developmental defects (numbers above bars represent the number of animals examined). Age-matched TKO animals are included for comparison. An example of a QKO E18.5 pup presenting with an omphalocele (asterisk), exencephaly (arrow), and spina bifida (arrowhead) is shown alongside an age-matched WT control. Scale bar = 5 mm.


Figure 2A ) Percentages of E18 . 5 QKO foetuses exhibiting the indicated defects when examined under a dissection microscope . Age - matched TKO animals are included for comparison . Numbers above bars represent number of animals examined . B - D ) Images depicting the hindlimbs ( HL ) , tail ( T ) , forelimbs ( FL ) , and ventral view of the palate ( P ) in a wild - type foetus at E18 . 5 of development . E - G ) Corresponding images of a representative age - matched QKO foetus . Panel G shows a cleft palate ( indicated by arrowheads ) . H , I ) Magnified images of panels E and F . Arrows indicate excess tissue at the anterior and posterior edges of the hindlimb and forelimb , respectively , of QKO foetuses . J ) Percentages of QKO animals exhibiting various aortic arch abnormalities and great vessel defects ( n = 19 ) . K ) Aortic arch and vessels in a wild - type foetus . A corresponding drawing to explain the images is shown on the right . L , M ) Images of QKO foetuses and accompanying illustration of vessel defects . Absence of the right subclavian artery ( L ) and persistent truncus arteriosus ( pTA ) with a right curving pulmonary trunk / ductus arteriosus ( PT / rcDA ; M ) .

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Figure 2A) Percentages of E18.5 QKO foetuses exhibiting the indicated defects when examined under a dissection microscope. Age-matched TKO animals are included for comparison. Numbers above bars represent number of animals examined.B-D) Images depicting the hindlimbs (HL), tail (T), forelimbs (FL), and ventral view of the palate (P) in a wild-type foetus at E18.5 of development. E-G) Corresponding images of a representative age-matched QKO foetus. Panel G shows a cleft palate (indicated by arrowheads). H, I) Magnified images of panels E and F. Arrows indicate excess tissue at the anterior and posterior edges of the hindlimb and forelimb, respectively, of QKO foetuses.J) Percentages of QKO animals exhibiting various aortic arch abnormalities and great vessel defects (n = 19).K) Aortic arch and vessels in a wild-type foetus. A corresponding drawing to explain the images is shown on the right.L, M) Images of QKO foetuses and accompanying illustration of vessel defects. Absence of the right subclavian artery (L) and persistent truncus arteriosus (pTA) with a right curving pulmonary trunk / ductus arteriosus (PT/rcDA; M).


Figure 3A - C ) Heads of E18 . 5 foetuses were serially sectioned in coronal orientation and stained with H & E . Representative images of rostro - occipital level - matched sections from age - matched wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( TKO ; n = 4 ) and QKO foetuses ( n = 4 ) depicting the forebrain , nasal cavity , and tongue are shown . BP , bony palate ; Cx , cerebral cortex ; ES , epidural space ; In , incisor ; LV ; lateral ventricle ; Ma , mandible ; MC , mouth cavity ; NC , nasal cavity ; NS , nasal septum ; St , striatum ; SVZ , subventricular zone ; To , tongue ; WP , whisker follicles . Scale bar = 850 µm ( A ) , 1200 µm ( B , C ) . D ) Morphometric assessment of indicated tissue areas . Data of individual animals are displayed as circles with means and standard error of the mean . Data were analysed by unpaired , two - tailed Student ' s t - test . Related data normalised to mean bodyweight are displayed in Figure EV1 .

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Figure 3A-C) Heads of E18.5 foetuses were serially sectioned in coronal orientation and stained with H&E. Representative images of rostro-occipital level-matched sections from age-matched wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (TKO; n = 4) and QKO foetuses (n = 4) depicting the forebrain, nasal cavity, and tongue are shown. BP, bony palate; Cx, cerebral cortex; ES, epidural space; In, incisor; LV; lateral ventricle; Ma, mandible; MC, mouth cavity; NC, nasal cavity; NS, nasal septum; St, striatum; SVZ, subventricular zone; To, tongue; WP, whisker follicles. Scale bar = 850 µm (A), 1200 µm (B, C).D) Morphometric assessment of indicated tissue areas. Data of individual animals are displayed as circles with means and standard error of the mean. Data were analysed by unpaired, two-tailed Student's t-test. Related data normalised to mean bodyweight are displayed in Figure EV1.


Figure 4A - C ) Representative H & E - stained coronal serial sections of the eye from E18 . 5 wild - type ( n = 2 ) , Bok - / - Bak - / - Bax - / - triple knockout ( n = 3 ) , and Bok - / - Bak - / - Bax - / - Bid - / - quadruple knockout ( n = 3 ) foetuses . D ) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial cells present . Data of individual animals are displayed as circles with means and standard error of the mean and were analysed by unpaired , two - tailed Student ' s t - test .

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Figure 4A-C) Representative H&E-stained coronal serial sections of the eye from E18.5 wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (n = 3), and Bok-/-Bak-/-Bax-/-Bid-/- quadruple knockout (n = 3) foetuses.D) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial cells present. Data of individual animals are displayed as circles with means and standard error of the mean and were analysed by unpaired, two-tailed Student's t-test.


Figure 9 ( A ) Estimation of BID protein levels in TKO MEFs . DKO MEFs re - expressing human BAK , TKO MEFs , and two human leukaemia cell lines RS4 ; 11 and MV4 ; 11 , were lysed and analysed by Western blot for BID levels , using a rat monoclonal antibody raised against human BID ( anti - hBID clone 2D1 ) that also recognises mouse BID . Also present are samples of the human FL - BID protein added at the equivalent concentrations used in ( B ) and ( C ) . On the right gel , duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells ( from B and C ) showing that endogenous mouse BID is cytosolic in these cells . Data shown are representative of 2 independent experiments . ( B , C ) Partial cytochrome c release by FL - BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID . DKO MEFs re - expressing human BAK or TKO MEFs were permeabilised , and then the cells incubated with the indicated concentrations of Bim BH3 peptide , cBID or FL - BID , alone or in the indicated combinations , at 30oC for 30 min . Cells were then co - stained for cytochrome c and TOM20 and analysed by flow cytometry . Left hand panels show median fluorescence data from replicate experiments ( squares denote experiment 1 , circles denote experiment 2 ) . Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 ( treatments are as listed in the left - hand panels ) .

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Figure 9(A) Estimation of BID protein levels in TKO MEFs. DKO MEFs re-expressing human BAK, TKO MEFs, and two human leukaemia cell lines RS4;11 and MV4;11, were lysed and analysed by Western blot for BID levels, using a rat monoclonal antibody raised against human BID (anti-hBID clone 2D1) that also recognises mouse BID. Also present are samples of the human FL-BID protein added at the equivalent concentrations used in (B) and (C). On the right gel, duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells (from B and C) showing that endogenous mouse BID is cytosolic in these cells. Data shown are representative of 2 independent experiments.(B, C) Partial cytochrome c release by FL-BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID. DKO MEFs re-expressing human BAK or TKO MEFs were permeabilised, and then the cells incubated with the indicated concentrations of Bim BH3 peptide, cBID or FL-BID, alone or in the indicated combinations, at 30oC for 30 min. Cells were then co-stained for cytochrome c and TOM20 and analysed by flow cytometry. Left hand panels show median fluorescence data from replicate experiments (squares denote experiment 1, circles denote experiment 2). Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 (treatments are as listed in the left-hand panels).


Figure 1 ( A ) GSCs contain the higher levels of stemness markers ( Nestin , SOX2 , Musashi - 1 , NANOG , and OCT4 ) compared with their DFCs . Total RNAs from GSCs and DFCs were subjected to real - time PCR . The average relative amounts of GSCs were normalized with those of DFCs . Shown are means + S . D . of the normalized mRNA levels of GSCs obtained from the three independent experiments . ( B ) Immunoblotting analysis of markers for stemness ( SOX2 , Nestin and Musashi - 1 ) and differentiation ( GFAP and TUJ1 ( Neuron - specific class III beta - tubulin ) ) in GSCs in comparison with GSC - derived cells at different passage numbers ( P1 - P7 ) . ( C ) Measurement of the cell doubling time . Approximately 3 × 104 cells for each of XO8 GSCs and XO8 DFCs were seeded in a 60 mm culture dish and counted every day . The cells were treated with trypsin - EDTA , and the number of viable cells was counted with a hemocytometer . The doubling time was calculated from the cell growth curve over 5 days . Shown are means ± S . D . obtained from three independent experiments . ( D ) FACS analysis of CD133 and GFAP using X08 GSCs and DFCs . Data are from representative experiments repeated at least three times . ( E ) Immunoblotting analysis of CD133 and GFAP in GSCs in comparison with DFCs . ( F ) Quantitation of ( G ) . ( G ) Equal numbers of XO8 GSCs and DFCs were injected orthotropically into mouse brains . After six weeks , brains were harvested and tumorigenic potential was assessed by evaluating tumor formation . Representative microphotographs of the brain sections of mice injected with XO8 GSCs and XO8 DFC controls .

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Figure 1(A) GSCs contain the higher levels of stemness markers (Nestin, SOX2, Musashi-1, NANOG, and OCT4) compared with their DFCs. Total RNAs from GSCs and DFCs were subjected to real-time PCR. The average relative amounts of GSCs were normalized with those of DFCs. Shown are means +S.D. of the normalized mRNA levels of GSCs obtained from the three independent experiments.(B) Immunoblotting analysis of markers for stemness (SOX2, Nestin and Musashi-1) and differentiation (GFAP and TUJ1 (Neuron-specific class III beta-tubulin)) in GSCs in comparison with GSC-derived cells at different passage numbers (P1-P7).(C) Measurement of the cell doubling time. Approximately 3×104 cells for each of XO8 GSCs and XO8 DFCs were seeded in a 60 mm culture dish and counted every day. The cells were treated with trypsin-EDTA, and the number of viable cells was counted with a hemocytometer. The doubling time was calculated from the cell growth curve over 5 days. Shown are means ±S.D. obtained from three independent experiments.(D) FACS analysis of CD133 and GFAP using X08 GSCs and DFCs. Data are from representative experiments repeated at least three times.(E) Immunoblotting analysis of CD133 and GFAP in GSCs in comparison with DFCs.(F) Quantitation of (G). (G) Equal numbers of XO8 GSCs and DFCs were injected orthotropically into mouse brains. After six weeks, brains were harvested and tumorigenic potential was assessed by evaluating tumor formation. Representative microphotographs of the brain sections of mice injected with XO8 GSCs and XO8 DFC controls.


Figure 2Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide ( A ) , cisplatin ( B ) , adriamycin ( C ) , taxol ( D ) , TRAIL ( E ) , bafilomycin A1 ( F ) , thapsigargin ( G ) , and MG132 ( H ) . Means ± S . D . from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO . ( I ) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs . Cell viability was measured using trypan blue exclusion assay . Error bars represent the mean ± SD from three independent experiments . For statistical analysis , student t - test ( two - sided , one type ) were used . ( * P < 0 . 05 ) . ( J ) XO8 GSCs were treated with MG132 were cultured in 0 . 3 % soft - agar as described in Materials and Methods . The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting . Bars , means + S . D . from three independent experiments .

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Figure 2Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO.(I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05).(J) XO8 GSCs were treated with MG132 were cultured in 0.3% soft-agar as described in Materials and Methods. The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting. Bars, means +S.D. from three independent experiments.


Figure 3 ( A ) XO8 GSCs and DFCs were differentially labeled with PKH67 ( green ) and PKH26 ( red ) , respectively , and mixed before plated on laminine - coated plates . The cells were treated with 50 nM MG132 and subjected to fluorescent microscopy . Scale bar , 100 μM . ( B ) Quantitation of ( A ) . Cell numbers from three independent experiments were normalized to those of control cells treated with DMSO and presented as means + S . D . ( * p = 0 . 0035 , n = 3 ) . Student t - test ( two - sided , one type ) were used for statistical analysis . ( C ) The harvested cells from ( A ) were subjected to FACS analysis . ( D ) XO8 GSCs were treated with various PIs ( salinosporamide A , epoxomicin , and PS341 ) , followed by cell viability assays . Bars , means ± S . D . from three independent experiments . ( E ) XO6 , XO8 , and XO10 GSCs were treated with MG132 and subjected to cell viability assay . Bars , means ± S . D . from three independent experiments . ( F ) XO6 , XO8 , and XO10 GSCs were treated with MG132 and subjected to cell viability assay in serum - containing media . Bars , means ± S . D . from three independent experiments .

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Figure 3(A) XO8 GSCs and DFCs were differentially labeled with PKH67 (green) and PKH26 (red), respectively, and mixed before plated on laminine-coated plates. The cells were treated with 50 nM MG132 and subjected to fluorescent microscopy. Scale bar, 100 μM. (B) Quantitation of (A). Cell numbers from three independent experiments were normalized to those of control cells treated with DMSO and presented as means +S.D. (*p=0.0035, n=3). Student t-test (two-sided, one type) were used for statistical analysis.(C) The harvested cells from (A) were subjected to FACS analysis.(D) XO8 GSCs were treated with various PIs (salinosporamide A, epoxomicin, and PS341), followed by cell viability assays. Bars, means ±S.D. from three independent experiments.(E) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay. Bars, means ±S.D. from three independent experiments. (F) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay in serum-containing media. Bars, means ±S.D. from three independent experiments.