( A ) Representative images of EC - Pik3caH1047R / WT P6 retinas isolated from mice treated with 0 . 125 mg / kg of 4 - OHT on P1 . Retinas were immunostained for blood vessels ( IB4 ) . Red asterisks show arteries and arterioles and yellow arrowheads veins . Scale bars : 150 μm ( left panel ) and 30 μm ( right , high magnification panels ) . ( B ) Representative images of P6 retinas from control and EC - Pik3caH1047R / WT mice treated with 0 . 125 mg / kg 4 - OHT on P1 , following immunostaining for p - S6 ( S235 / 236 ) and blood vessels ( IB4 ) . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( C ) Representative control and EC - Pik3caH1047R / WT P6 retinas immunostained for blood vessels ( IB4 ) , EC nuclei ( Erg ) and EdU . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( D - G ) Quantification of ( D ) p - S6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) , ( E ) EC proliferation by EdU staining , ( F ) EC number by Erg positive cells and ( G ) retinal vascularity by IB4 - positive area in control and EC - Pik3caH1047R / WT retinas . Error bars are s . e . m . n > 5 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 was considered statistically 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.


( B , C ) Representative images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panel ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically 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

(B, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 3 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically 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

(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 were considered statistically 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

(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.


( B ) Representative sequencing chromatograms of PIK3CA and TEK mutant VM - derived ECs . Arrows show the detected point mutations .

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

(B) Representative sequencing chromatograms of PIK3CA and TEK mutant VM-derived ECs. Arrows show the detected point mutations.


( C ) Representative confocal images of PIK3CA and TEK patient - derived ECs immunostained for VE - cadherin ( EC - specific junctional protein ) and ERG ( EC - specific transcription factor ) . Cell nuclei were visualised with DAPI . Scale bars : 30 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

(C) Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific transcription factor). Cell nuclei were visualised with DAPI. Scale bars: 30 μm.


( D ) Immunoblot showing the activation of PI3K / AKT / mTORC1 pathway ( by assessing the levels of p - AKT and p - S6 ) among different PIK3CA and TEK patient - derived ECs . Primary HUVEC were used as wild - type control .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control.


( E - F ) Immunoblot showing the impact of miransertib at increasing doses on PI3K / AKT / mTORC1 pathway ( by assessing p - AKT and p - S6 levels ) in ( E ) PIK3CA and ( F ) TEK mutant patient - derived ECs .

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

(E-F) Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK mutant patient-derived ECs.


( G ) Wild - type ( HUVECs ) , PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay . Fitting curves and 95 % CI IC50 values for wild - type ( HUVECs ) , PIK3CA and TEK ECs are shown . For PIK3CA and TEK - mutant ECs n = 3 biological replicates ( patients ) ; for wild - type ECs ( HUVECs ) n = 4 technical replicates PIK3CA - mutant vs . TEK - mutant p = 0 . 501 ; PIK3CA - mutant vs . HUVECs p = 0 . 0815 ; TEK - mutant vs . HUVECs : p = 0 . 4085 . Statistical analysis was performed by comparison of best - fit values using the extra sum - of - squares F test .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wild-type (HUVECs), PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.


Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL / 6 mouse lines . Tissues were isolated from indicated C57BL / 6 or iNKT TN mouse lines and stained with anti - CD3 and CD1d ( PBS57 ) tetramer .

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

Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.


Spleen , mLN , sdLN , liver , adipose , and lung lymphocytes from Jα18 - / - or Vα14 mice were stimulated in vitro with RAW - CD1d cells and 1μg α - GalCer . An additional sample of Vα14 lymphocytes from each organ was plated with RAW - CD1d cells but no α - GalCer . Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array . Error bars are SD of mean values from three different mice per group . Results shown are representative of two independent experiments where n = 3 biological replicates .

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

Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.


Lymphocytes from the indicated tissues of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer , before they were fixed , permeabilized , and stained with antibodies to T - bet , RORγt , and PLZF . Results shown are gated on CD3 + CD1d - tetramer + cells .

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

Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells.


The percentage of CD3 + CD1d - tetramer + iNKT cells in each organ that stained positively for PLZF , T - bet , and RORγt are shown . * * p < 0 . 01 , Mann - Whitney test . Error bars are SD .

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

The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **p<0.01, Mann-Whitney test. Error bars are SD.


Lymphocytes from the Peyer ' s patches of C57BL / 6 and Vα14 mice were stained with anti - CD3 and CD1d ( PBS57 ) tetramer . Percentage of lymphocytes that were CD3 + CD1d - tetramer + iNKT cells among the Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 22 ; Vα14 n = 16 ; Jα18 - / - n = 8 .

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

Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.


( C - E ) Mice were analyzed for total fecal IgA by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 .

(C-E)Mice were analyzed for total fecal IgA by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.


( C - E ) Mice were analyzed for total fecal IgG1 ( D ) or total serum IgG1 ( E ) by ELISA . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E)Mice were analyzed for total fecal IgG1 (D) or total serum IgG1 (E) by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.


Percentages of total B cells that were IgG1 + in the spleen , mLN , and Peyer ' s patches of C57BL / 6 , Vα14 , and Jα18 - / - mice are shown . Mann - Whitney test . Error bars are SD . C57BL / 6 n = 15 ; Vα14 n = 15 ; Jα18 - / - n =

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

Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=15; Vα14 n=15; Jα18-/- n=5


CD1d ( PBS57 ) tetramer + CD3 + cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4 + CD3 + CD1d tetramer - cells from PP ( PP CD4 ) . RNAseq was performed . Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample .

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

CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.


Spleen , mLN , and PP lymphocytes from Vα14 and C57BL / 6 mice were stimulated with PMA and ionomycin . Lymphocytes were stained with anti - CD3 , CD1d ( PBS57 ) tetramer , anti - IL - 4 , and anti - IFNγ . Percentages of iNKT cells and non - iNKT T cells within the population of CD3 + IL - 4 + cells and CD3 + IFNγ + cells , n = 5 mice for C57BL / 6 group and n = 4 mice for Vα14 group . Error bars are SD .

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

Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD.


Vα14 TN mice were administered α - GalCer either 2μg intravenously or 5μg by oral gavage . Mice were given Brefeldin A intraperitoneally after 30 min , and tissues were harvested 3 hours later . Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with antibodies to IL - 4 , IFNγ , and IL - 17 . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells .

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

Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.


Quantification of data from C , n = 2 mice per group . Mann - Whitney test . Error bars are SEM .

Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.


Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α - GalCer and with or without agnostic anti - CD40 . IgM was measured by ELISA of culture supernatants 4 days later .

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

Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.


Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti - CD40 activated wild type B cells with or without 1μg α - GalCer and with blocking antibody to CD1d ( 1μg / mL , clone 1B1 ) or isotype control as indicated . IgM , IgG1 , and IgA were measured by ELISA of 4 day culture supernatants .

O 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 I-CONTROLLED_VAR I-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

Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . IL - 4 were measured by ELISA of culture supernatants .

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

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants.


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 ( D - E ) were measured by ELISA of culture supernatants .

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

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants.


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + and IgA + class switched B cells were enumerated by flow cytometry after 4 days of

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

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


Spleen , mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B cells . 1μg α - GalCer was added to the cultures to specifically activate iNKT cells . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + class switched B cells were enumerated by flow cytometry after 4 days of

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Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


OVA - specific antibody titers from feces were determined by ELISA from the mice in ( A ) . Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD .

OVA-specific antibody titers from feces were determined by ELISA from the mice in (A). Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.


OVA - specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after the final boost ) . Error bars are SD .

OVA-specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.


Cells from spleen , mesenteric lymph node , and Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , and stained with the indicated antibodies . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT cells . Quantification of data from E , n = 3 mice per group . Mann - Whitney test . Error bars are SEM . * p < 0 . 05 .

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Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.


Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP - iNKT cells .

Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP-iNKT cells.


Heatmap of FPKM values from genes uniquely upregulated in PP - iNKT cells as compared to both spleen iNKT and PP CD4 cells . Red indicates higher expression and blue indicates lower expression .

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Heatmap of FPKM values from genes uniquely upregulated in PP-iNKT cells as compared to both spleen iNKT and PP CD4 cells. Red indicates higher expression and blue indicates lower expression.


Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies , analyzed by flow cytometry and gated on CD1d ( PBS57 ) tetramer + CD3 + cells .

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Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.


Quantification of data from D , n = 11 mice per group . Mann - Whitney test . Error bars are SEM .

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Quantification of data from D, n=11 mice per group. Mann-Whitney test. Error bars are SEM.


( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 μM ecdysone for 24 h . ( a ) The cell viability was assayed by trypan blue staining . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) . ( b ) Cell count and viability assay using Muse ( Millipore , USA ) . Data are mean from three independent experiments , with error bars representing s . d . * P0 . 05 ( Student ' s t - test ) .

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 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

(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (a) The cell viability was assayed by trypan blue staining. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test). (b) Cell count and viability assay using Muse (Millipore, USA). Data are mean from three independent experiments, with error bars representing s.d. *P0.05 (Student's t-test).


( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 µM ecdysone for 24 h . ( c ) Caspase activity was measured from total cell lysate on DEVD - AMC , represented as fold increase relative to control RNAi . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

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(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 µM ecdysone for 24 h. (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic cell death . SL2 cells were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h and cell death was induced with 10 μM ecdysone for 24 h . ( d ) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49 . Relative dUTX expression levels following knockdown using the two independent dsRNA are shown ( n = 3 ) with error bars representing s . d . * P0 . 05 ( Student ' s t - test ) .

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(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (d) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49. Relative dUTX expression levels following knockdown using the two independent dsRNA are shown (n=3) with error bars representing s.d. *P0.05 (Student's t-test).


( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( a ) GST and GST - dUTX - NR containing the nuclear hormone domain were used in pull - down experiments with 35S - labelled EcR and Usp in the presence of ethanol control ( − ) or 10 μM ecdysone ( + ) . The GST input proteins are shown in Coomassie Brilliant blue - stained gel , GST is labelled with arrow head and dUTX - NR with an asterisk . The EcR and Usp inputs are shown labelled with arrows .

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-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

(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX-NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX-NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows.


( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( b ) Immunoprecipitation ( IP ) of EcR and dUTX in SL2 cells . Cells were transfected with HA - dUTX and the cell lysates were immunoprecipitated using anti - HA or a control IgG isotype antibody . The immunoprecipitates were analysed by western blotting using the anti - EcR and anti - HA antibodies .

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

(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies.


( c ) Colocalization of dUTX and EcR on salivary gland polytene chromosomes . Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG - dUTX with dUTX ( green ) and EcR ( red ) , with Hoechst - stained DNA ( blue ) . Arrows indicate examples of colocalized bands . No signal was detected when immunostaining with secondary antibody only . Scale bar represent 5 μm .

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(c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.


( a ) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF . Ovals indicate the position of salivary glands and fragments . Scale bar represent 50 μm . Quantification of the salivary gland phenotypic data was at 24 h RPF .

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(a) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data was at 24 h RPF.


( b , c ) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF . ( b ) Cleaved caspase - 3 antibody ( green ) and Lamin antibody ( red ) staining are shown in control and dUTX1 salivary glands at 14 h RPF . Scale bar represents 50 μm . ( c ) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD - AMC as a substrate . Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts . The error bars represent s . e . m . * P - value0 . 05 ( Student ' s t - test ) .

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(b,c) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF. (b) Cleaved caspase-3 antibody (green) and Lamin antibody (red) staining are shown in control and dUTX1 salivary glands at 14 h RPF. Scale bar represents 50 μm. (c) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD-AMC as a substrate. Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts. The error bars represent s.e.m. *P-value0.05 (Student's t-test).


( d ) Autophagy puncta examined using mCherry - Atg8a ( red ) and nuclei stained with Hoechst ( blue ) . Scale bar represents 100 μm . The quantitation of the mCherry - Atg8a puncta as the mean fluorescent pixel intensity per cell ( control n = 242 , dUTX1 n = 224 ) , with error bars representing s . e . m . * P - value0 . 05 ( Student ' s t - test ) .

(d) Autophagy puncta examined using mCherry-Atg8a (red) and nuclei stained with Hoechst (blue). Scale bar represents 100 μm. The quantitation of the mCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell (control n=242, dUTX1 n=224), with error bars representing s.e.m. *P-value0.05 (Student's t-test).


( a ) Levels of dark , dronc , drice and rpr transcripts were measured from third instar larvae ( L3 ) , 6 h and 14 h RPF salivary glands from control ( blue ) and dUTX1 ( red ) by qPCR and expressed relative to the internal control gene rp49 .

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(a) Levels of dark, dronc, drice and rpr transcripts were measured from third instar larvae (L3), 6 h and 14 h RPF salivary glands from control (blue) and dUTX1 (red) by qPCR and expressed relative to the internal control gene rp49.


( b ) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control ( blue ) and dUTX1 ( red ) were analysed by qPCR at L3 , 6 h and 14 h RPF . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

(b) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control (blue) and dUTX1 (red) were analysed by qPCR at L3, 6 h and 14 h RPF. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


( a , b ) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation ( ChIP ) . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess the binding to the promoters of dark , dronc , drice and rpr ( a )

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(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of dark, dronc, drice and rpr (a)


( a , b ) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation ( ChIP ) . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess the binding to the promoters of Atg1 , Atg2 , Atg3 , Atg4 , Atg5 , Atg6 , Atg7 , Atg8a , Atg9 , Atg12 and Atg18 ( b ) , expressed relative to the internal control gene Gapdh . Results are shown as the fold enrichment of % input compared with IgG control .

(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of Atg1, Atg2, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg9, Atg12 and Atg18 (b), expressed relative to the internal control gene Gapdh. Results are shown as the fold enrichment of % input compared with IgG control.


( c ) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP . SL2 cells knocked down for dUTX were chromatin - immunoprecipitated with anti - H3K27me3 and anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with RNAi control .

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(c) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP. SL2 cells knocked down for dUTX were chromatin-immunoprecipitated with anti-H3K27me3 and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control.


( d ) The occupancy of EcR on the promoter regions of dark , dronc and Atg1 by ChIP expressed relative to the internal control gene rp49 . Chromatin was immunoprecipitated with anti - EcR - B1 and anti - GFP ( IgG control ) antibodies from SL2 cell following ecdysone treatment . Results are shown as the fold enrichment of % input compared with IgG control . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - test ) .

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(d) The occupancy of EcR on the promoter regions of dark, dronc and Atg1 by ChIP expressed relative to the internal control gene rp49. Chromatin was immunoprecipitated with anti-EcR-B1 and anti-GFP (IgG control) antibodies from SL2 cell following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


( a ) Levels of BR - C and E93 transcripts were measured in salivary glands isolated from third instar larvae ( L3 ) , 6 h and 14 h RPF control ( blue line ) and dUTX1 ( red line ) using qPCR and expressed relative to the internal control gene rp49 .

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(a) Levels of BR-C and E93 transcripts were measured in salivary glands isolated from third instar larvae (L3), 6 h and 14 h RPF control (blue line) and dUTX1 (red line) using qPCR and expressed relative to the internal control gene rp49.


( b ) The occupancy of dUTX on the promoter regions of BR - C and E93 genes detected by ChIP . SL2 cells overexpressing HA - dUTX were chromatin - immunoprecipitated with anti - HA and anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with IgG control . In the right hand panel abundance of H3K27me3 on the promoters of BR - C and E93 genes as detected by ChIP is shown . SL2 cells knockdown for dUTX ( dUTX RNAi ) were chromatin - immunoprecipitated with anti - H3K27me3 and rabbit IgG ( control ) antibodies following ecdysone treatment . Results are shown as the fold enrichment of % input compared with RNAi control . Data in all panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Students t - test ) .

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(b) The occupancy of dUTX on the promoter regions of BR-C and E93 genes detected by ChIP. SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. In the right hand panel abundance of H3K27me3 on the promoters of BR-C and E93 genes as detected by ChIP is shown. SL2 cells knockdown for dUTX (dUTX RNAi) were chromatin-immunoprecipitated with anti-H3K27me3 and rabbit IgG (control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Students t-test).


( a ) Mosaic clones of dUTX1 ( hsFLP ; ubi - GFP FRT40A / dUTX1 FRT40A ) from third instar larval salivary glands with wild - type cells marked by nuclear - localized GFP ( green ) . dUTX1 clones have high levels of H3K27me3 ( red ) compared with the neighbouring control cells marked by GFP . Scale bar represents 50 μm .

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(a) Mosaic clones of dUTX1 (hsFLP; ubi-GFP FRT40A/dUTX1 FRT40A) from third instar larval salivary glands with wild-type cells marked by nuclear-localized GFP (green). dUTX1 clones have high levels of H3K27me3 (red) compared with the neighbouring control cells marked by GFP. Scale bar represents 50 μm.


( b ) Western blot analysis of the level of H3K27me3 in control and dUTX1 larval salivary glands . Alpha - tubulin and H3 serve as loading controls .

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(b) Western blot analysis of the level of H3K27me3 in control and dUTX1 larval salivary glands. Alpha-tubulin and H3 serve as loading controls.


( c ) Expression of dUTX wild - type ( dUTXWT ) and dUTX catalytic mutant ( dUTXJmj ) in the salivary glands ( using Sgs3 - GAL4 driver ) of dUTX1 . Histology analysis of paraffin sections at 24 h RPF shows intact salivary glands present in dUTX1 and in dUTX1 ; dUTXJmj * compared with control and dUTX1 ; dUTXWT at 24 h RPF . Ovals indicate the position of salivary glands and fragments . Scale bar represent 50 μm . Quantification of the salivary gland phenotypic data at 24 h RPF is shown on the right .

(c) Expression of dUTX wild-type (dUTXWT) and dUTX catalytic mutant (dUTXJmj) in the salivary glands (using Sgs3-GAL4 driver) of dUTX1. Histology analysis of paraffin sections at 24 h RPF shows intact salivary glands present in dUTX1 and in dUTX1; dUTXJmj* compared with control and dUTX1; dUTXWT at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data at 24 h RPF is shown on the right.


( B ) Scatterplots represent SILAC / SUMO - IP quantification after EGF treatment from three biological replicates . Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells ( dark blue dots ) or is significantly more present in one of the samples ( red and yellow dots ) .

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(B) Scatterplots represent SILAC/SUMO-IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots).


( C ) Left panel : Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation ( significant hits with P < 0 . 0001 ) , as well as the three non - changing proteins SUMO2 , RanGAP1 and TRIM28 . Right panel : Proteins highlighted in bold in the left panel were validated by SUMO - IP / immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation .

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(C) Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P<0.0001), as well as the three non-changing proteins SUMO2, RanGAP1 and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO-IP/immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation.


( D ) Time course experiment : serum starved HeLa cells were treated with 100 nM EGF , samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies . IRF2BP1 , IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment .

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(D) Time course experiment: serum starved HeLa cells were treated with 100 nM EGF, samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.


( B ) Identification of the endogenous SUMO - sites in IRF2BP1 ( left panel ) and IRF2BP2 ( right panel ) . HeLa cells were transfected with HA - tagged wildtype ( WT ) or mutant ( KR ) proteins , endogenous SUMO - IPs were performed and the HA - signal was analyzed by immunoblotting . Mutation of the C - terminal SUMO - site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells .

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(B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells.


( E ) Stable HeLa cells expressing pIRES - empty vector , IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non - targeting ( nt ) siRNA . Exogenous siRNA - resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1 . * refers to an unspecific band .

(E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band.


( F ) Wt and mutant IRF2BP1 localizes in the nucleus . After knockdown of endogenous IRF2BP1 , stable IRF2BP1 ( WT or K579R ) cell lines were immunostained for IRF2BP1 . Exogenous IRF2BP1 variants show a similar nuclear localization . Scale bar = 10 µm .

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(F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm.


( G ) IRF2BP1 wt and mutant associate with chromatin to a similar extent . HeLa cells were lysed in 0 . 075 % NP40 ( Input ) . After centrifugation , the nuclei were incubated and vortexed with a nuclear extract ( NE ) buffer containing 170 mM NaCl . The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations , first 290 mM , then 420 mM . Wildtype IRF2BP1 , the SUMO - deficient K579R mutant and the SUMOylated wildtype form ( * ) all behave similarly .

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(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.


( A ) Stable IRF2BP1 cell lines ( knocked - down for endogenous IRF2BP1 ) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour . A subset of 38 EGF - dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines ( at least 1 . 5 - fold ) . Among them are DUSP1 ( arrow ) , ATF3 , Fos and Egr2 . Each microarray was performed in triplicates , the bars show log2 values of the fold changes for wt cells ( black bars ) and for KR cells ( grey bars ) .

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(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars).


( B ) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells . Gene architecture of human DUSP1 . The primers at - 243 / - 67 ( " - 67 " ) , - 473 / - 224 ( " - 224 " ) and - 1170 / - 961 ( " - 916 " ) relative to the TSS were used for ChIP experiments . IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides - 243 and - 67 .

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(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67.


( C ) ChIP / qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between - 243 and - 67 . Data show mean ( bar ) and individual data points from 2 biological replicates .

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(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates.


( D ) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF . Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked - down for endogenous IRF2BP1 , followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region - 243 and - 67 . Data show means + / - SEM from 3 biological replicates .

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(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates.


( E ) Q - PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / - SEM from 3 biological replicates .

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(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates.


( F ) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q - PCR ( left panel ) , data show means + / − SEM from 5 biological replicates ( left panel ) , and IRF2BP1 protein levels . As expected , IRF2BP1 V578A is not SUMOylated ( right panel ) . * refers to an unspecific band .

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(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band.


( G ) Q - PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / − SEM from 4 biological replicates .

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(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.


( A ) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1 . Uba2 and beta - actin serve as independent loading controls .

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(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls.


( C ) Wound healing assay : HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days , grown to 90 % confluency and analysed for wound closure with or without addition of 100 ng / ml EGF . Data show means of the relative wound density + / - SEM from 4 biological replicates .

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(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates.


( D ) Proliferation assay : HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium . One representative biological experiment is shown with means + / - SEM from 5 technical replicates .

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(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates.


( E ) IRF2BP1 , IRF2BP2 , IRF2BP1 + IRF2BP2 were knocked - down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis ; non - targeting siRNAs were used as a control . IRF2BP1 and IRF2BP2 have distinct and overlapping functions . Experiments were done in triplicates .

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(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates.


( G ) Validation of two candidate genes : IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells , and cell lysates were analyzed by immunoblotting with the indicated antibodies .

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(G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies.


( H ) FACS - based analysis of EGFR surface expression : IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells , cells were serum starved for 16 hours , stained with fluorescent anti - EGFR antibodies and analyzed by flow cytometry . Shown are means of 3 independent experiments + / - SEM .

(H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM.


A . FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3 - L1 adipocytes . Cells were cultured in RAW - CM to induce inflammation and insulin resistance , while vehicle , FGF21 or FGF21SS were added simultaneously . The insulin sensitivity was indicated by insulin - induced AKT phosphorylation using western blotting analysis . Error bars show the SEM of four independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) vs RAW - CM treated only group .

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A. FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3-L1 adipocytes. Cells were cultured in RAW-CM to induce inflammation and insulin resistance, while vehicle, FGF21 or FGF21SS were added simultaneously. The insulin sensitivity was indicated by insulin-induced AKT phosphorylation using western blotting analysis. Error bars show the SEM of four independent experiments. *p < 0.05, **p < 0.01 (Student's t-test) vs RAW-CM treated only group.


Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . Body weight ( B ) was measured Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. Body weight (B) was measured Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . plasma glucose ( D ) was measured every day . Plasma insulin ( C ) was analyzed at day 7 using insulin ELISA kit . Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. plasma glucose (D) was measured every day. Plasma insulin (C) was analyzed at day 7 using insulin ELISA kit. Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


( a ) Identification of biomarkers modulated upon VCP silencing . HeLa , HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h . Protein cell extracts were resolved by SDS - PAGE , and filters were probed with the indicated antibodies . The extent of biomarker modulation correlated with the efficiency of silencing ( oligo 4 versus pool ) . VCP silencing determined stabilization of cyclin E ; induction of the endoplasmic reticulum chaperone GRP78 , CHOP transcription factor and its downstream target GADD - 34 ; conversion of the autophagy regulator ATG8 / light chain 3 ( LC3B ) from the free to the lipidated form ; activation of the effector caspase - 3 ; and cleavage of the caspase target PARP - 1 . A graphical representation of the cell cycle distribution ( percentage of cells ) obtained from the cell cycle analysis ( Supplementary Fig . 3b ) is reported at the bottom of each sample . C , control ; NT , nontargeting oligo ; P , oligonucleotide pool .

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(a) Identification of biomarkers modulated upon VCP silencing. HeLa, HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h. Protein cell extracts were resolved by SDS-PAGE, and filters were probed with the indicated antibodies. The extent of biomarker modulation correlated with the efficiency of silencing (oligo 4 versus pool). VCP silencing determined stabilization of cyclin E; induction of the endoplasmic reticulum chaperone GRP78, CHOP transcription factor and its downstream target GADD-34; conversion of the autophagy regulator ATG8/light chain 3 (LC3B) from the free to the lipidated form; activation of the effector caspase-3; and cleavage of the caspase target PARP-1. A graphical representation of the cell cycle distribution (percentage of cells) obtained from the cell cycle analysis (Supplementary Fig. 3b) is reported at the bottom of each sample. C, control; NT, nontargeting oligo; P, oligonucleotide pool.


( b ) Effect of VCP silencing on cell growth . HeLa , HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter ( data represent mean values of two experiments ) .

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(b) Effect of VCP silencing on cell growth. HeLa, HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter (data represent mean values of two experiments).


( c ) Combination of VCP siRNA with standard - of - care therapeutics . HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose - response analysis with a variety of chemotherapeutic agents .

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(c) Combination of VCP siRNA with standard-of-care therapeutics. HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose-response analysis with a variety of chemotherapeutic agents.


( b ) LC / MS analysis of VCP after 1 h incubation at 20 ° C with 1 % DMSO ( top ) or 100 μM NMS - 859 ( bottom ) , showing a 313 - Da increase corresponding to the addition of NMS - 859 lacking the Cl atom .

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(b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom.


( a ) Activity of VCP inhibitors NMS - 249 and NMS - 873 on wild - type VCP and mutants . Data represent mean values of at least three independent experiments .

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(a) Activity of VCP inhibitors NMS-249 and NMS-873 on wild-type VCP and mutants. Data represent mean values of at least three independent experiments.


( b ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP in the presence of different allosteric inhibitors or ADP .

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(b) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP in the presence of different allosteric inhibitors or ADP.


( c ) Binding of the EDA - ADP - ATTO 590 probe to wild - type ( WT ) VCP and the K615V and N616F mutants in the presence of NMS - 249 . Minor differences in the mP values are due to the use of different enzyme batches .

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(c) Binding of the EDA-ADP-ATTO 590 probe to wild-type (WT) VCP and the K615V and N616F mutants in the presence of NMS-249. Minor differences in the mP values are due to the use of different enzyme batches.


( d ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP and Walker A mutants in the presence of NMS - 249 . Data shown are representative results of one of three independent experiments : each determination was done in triplicate , data points denote the average value , and error bars represent s . d .

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(d) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP and Walker A mutants in the presence of NMS-249. Data shown are representative results of one of three independent experiments: each determination was done in triplicate, data points denote the average value, and error bars represent s.d.


( a ) Western blot analysis of biomarker modulation in the HCT116 cell line . HCT116 cells were treated with the indicated doses of inhibitors for 8 h ( top ) or 24 h ( bottom ) , and protein cell extracts were resolved on SDS - PAGE gel . Filters were probed with the indicated antibodies . C , control ; B , bortezomib ( 0 . 01 μM ) ; T , thapsigargin ( 0 . 1 μM ) ; Mg , MG132 ( 0 . 1 μM ) .

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(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM).


( b ) NMS - 873 compound - mediated stabilization of Mcl - 1 in live cells . Top , confocal time - lapse images of live stable , inducible T - REx - 293 cells expressing a bicistronic construct of mCherry - BimS ( 2A ) ΔC ( red ) with eGFP - Mcl - 1 ( green ) after doxycycline treatment . Cells were treated with cycloheximide ( CHX , 50 μg ml − 1 ) ± 2 . 5 μM of NMS - 873 for 6 h . Bottom , total intensity of eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC was measured for time - lapse series shown at top . A decrease in eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control . WT , wild type . Scale bar , 10 μm .

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(b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A)ΔC (red) with eGFP-Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP-Mcl-1 and mCherry-BimS(2A)ΔC was measured for time-lapse series shown at top. A decrease in eGFP-Mcl-1 and mCherry-BimS(2A)ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm.


( c ) High - content analysis of poly - Ub and CHOP induction upon treatment with VCP inhibitors . HeLa cells were treated with increasing doses of the indicated compounds . Cells were then fixed and immunostained with the indicated antibodies . Cell numbers and mean fluorescence intensity ( MFI ) were determined using the ArrayScan platform . Data were collected in triplicate ( n = 3 ) , and error bars represent s . d . AU , arbitrary units .

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(c) High-content analysis of poly-Ub and CHOP induction upon treatment with VCP inhibitors. HeLa cells were treated with increasing doses of the indicated compounds. Cells were then fixed and immunostained with the indicated antibodies. Cell numbers and mean fluorescence intensity (MFI) were determined using the ArrayScan platform. Data were collected in triplicate (n = 3), and error bars represent s.d. AU, arbitrary units.


Representative flow cytometric analysis of BAL from Csf2ra - / - recipients 8 weeks after neonatal i . n . transfer of YS EMP from CD45 . 1 + E10 . 5 embryos , 8 - week old un - transferred Csf2ra - / - , and WT ( CD45 . 2 ) mice . Pre - gated viable CD45 + singlets .

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Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 8 weeks after neonatal i.n. transfer of YS EMP from CD45.1+ E10.5 embryos, 8-week old un-transferred Csf2ra-/-, and WT (CD45.2) mice. Pre-gated viable CD45+ singlets.


Numbers of EMP effectively transferred in neonates ( white circle ) and numbers of EMP - derived AM ( red circles ) in BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) .

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Numbers of EMP effectively transferred in neonates (white circle) and numbers of EMP-derived AM (red circles) in BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice).


Numbers of EMP - derived AM in the BAL ( left ) and lung ( right ) ( C ) in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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Numbers of EMP-derived AM in the BAL (left) and lung (right) (C) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


total protein in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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total protein in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


total eFluor780 + events ( E ) in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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total eFluor780+ events (E) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with E17 . 5 fetal liver ( FeLi ) monocytes ( Mo ) ( CD45 . 1 + ) , E14 . 5 FeLi Mo ( CD45 . 1 + CD45 . 2 + ) , and E10 . 5 YS EMP ( CD45 . 2 + ) ( F ) or E17 . 5 fetal lung ( FeLu ) Mo ( CD45 . 1 + ) , E14 . 5 FeLu Mo ( CD45 . 1 + CD45 . 2 + ) , and E10 . 5 YS EMP ( CD45 . 2 + ) ( G ) ( n = 3 mice ) .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with E17.5 fetal liver (FeLi) monocytes (Mo) (CD45.1+), E14.5 FeLi Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (F) or E17.5 fetal lung (FeLu) Mo (CD45.1+), E14.5 FeLu Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (G) (n = 3 mice).


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL ( top ) and lung ( bottom ) from Csf2ra - / - recipients 10 weeks after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo and CD45 . 2 + E14 . 5 FeLi pMΦ ( Group 1 ) or CD45 . 1 + E14 . 5 FeLi pMΦ and CD45 . 2 + FeLi Mo ( Group 2 ) ( n = 3 mice ) . The expansion fold of donor cells in Csf2ra - / - recipients 10 weeks after neonatal transfer of cells described above in A ( n = 3 mice ) .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL (top) and lung (bottom) from Csf2ra-/- recipients 10 weeks after neonatal transfer of 1:1 mixture with CD45.1+ E14.5 FeLi Mo and CD45.2+ E14.5 FeLi pMΦ (Group 1) or CD45.1+ E14.5 FeLi pMΦ and CD45.2+ FeLi Mo (Group 2) (n = 3 mice). The expansion fold of donor cells in Csf2ra-/- recipients 10 weeks after neonatal transfer of cells described above in A (n = 3 mice).


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) of BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLi pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLi pMΦ , and CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in the BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer of cells described above in C ( n = 3 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) of BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with CD45.1+ E17.5 FeLi pMΦ, CD45.1+CD45.2+ E14.5 FeLi pMΦ, and CD45.2+ E10.5 YS pMΦ (n = 3 mice). Total numbers of donor-derived AM in the BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer of cells described above in C (n = 3 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) of BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLu pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLu pMΦ , and CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in the BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer of cells described above in E ( n = 3 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) of BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with CD45.1+ E17.5 FeLu pMΦ, CD45.1+CD45.2+ E14.5 FeLu pMΦ, and CD45.2+ E10.5 YS pMΦ (n = 3 mice). Total numbers of donor-derived AM in the BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer of cells described above in E (n = 3 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL and lung from Csf2ra - / - recipients 7 weeks after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo from WT embryos and CD45 . 2 + E14 . 5 FeLi pMΦ from Myb - / - ( G ) or Myb + / + ( H ) embryos ( n = 3 mice ) .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 7 weeks after neonatal transfer of 1:1 mixture with CD45.1+ E14.5 FeLi Mo from WT embryos and CD45.2+ E14.5 FeLi pMΦ from Myb-/- (G) or Myb+/+ (H) embryos (n = 3 mice).


Representative flow cytometric analysis of BAL from Csf2ra - / - recipients 7 weeks after neonatal i . n . transfer of pMΦ or fetal Mo from CD45 . 1 + E14 . 5 fetal livers , 7 - week old un - transferred Csf2ra - / - , WT ( CD45 . 2 ) mice . Pre - gated viable CD45 + singlets .

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Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 7 weeks after neonatal i.n. transfer of pMΦ or fetal Mo from CD45.1+ E14.5 fetal livers, 7-week old un-transferred Csf2ra-/-, WT (CD45.2) mice. Pre-gated viable CD45+ singlets.


Numbers of donor - derived AM in the BAL ( left ) and lung ( right ) of adult Csf2ra - / - recipients 7 weeks ( B ) or 1 year ( C ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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Numbers of donor-derived AM in the BAL (left) and lung (right) of adult Csf2ra-/- recipients 7 weeks (B) or 1 year (C) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Total protein in the BAL of adult Csf2ra - / - recipients 7 weeks after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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Total protein in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


total eFluor780 + events in the BAL of adult Csf2ra - / - recipients 7 weeks after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Total protein in the BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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Total protein in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


total eFluor780 + events in the BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Body weight measurement of influenza virus ( 20 pfu of PR8 strain ) - infected Csf2ra - / - recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14 . 5 fetal livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , and WT ( n = 8 ) .

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Body weight measurement of influenza virus (20 pfu of PR8 strain)-infected Csf2ra-/- recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14.5 fetal livers (n = 7 mice from each transferred group), 24-week old un-transferred Csf2ra-/- (n = 5), and WT (n = 8).


temperature ( B ) measurement of influenza virus ( 20 pfu of PR8 strain ) - infected Csf2ra - / - recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14 . 5 fetal livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , and WT ( n = 8 ) .

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temperature (B) measurement of influenza virus (20 pfu of PR8 strain)-infected Csf2ra-/- recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14.5 fetal livers (n = 7 mice from each transferred group), 24-week old un-transferred Csf2ra-/- (n = 5), and WT (n = 8).


Oxygen ( O2 ) saturation in influenza - infected mice described in A , B 7 days post - infection ( p . i . ) ( n = 5 mice from Csf2ra - / - group ; n = 7 mice from each transferred group ; n = 8 from WT group ) .

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Oxygen (O2) saturation in influenza-infected mice described in A, B 7 days post-infection (p.i.) (n = 5 mice from Csf2ra-/- group; n = 7 mice from each transferred group; n=8 from WT group).


Survival curve of influenza - infected mice described in A , B ( n = 5 mice from Csf2ra - / - group ; n = 7 mice from each transferred group ; n = 8 from WT group ) .

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Survival curve of influenza-infected mice described in A, B (n = 5 mice from Csf2ra-/- group; n = 7 mice from each transferred group; n=8 from WT group).


Total eFluor780 + events in the BAL of influenza - infected mice described in A , B 10 days p . i . ( n = 3 mice from each group ) .

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Total eFluor780+ events in the BAL of influenza-infected mice described in A, B 10 days p.i. (n = 3 mice from each group).


Representative flow cytometric analysis of Csf2ra ( CD116 ) expression on different fetal precursors from WT embryos . The solid gray histogram represents the fluorescence minus one ( FMO ) controls , the open black histogram represents stained samples . The gray number represents mean fluorescence intensity ( MFI ) of FMO controls , the black number represents MFI of stained samples . E10 . 5 YS cells were pooled from 8 - 10 embryos , E14 . 5 lung cells were pooled from 3 - 4 embryos , and E14 . 5 liver cells were from individual embryo .

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Representative flow cytometric analysis of Csf2ra (CD116) expression on different fetal precursors from WT embryos. The solid gray histogram represents the fluorescence minus one (FMO) controls, the open black histogram represents stained samples. The gray number represents mean fluorescence intensity (MFI) of FMO controls, the black number represents MFI of stained samples. E10.5 YS cells were pooled from 8-10 embryos, E14.5 lung cells were pooled from 3-4 embryos, and E14.5 liver cells were from individual embryo.


Representative flow cytometric analysis of Csf2ra expressions on pMΦ - and fetal Mo - derived AM in Csf2ra - / - recipients 4 weeks after transfer of pMΦ or fetal Mo from E14 . 5 fetal livers . AM from 4 - week old WT mice were included as controls .

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Representative flow cytometric analysis of Csf2ra expressions on pMΦ- and fetal Mo-derived AM in Csf2ra-/- recipients 4 weeks after transfer of pMΦ or fetal Mo from E14.5 fetal livers. AM from 4-week old WT mice were included as controls.


Heatmap of AM signature - up genes ( D ) , AM signature - down genes ( E ) , and AM - specific transcription factors ( F ) in the precursors and mature AM described above in A . the data are from one RNA - seq experiment ( 1 - 2 biological replicates per group ) .

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Heatmap of AM signature-up genes (D), AM signature-down genes (E), and AM-specific transcription factors (F) in the precursors and mature AM described above in A. the data are from one RNA-seq experiment (1 - 2 biological replicates per group).


qPCR ( n = 3 biological replicates ) analysis of Myb , Maf , Mafb , and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice .

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qPCR (n = 3 biological replicates) analysis of Myb, Maf, Mafb, and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice.


Oxygen consumption rate ( OCR ) of pMΦ and fetal Mo from E14 . 5 WT fetal liver at baseline and after treatment with oligomycin ( Oligo ) , FCCP or rotenone ( Rot ) and antimycin A ( AA ) .

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Oxygen consumption rate (OCR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with oligomycin (Oligo), FCCP or rotenone (Rot) and antimycin A (AA).


Extracellular acidification rate ( ECAR ) of pMΦ and fetal Mo from E14 . 5 WT fetal liver at baseline and after treatment with glucose , Oligo , or 2 - Deoxy - D - glucose ( 2 - DG ) .

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Extracellular acidification rate (ECAR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with glucose, Oligo, or 2-Deoxy-D-glucose (2-DG).


RNA - seq ( C , n = 2 biological replicates ) analysis of Eno1 , Slc2a6 , Txn1 , and Txnip gene expression in pMΦ and fetal Mo from E14 . 5 WT fetal liver .

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RNA-seq (C, n = 2 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


qPCR ( D , n = 3 biological replicates ) analysis of Eno1 , Slc2a6 , Txn1 , and Txnip gene expression in pMΦ and fetal Mo from E14 . 5 WT fetal liver .

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qPCR (D, n = 3 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and p53 - depleted ( si - p53 ; si - p53 _ 1 ) ( C ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

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C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and p53-depleted (si-p53; si-p53_1) (C) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and YAP - depleted ( si - YAP ; si - YAP _ 1 ) ( D ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and TAZ - depleted ( si - TAZ ) ( E ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

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C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and TAZ-depleted (si-TAZ) (E) MDA-MB-231 and CAL27 cell lines. Representative images are shown.