Figure 5RT - PCR results showing that YAP stimulates the mRNA expressions of EGFR , AREG , and TGF - α in ME180 cervical cancer cell . Data were analyzed for significance using one - way ANOVA in GraphPad Prism 5 with Tukey ' s post hoc tests . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( YAP mRNA : MXIV vs . YAP , P = 0 . 0003 ; MXIV vs . YAPS127A , P = 0 . 0001 ; EGFR mRNA : MXIV vs . YAP , P = 0 . 0066 ; MXIV vs . YAPS127A , P = 0 . 0003 ; TGF - α mRNA : MXIV vs . YAP , P = 0 . 0235 ; MXIV vs . YAPS127A , P = 0 . 0040 ; AREG mRNA : MXIV vs . YAP , P = 0 . 0002 ; MXIV vs . YAPS127A , P < 0 . 0001 ) . Proliferation of ME180 cells incubated in medium containing 1 % FBS in the absence ( control ) or presence of 10 ng / ml TGF - α . Data were analyzed with unpaired t - test in GraphPad Prism 5 with Welch ' s correction . Each point represents the mean ± SEM of four independent repeats . * * * P < 0 . 0001 versus control on day 5 . TGF - α treatment ( 10 ng / ml , 24 h ) promotes ME180 cell cycle progression . G1 , S , and G2 indicate cells in G1 phase , DNA synthesis phase , and the G2 / M phase , respectively , of cell cycle . Apo : apoptotic cells . Representative images showing the morphology of ME180 cells with or without TGF - α ( 10 ng / ml ) treatment for 24 h ( scale bar : 50 μm ) or 108 h ( scale bar : 25 μm ) . Please note the elongation of ME180 cells after TGF - α treatment for 24 h ( TGF - α , 24 h ) and the formation of multiple layers in ME180 cells after TGF - α treatment for 108 h ( TGF - α , 108 h ) . Effect of TGF - α on the migration of ME180 cells . TGF - α treatment ( 100 ng / ml , 10 h ) drastically stimulated the migration of ME180 cells .

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

Figure 5RT-PCR results showing that YAP stimulates the mRNA expressions of EGFR, AREG, and TGF-α in ME180 cervical cancer cell. Data were analyzed for significance using one-way ANOVA in GraphPad Prism 5 with Tukey's post hoc tests. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (YAP mRNA: MXIV vs. YAP, P = 0.0003; MXIV vs. YAPS127A, P = 0.0001; EGFR mRNA: MXIV vs. YAP, P = 0.0066; MXIV vs. YAPS127A, P = 0.0003; TGF-α mRNA: MXIV vs. YAP, P = 0.0235; MXIV vs. YAPS127A, P = 0.0040; AREG mRNA: MXIV vs. YAP, P = 0.0002; MXIV vs. YAPS127A, P < 0.0001).Proliferation of ME180 cells incubated in medium containing 1% FBS in the absence (control) or presence of 10 ng/ml TGF-α. Data were analyzed with unpaired t-test in GraphPad Prism 5 with Welch's correction. Each point represents the mean ± SEM of four independent repeats. ***P < 0.0001 versus control on day 5.TGF-α treatment (10 ng/ml, 24 h) promotes ME180 cell cycle progression. G1, S, and G2 indicate cells in G1 phase, DNA synthesis phase, and the G2/M phase, respectively, of cell cycle. Apo: apoptotic cells.Representative images showing the morphology of ME180 cells with or without TGF-α (10 ng/ml) treatment for 24 h (scale bar: 50 μm) or 108 h (scale bar: 25 μm). Please note the elongation of ME180 cells after TGF-α treatment for 24 h (TGF-α, 24 h) and the formation of multiple layers in ME180 cells after TGF-α treatment for 108 h (TGF-α, 108 h).Effect of TGF-α on the migration of ME180 cells. TGF-α treatment (100 ng/ml, 10 h) drastically stimulated the migration of ME180 cells.


Figure 6Western blot analysis showing effects of TGF - α on phosphorylation of EGFR , YAP , AKT , and ERK1 / 2 . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with TGF - α ( 100 ng / ml ) for 0 , 5 , 10 , 30 , 60 or 120 min . Western blot analysis showing effects of TGF - α on the phosphorylation of the components of the Hippo signaling pathway . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with TGF - α ( 100 ng / ml ) for 0 , 30 , 60 min . Real - time PCR determines the action of TGF - α and YAP levels on the mRNA expression of AREG . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( Ctrl vs . TGF - α , P < 0 . 0001 ; MXIV vs . YAP , P < 0 . 0001 ; MXIV vs . YAPS127A , P < 0 . 0001 ) . Real - time PCR analysis showing that knockdown of YAP with YAP siRNA ( siYAP ) significantly suppressed TGF - α - induced ( FBS 10 % , TGF - α : 10 ng / ml for 48 h ) AREG expression in ME180 cells . siCTRL , a non - targeting siRNA , was used as a negative control . Each bar represents the mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0067 ) . Proliferation of ME180 cells ( FBS 1 % ) treated with control ( siCTRL ) or YAP ( siYAP ) prior to treatment with control medium or TGF - α ( 10 ng / ml ) for 108 h . Each bar represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P = 0 . 0058 ; siYAP + CTRL vs . siYAP + TGF - α , P = 0 . 1840 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0013 ) . Quantitative data of the wound - healing assay showing the migration of ME180 cells that were treated with control ( siCTRL ) or YAP siRNA ( siYAP ) prior to treatment with or without TGF - α for 12 h . Each bar in bar graphs represents the mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siCTRL + TGF - α , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + TGF - α , P < 0 . 0001 ; siCTRL + TGF - α vs . siYAP + TGF - α , P = 0 . 0041 ) .

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

Figure 6Western blot analysis showing effects of TGF-α on phosphorylation of EGFR, YAP, AKT, and ERK1/2. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 5, 10, 30, 60 or 120 min.Western blot analysis showing effects of TGF-α on the phosphorylation of the components of the Hippo signaling pathway. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with TGF-α (100 ng/ml) for 0, 30, 60 min.Real-time PCR determines the action of TGF-α and YAP levels on the mRNA expression of AREG. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (Ctrl vs. TGF-α, P < 0.0001; MXIV vs. YAP, P < 0.0001; MXIV vs. YAPS127A, P < 0.0001).Real-time PCR analysis showing that knockdown of YAP with YAP siRNA (siYAP) significantly suppressed TGF-α-induced (FBS 10%, TGF-α: 10 ng/ml for 48 h) AREG expression in ME180 cells. siCTRL, a non-targeting siRNA, was used as a negative control. Each bar represents the mean ± SEM (n = 5). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0067).Proliferation of ME180 cells (FBS 1%) treated with control (siCTRL) or YAP (siYAP) prior to treatment with control medium or TGF-α (10 ng/ml) for 108 h. Each bar represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P = 0.0058; siYAP+CTRL vs. siYAP+TGF-α, P = 0.1840; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0013).Quantitative data of the wound-healing assay showing the migration of ME180 cells that were treated with control (siCTRL) or YAP siRNA (siYAP) prior to treatment with or without TGF-α for 12 h. Each bar in bar graphs represents the mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siCTRL+TGF-α, P < 0.0001; siYAP+CTRL vs. siYAP+TGF-α, P < 0.0001; siCTRL+TGF-α vs. siYAP+TGF-α, P = 0.0041).


Figure 7Western blot analysis showing that AREG treatment induced dephosphorylation of LATS1 , MOB1 , and YAP . ME180 cells were starved for 6 h after reaching cell confluence , then cells were treated with AREG ( 50 ng / ml ) for 0 , 30 , 60 min . AREG ( 50 ng / ml ) treatment induced appearance of elongated cells in ME180 cells ( top and middle panels ) and significantly stimulated ME180 cell proliferation ( lower panel ) . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 003 ) . Scale bar : 100 μm . Wound - healing assay showing that AREG stimulates migration of ME180 cervical cancer cells within 10 h in serum - free medium . Scale bar : 200 μm . Real - time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression . Each bar represents mean ± SEM ( n = 9 ) . Bars with different letters are significantly different from each other ( P < 0 . 0001 ) . Real - time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA ( siYAP ) significantly suppressed AREG - induced AREG mRNA expression . siCTRL ( non - target siRNA ) was used as a siRNA control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCTRL vs . siCTRL + AREG , P < 0 . 0001 ; siYAP + CTRL vs . siYAP + AREG , P < 0 . 0001 ; siCTRL + AREG vs . siYAP + AREG , P = 0 . 0037 ) .

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

Figure 7Western blot analysis showing that AREG treatment induced dephosphorylation of LATS1, MOB1, and YAP. ME180 cells were starved for 6 h after reaching cell confluence, then cells were treated with AREG (50 ng/ml) for 0, 30, 60 min.AREG (50 ng/ml) treatment induced appearance of elongated cells in ME180 cells (top and middle panels) and significantly stimulated ME180 cell proliferation (lower panel). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.003). Scale bar: 100 μm.Wound-healing assay showing that AREG stimulates migration of ME180 cervical cancer cells within 10 h in serum-free medium. Scale bar: 200 μm.Real-time PCR showing that treatment of ME180 cells with AREG for 24 h significantly increased AREG mRNA expression. Each bar represents mean ± SEM (n = 9). Bars with different letters are significantly different from each other (P < 0.0001).Real-time PCR showing that knockdown of YAP in ME180 cells with YAP siRNA (siYAP) significantly suppressed AREG-induced AREG mRNA expression. siCTRL (non-target siRNA) was used as a siRNA control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCTRL vs. siCTRL+AREG, P < 0.0001; siYAP+CTRL vs. siYAP+AREG, P < 0.0001; siCTRL+AREG vs. siYAP+AREG, P = 0.0037).


Figure 8Representative images showing the morphology of spheroids derived from ME180 - siCtrl and ME180 - siLATS1 / 2 cells growing in a 3D hanging - drop culture system for 10 days . Scale bar : 1 . 0 mm . Quantitative data showing changes in the volume of spheroids derived from ME180 - siCTRL and ME180 - siLATS1 / 2 cells growing in a 3D hanging - drop culture system . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0004 ) . Quantitative data showing changes in the volume of spheroids derived from ME180 cells growing in a 3D hanging - drop culture system in the absence or presence of AREG ( 20 ng / ml , 8 days ) . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0030 ) . Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180 - MXIV , ME180 - YAP and ME180 - YAPS127A cells . Scale bar : 500 μm . Representative images showing the effect of AREG , EGFR inhibitor ( AG1478 ) , and YAP antagonist verteporfin ( VTPF ) on the growth of ME180 cell in a 3D hanging - drop culture system . ME180 cells were incubated in the 3D hanging - drop culture system for 10 days in the absence or presence of AREG , AG1478 or verteporfin for 8 days . Scale bar : 1 . 0 mm .

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

Figure 8Representative images showing the morphology of spheroids derived from ME180-siCtrl and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system for 10 days. Scale bar: 1.0 mm.Quantitative data showing changes in the volume of spheroids derived from ME180-siCTRL and ME180-siLATS1/2 cells growing in a 3D hanging-drop culture system. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0004).Quantitative data showing changes in the volume of spheroids derived from ME180 cells growing in a 3D hanging-drop culture system in the absence or presence of AREG (20 ng/ml, 8 days). Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0030).Soft agar assay showing the effect of AG1478 and VTPF on colony formation in ME180-MXIV, ME180-YAP and ME180-YAPS127A cells. Scale bar: 500 μm.Representative images showing the effect of AREG, EGFR inhibitor (AG1478), and YAP antagonist verteporfin (VTPF) on the growth of ME180 cell in a 3D hanging-drop culture system. ME180 cells were incubated in the 3D hanging-drop culture system for 10 days in the absence or presence of AREG, AG1478 or verteporfin for 8 days. Scale bar: 1.0 mm.


Figure 9Effect of recombinant HPV16 E6 protein on the proliferation of HT3 cells . Each point represents mean ± SEM ( n = 5 ) . * * * P = 0 . 0001 compared with control ( Ctrl ) on day 4 . HT3 cells were cultured in serum - reduced medium in the presence or absence of recombinant HPV16 E6 ( 400 nM ) . Recombinant HPV16 E6 protein increased YAP protein level , but had no effect on β - actin protein level in HT3 cells . Cell culture and treatment procedure are the same as described in ( A ) . AREG mRNA levels in HT3 cells incubated for 48 h with or without recombinant HPV E6 . Each bar represents mean ± SEM ( n = 5 ) . Bars with different letters are significantly different from each other ( P = 0 . 0042 ) . Western blotting analysis showing the effect of HPV16E6 ( 400 nM , 48 h ) on YAP protein levels in HT3 cells transfected with non - targeting control siRNA ( siCtrl ) or YAP siRNA ( siYAP ) . Effect of YAP on HPV16 E6 stimulation of HT3 cell proliferation . siCtrl : non - targeting control siRNA ; siYAP : YAP siRNA ; E6 : 400 nM recombinant HPV16 E6 , 48 h . Each bar represents mean ± SEM ( n = 4 ) . Bars with the same letters are not significantly different from each other ( siCtrl vs . siCtrl + E6 , P = 0 . 0232 ; siCtrl + E6 vs . siYAP + E6 , P = 0 . 0011 ) . Knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA ( siE6 ) reduced YAP protein in HeLa cells , while knockdown of YAP with YAP siRNA ( siYAP ) in these cells had no effect on the mRNA level of HPV18 E6 . siCtrl : non - targeting control siRNA . Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6 - specific siRNA ( siE6 ) significantly suppressed mRNA expression of AREG . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( siCtrl vs . siHPV18E6 , P = 0 . 0181 ; siCtrl vs . siYAP , P = 0 . 0005 ) . Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6 - specific siRNA ( siE6 ) significantly suppressed cell growth ( n = 9 , siCtrl vs . siHPV18E6 , P = 0 . 0002 ; siCtrl vs . siYAP , P = 0 . 0004 ) . Concentrations of AREG in the culture medium of HeLa cells transfected with non - targeting control siRNA ( siCTRL ) or HPV18 E6 siRNA ( siHPV18E6 ) . Each bar represents mean ± SEM ( n = 6 ) . Bars with different letters are significantly different from each other ( P = 0 . 0461 ) .

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

Figure 9Effect of recombinant HPV16 E6 protein on the proliferation of HT3 cells. Each point represents mean ± SEM (n = 5). ***P = 0.0001 compared with control (Ctrl) on day 4. HT3 cells were cultured in serum-reduced medium in the presence or absence of recombinant HPV16 E6 (400 nM).Recombinant HPV16 E6 protein increased YAP protein level, but had no effect on β-actin protein level in HT3 cells. Cell culture and treatment procedure are the same as described in (A).AREG mRNA levels in HT3 cells incubated for 48 h with or without recombinant HPV E6. Each bar represents mean ± SEM (n = 5). Bars with different letters are significantly different from each other (P = 0.0042).Western blotting analysis showing the effect of HPV16E6 (400 nM, 48 h) on YAP protein levels in HT3 cells transfected with non-targeting control siRNA (siCtrl) or YAP siRNA (siYAP).Effect of YAP on HPV16 E6 stimulation of HT3 cell proliferation. siCtrl: non-targeting control siRNA; siYAP: YAP siRNA; E6: 400 nM recombinant HPV16 E6, 48 h. Each bar represents mean ± SEM (n = 4). Bars with the same letters are not significantly different from each other (siCtrl vs. siCtrl+E6, P = 0.0232; siCtrl+E6 vs. siYAP+E6, P = 0.0011).Knockdown of endogenous E6 in HeLa cells with HPV18 E6 siRNA (siE6) reduced YAP protein in HeLa cells, while knockdown of YAP with YAP siRNA (siYAP) in these cells had no effect on the mRNA level of HPV18 E6. siCtrl: non-targeting control siRNA.Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6-specific siRNA (siE6) significantly suppressed mRNA expression of AREG. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (siCtrl vs. siHPV18E6, P = 0.0181; siCtrl vs. siYAP, P = 0.0005).Knockdown of endogenous HPV18 E6 in HeLa cells with HPV18 E6-specific siRNA (siE6) significantly suppressed cell growth (n = 9, siCtrl vs. siHPV18E6, P = 0.0002; siCtrl vs. siYAP, P = 0.0004).Concentrations of AREG in the culture medium of HeLa cells transfected with non-targeting control siRNA (siCTRL) or HPV18 E6 siRNA (siHPV18E6). Each bar represents mean ± SEM (n = 6). Bars with different letters are significantly different from each other (P = 0.0461).


Figure 10Effect of YAP mRNA levels in HT3 cells treated with or without E6 ( 400 nM ) for 60 min in McCoy ' s 5A with 1 % serum . Data were normalized with 18S mRNA . Bars with same letters are not significantly different from each other ( P > 0 . 05 ) . n = 4 , P = 0 . 9407 . YAP mRNA levels in HT3 cells treated with or without E6 ( 400 nM ) for 48 h in McCoy ' s 5A with 1 % serum . mRNA levels were measured with real - time PCR . Data were normalized with 18S mRNA . Each bar represents mean ± SEM ( n = 5 ) . Bars with same letters are not significantly different from each other ( P > 0 . 05 ) ( P = 0 . 3233 ) . Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide ( CHX ) for 4 h or 8 h . β - actin was used as a protein loading control . Confluent HT3 cells were starved for 4 h before treatment with MG132 ( 10 μM ) or CHX ( 20 μg / ml ) for 0 , 4 or 8 h . HPV16E6 protein prevented YAP protein from degradation . Confluent HT3 cells were starved for 4 h before treatment with or without CHX ( 20 μg / ml ) , HPV16E6 , or CHX combined with HPV16E6 for 8 h . Treatment of starved HT3 cells with HPV16E6 ( 400 nM ) for 8 h suppressed degradation of YAP , but not EGFR protein . Quantitative data showing relative YAP protein levels in ( D ) . Protein levels were normalized with β - actin and presented as ratios relative to that of control . Each bar represents mean ± SEM ( n = 4 ) . Bars with different letters are significantly different from each other ( Ctrl vs . CHX , P = 0 . 0003 ; Ctrl vs . CHX + E6 , P = 0 . 2665 ; Ctrl vs . E6 , P = 0 . 0258 ) . Western blot analysis showing that expression of HPV16E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε , but decreased the protein level of SOCS6 . Importantly , HPV16E6 increased YAP phosphorylation at serine 127 , but suppressed its phosphorylation at serine 397 . HPV16E6 had no effect on the level of β - TrcP , LATS1 / 2 and MOB1 in HT3cervical cancer cells . Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18E6 siRNA ( siE6 ) reduced YAP protein levels , but increased SOCS6 protein levels . Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA ( siSOCS # 1 and siSOCS6 # 2 ) increased total YAP protein , enhanced phosphorylation of YAP protein at serine 127 , but suppressed phosphorylation of YAP at serine 397 .

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

Figure 10Effect of YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 60 min in McCoy's 5A with 1% serum. Data were normalized with 18S mRNA. Bars with same letters are not significantly different from each other (P > 0.05). n = 4, P = 0.9407.YAP mRNA levels in HT3 cells treated with or without E6 (400 nM) for 48 h in McCoy's 5A with 1% serum. mRNA levels were measured with real-time PCR. Data were normalized with 18S mRNA. Each bar represents mean ± SEM (n = 5). Bars with same letters are not significantly different from each other (P > 0.05) (P = 0.3233).Western blot results showing YAP and EGFR protein levels after treatment with MG132 or cycloheximide (CHX) for 4 h or 8 h. β-actin was used as a protein loading control. Confluent HT3 cells were starved for 4 h before treatment with MG132 (10 μM) or CHX (20 μg/ml) for 0, 4 or 8 h.HPV16E6 protein prevented YAP protein from degradation. Confluent HT3 cells were starved for 4 h before treatment with or without CHX (20 μg/ml), HPV16E6, or CHX combined with HPV16E6 for 8 h. Treatment of starved HT3 cells with HPV16E6 (400 nM) for 8 h suppressed degradation of YAP, but not EGFR protein.Quantitative data showing relative YAP protein levels in (D). Protein levels were normalized with β-actin and presented as ratios relative to that of control. Each bar represents mean ± SEM (n = 4). Bars with different letters are significantly different from each other (Ctrl vs. CHX, P = 0.0003; Ctrl vs. CHX+E6, P = 0.2665; Ctrl vs. E6, P = 0.0258).Western blot analysis showing that expression of HPV16E6 in HT3 cells increased the protein level of total YAP and casein kinase Iε, but decreased the protein level of SOCS6. Importantly, HPV16E6 increased YAP phosphorylation at serine 127, but suppressed its phosphorylation at serine 397. HPV16E6 had no effect on the level of β-TrcP, LATS1/2 and MOB1 in HT3cervical cancer cells.Western blot results showing that knockdown of endogenous E6 in HeLa cells with HPV18E6 siRNA (siE6) reduced YAP protein levels, but increased SOCS6 protein levels.Western blot analysis showing that knockdown of SOCS6 in HT3 cells with SOCS6 siRNA (siSOCS#1 and siSOCS6#2) increased total YAP protein, enhanced phosphorylation of YAP protein at serine 127, but suppressed phosphorylation of YAP at serine 397.


Figure 11A - F Representative images showing expression of YAP ( in red ) in wild - type mousecervical tissues ( n = 5 ) ( A ) , HPV16E6 - induced ( C ) and HPV16E6 / E7 - induced ( E ) mousecervicaltumor tissue ( n = 4 each ) . Scale bars for ( A ) , ( C ) , and ( E ) : 100 μm . High - resolution images showing the expression and cellular location of YAP in normal cervical tissues ( B ) , HPV16E6 - induced ( D ) and HPV16E6 / E7 - induced ( F ) mousecervicaltumor tissue . Scale bars for ( B ) , ( D ) , and ( F ) : 25 μm . G , H Representative images showing expression of YAP ( in red ) in normal ( G ) and HPV16 - containing humanforeskinkeratinocyte raft cultures ( n = 5 each ) ( H ) . Scale bars : 100 μm . I , J High - resolution images showing the expression and cellular location of YAP in normal ( I ) and HPV16 - containing humanforeskinkeratinocytes raft cultures ( J ) . Scale bars : 20 μm .

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

Figure 11A-F Representative images showing expression of YAP (in red) in wild-type mousecervical tissues (n = 5) (A), HPV16E6-induced (C) and HPV16E6/E7-induced (E) mousecervicaltumor tissue (n = 4 each). Scale bars for (A), (C), and (E): 100 μm. High-resolution images showing the expression and cellular location of YAP in normal cervical tissues (B), HPV16E6-induced (D) and HPV16E6/E7-induced (F) mousecervicaltumor tissue. Scale bars for (B), (D), and (F): 25 μm.G, H Representative images showing expression of YAP (in red) in normal (G) and HPV16-containing humanforeskinkeratinocyte raft cultures (n = 5 each) (H). Scale bars: 100 μm.I, J High-resolution images showing the expression and cellular location of YAP in normal (I) and HPV16-containing humanforeskinkeratinocytes raft cultures (J). Scale bars: 20 μm.


Figure 1 ( A ) miR - 155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB / c mice 6 weeks postinfection ( B ) Murine bone marrow - derived macrophages ( BMDMs ) were infected with BCG at an MOI of 5 for the indicated times , and the expression levels of miR - 155 were measured by real - time PCR . ( C and D ) RAW264 . 7 cells were infected with BCG at an MOI of 5 for the indicated time points ( C ) or at indicated MOIs for 24 h ( D ) . The expression levels of miR - 155 were examined by real - time PCR . ( E and F ) RAW264 . 7 cells were infected with H37Ra at an MOI of 5 for the indicated time ( E ) or at indicated MOI for 24 h ( F ) . The expression levels of miR - 155 were examined by real - time PCR . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Figure 1(A) miR-155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB/c mice 6 weeks postinfection(B) Murine bone marrow-derived macrophages (BMDMs) were infected with BCG at an MOI of 5 for the indicated times, and the expression levels of miR-155 were measured by real-time PCR.(C and D) RAW264.7 cells were infected with BCG at an MOI of 5 for the indicated time points (C) or at indicated MOIs for 24 h (D). The expression levels of miR-155 were examined by real-time PCR.(E and F) RAW264.7 cells were infected with H37Ra at an MOI of 5 for the indicated time (E) or at indicated MOI for 24 h (F). The expression levels of miR-155 were examined by real-time PCR. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.


Figure 2 ( A - C ) RAW264 . 7 cells were transfected with control or miR - 155 mimic for 24 h followed by BCG ( A ) , H37Ra ( B ) or H37Rv ( C ) infection at an MOI of 10 for 1 h , and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints . ( D - F ) RAW264 . 7 cells were transfected with control or miR - 155 inhibitor for 24 h followed by BCG ( D ) , H37Ra ( E ) or H37Rv ( F ) infection at an MOI of 10 for 1 h , and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints . ( G and H ) RAW264 . 7 cells were transfected with miR - 155 mimic ( G ) or inhibitor ( H ) for 24 h followed by Texas Red - labeled BCG infection at an MOI of 10 for 1 h . Phagocytosis of mycobacteria was determined by flow cytometry . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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

Figure 2(A-C) RAW264.7 cells were transfected with control or miR-155 mimic for 24 h followed by BCG (A), H37Ra (B) or H37Rv (C) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints.(D-F) RAW264.7 cells were transfected with control or miR-155 inhibitor for 24 h followed by BCG (D), H37Ra (E) or H37Rv (F) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints.(G and H) RAW264.7 cells were transfected with miR-155 mimic (G) or inhibitor (H) for 24 h followed by Texas Red-labeled BCG infection at an MOI of 10 for 1 h. Phagocytosis of mycobacteria was determined by flow cytometry. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01.


Figure 3RAW 264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h and then infected with Texas Red - labeled BCG for 1 h . Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG antibody ( A ) The colocalization of BCG with lysosome was detected by confocal microscopy . The percentage of co - localization of BCG with CD63 - positive ( B ) RAW 264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h and then infected with Texas Red - labeled BCG for 1 h . Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG antibody ( A ) , or labeled with a fluorogenic substrate for proteases , DQ - Green ( C ) . The colocalization of BCG with lysosome was detected by confocal microscopy . The percentage of co - localization of BCG with CD63 - positive ( B ) or DQ - Green labeled ( D ) lysosomes was quantified , respectively . Cells treated with rapamycin were used as a positive control . Arrows indicate the co - localization of BCG with lysosomes ; scale bar = 5 µm . Quantification of data are shown as the mean ± SEM of three independent experiments ( n = 100 phagosomes ) . * * , p < 0 . 01 .

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

Figure 3RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A)The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B)RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A), or labeled with a fluorogenic substrate for proteases, DQ-Green (C). The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B) or DQ-Green labeled (D) lysosomes was quantified, respectively. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with lysosomes; scale bar = 5 µm. Quantification of data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01.


Figure 4 ( A - D ) RAW264 . 7 cells were transfected with miR - 155 mimic ( A and C ) or inhibitor ( B and D ) for 24 h and then either left uninfected or infected with BCG . Expression levels of miR - 155 were detected by real - time PCR ( A and B ) . ( A - D ) RAW264 . 7 cells were transfected with miR - 155 mimic ( A and C ) or inhibitor ( B and D ) for 24 h and then either left uninfected or infected with BCG . Expression levels of miR - 155 were detected by real - time PCR ( A and B ) . The LC3 levels were detected by Western - blot , and the ratios of LC3 - II / β - actin were calculated as shown below the representative blot ( C and D ) ( E and F ) RAW264 . 7 cells transfected with miR - 155 mimic ( E ) or inhibitor ( F ) was incubated with DMSO or bafilomycin A1 ( BafA . ) at a concentration of 100 nM for 2 h , and then LC3 levels were detected by Western - blot . The ratios of LC3 - II / β - actin were calculated as shown below the representative blot . * * * , p < 0 . 001 .

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

Figure 4(A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B).(A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B). The LC3 levels were detected by Western-blot, and the ratios of LC3-II/β-actin were calculated as shown below the representative blot (C and D)(E and F) RAW264.7 cells transfected with miR-155 mimic (E) or inhibitor (F) was incubated with DMSO or bafilomycin A1 (BafA.) at a concentration of 100 nM for 2 h, and then LC3 levels were detected by Western-blot. The ratios of LC3-II/β-actin were calculated as shown below the representative blot. ***, p<0.001.


Figure 5 ( A and B ) RAW264 . 7 cells stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic for 24 h , and GFP - LC3 puncta ( > 1 µm ) were detected by confocal microscopy ( A ) and quantified ( B ) . RAW264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h . Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( Green ) , and LC3 puncta ( > 1 µm ) were detected by confocal microscopy ( C ) and quantified ( D ) . ( E and F ) RAW264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h , and then incubated with a specific fluorescent dye MDC ( 50 µM ) . The MDC - labeled autophagic vacuoles were detected by confocal microscopy ( E ) and cells with MDC - positive autophagic vacuoles were quantified ( F ) . Cells treated with rapamycin were used as a positive control . Arrows indicate the GFP - LC3 puncta or autophagic vacuoles labeled with MDC ; scale bar = 5 µm . Data are shown as the mean ± SEM of three independent experiments ( n = 300 cells ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Figure 5(A and B) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic for 24 h, and GFP-LC3 puncta (>1 µm) were detected by confocal microscopy (A) and quantified (B).RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green), and LC3 puncta (>1 µm) were detected by confocal microscopy (C) and quantified (D).(E and F) RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with a specific fluorescent dye MDC (50 µM). The MDC-labeled autophagic vacuoles were detected by confocal microscopy (E) and cells with MDC-positive autophagic vacuoles were quantified (F). Cells treated with rapamycin were used as a positive control. Arrows indicate the GFP-LC3 puncta or autophagic vacuoles labeled with MDC; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 300 cells). **, p<0.01; ***, p<0.001.


Figure 6 ( A ) RAW264 . 7 cells stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic and then infected with Texas Red - labeled BCG for 1 h . The co - localization of BCG with LC3 was detected by confocal microscopy . ( B ) Quantification of the co - localization of BCG with LC3 - positive autophagosomes is shown . ( C ) RAW264 . 7 cells were transiently transfected with control or miR - 155 mimic , and then infected with Texas Red - labeled BCG for 1 h . Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( Green ) . The co - localization of BCG with endogenous LC3 was detected by confocal microscopy . ( D ) Quantification of the co - localization of BCG with LC3 - positive autophagosomes is shown . ( E ) After transient transfection with control or miR - 155 mimic , RAW264 . 7 cells were infected with Texas Red - labeled BCG for 1 h , and then were labeled with a specific fluorescent dye MDC ( 50 µM ) for autophagic vacuoles . The co - localization of BCG with MDC - positive autophagic vacuoles was detected by confocal microscopy . ( F ) Quantification of the co - localization of BCG with MDC - positive autophagosomes is shown . Cells treated with rapamycin were used as a positive control . Arrows indicate the co - localization of BCG with autophagosomes ; scale bar = 5 µm . Data are shown as the mean ± SEM of three independent experiments ( n = 100 phagosomes ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

Figure 6(A) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG with LC3 was detected by confocal microscopy. (B) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown.(C) RAW264.7 cells were transiently transfected with control or miR-155 mimic, and then infected with Texas Red-labeled BCG for 1 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green). The co-localization of BCG with endogenous LC3 was detected by confocal microscopy. (D) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown.(E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.


Figure 7 ( A and B ) RAW 264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h , and then incubated with DMSO or 3 - MA for 2 h . Protein levels of LC3 were detected by Western - blot in uninfected cells ( A ) Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h ( B ) ( C and D ) RAW 264 . 7 cells were transiently co - transfected with control or miR - 155 mimic together with a control siRNA or Atg7 siRNA . The expression levels of Atg7 and LC3 were detected by Western - blot ( C ) . Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h ( D ) . Values of LC3 - II / β - actin ratios are indicated below the representative blot . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

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

Figure 7(A and B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with DMSO or 3-MA for 2 h. Protein levels of LC3 were detected by Western-blot in uninfected cells (A)Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h (B)(C and D) RAW 264.7 cells were transiently co-transfected with control or miR-155 mimic together with a control siRNA or Atg7 siRNA. The expression levels of Atg7 and LC3 were detected by Western-blot (C).Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h (D). Values of LC3-II/β-actin ratios are indicated below the representative blot. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.


Figure 8 ( A ) Sequences of mouse and human miR - 155 and their predicted interactions with conserved 7 - mer 1A miR - 155 seeds found within the Rheb 3 ′ UTRs of different species are shown . The sequence of the Rheb 3 ′ UTR seed mutant used for the reporter assays and the predicted disruption of the miR - 155 interaction are also shown . B ) RAW264 . 7 cells were co - transfected with control or miR - 155 mimic and a wild - type ( WT - Rheb ) or mutated Rheb 3 ′ UTR ( mut - Rheb ) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection . Data are shown as the mean ± SEM of three independent experiments . * * * , p < 0 . 001 ; NS , not significant . ( C - F ) RAW264 . 7 cells were transfected with miR - 155 mimic ( C and E ) or inhibitor ( D and F ) for 24 h and either left uninfected or infected with BCG . Protein expression levels of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below the representative blot ( C and D ) ( C - F ) RAW264 . 7 cells were transfected with miR - 155 mimic ( C and E ) or inhibitor ( D and F ) for 24 h and either left uninfected or infected with BCG . Protein expression levels of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below the representative blot ( C and D ) , and expression levels of Rheb mRNA were detected by RT - PCR ( E and F ) .

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

Figure 8(A) Sequences of mouse and human miR-155 and their predicted interactions with conserved 7-mer 1A miR-155 seeds found within the Rheb 3′UTRs of different species are shown. The sequence of the Rheb 3′UTR seed mutant used for the reporter assays and the predicted disruption of the miR-155 interaction are also shown.B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant.(C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D)(C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D), and expression levels of Rheb mRNA were detected by RT-PCR (E and F).


Figure 9 ( A ) RAW264 . 7 cells were transfected with a plasmid expressing Rheb for 24 h , and then incubated with DMSO or BafA . for 2 h . The expression levels of Rheb and LC3 were detected by Western - blot . Values of LC3 - II / β - actin ratios are indicated below the representative blot . ( B and C ) RAW264 . 7 cells were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h , and then infected with Texas Red - labeled BCG for 1 h . The co - localization of BCG and MDC - labeled autophagosome was detected by confocal microscopy . Arrows indicate Quantification of BCG co - localization with autophagosomes described in B is shown ( C ) . D ) RAW264 . 7 cells were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h . Cells were infected with BCG for 1 h , and washed for three times to remove extracellular mycobacteria . Intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

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

Figure 9(A) RAW264.7 cells were transfected with a plasmid expressing Rheb for 24 h, and then incubated with DMSO or BafA. for 2 h. The expression levels of Rheb and LC3 were detected by Western-blot. Values of LC3-II/β-actin ratios are indicated below the representative blot.(B and C) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h, and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG and MDC-labeled autophagosome was detected by confocal microscopy. Arrows indicate Quantification of BCG co-localization with autophagosomes described in B is shown (C).D) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h. Cells were infected with BCG for 1 h, and washed for three times to remove extracellular mycobacteria. Intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.


Figure 1A . Representative spinning disk confocal live - cell imaging time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , treated with control and KIF4A siRNAs . White arrowheads highlight poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec . B . Representative spinning disk confocal live - cell imaging time series images of S - trityl - L - cysteine ( STLC ) - treated U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) . Note that MT - flux is abrogated in presence of taxol ( lower panel ) . Scale bars , 10 µm . Time , min : sec . C . Illustration of 405nm laser - photoactivated regions in monopolar spindles ( blue circles , left ) . The effect of photoactivation and region selected for kymograph generation ( dashed white rectangle , right ) . Scale bar , 10 µm . D . Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 30 sec . Quantification of MT - flux in bipolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) : - bipolar spindles : siControl ( 49 , 5 ) , STLC ( 31 , 3 ) , siKIF15 ( 39 , 3 ) , siKID ( 32 , 3 ) , siKIF4A ( 28 , 3 ) , siKIF2A ( 36 , 3 ) , siCLASPs ( 21 , 2 ) , GSK923295 ( 44 , 3 ) Quantification of MT - flux in monopolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) monopolar spindles : siControl ( 35 , 3 ) , siKIF15 ( 44 , 3 ) , siKID ( 38 , 3 ) , siKIF4A ( 44 , 3 ) , siKIF2A ( 38 , 3 ) , siCLASPs ( 12 , 2 ) , GSK923295 ( 33 , 3 ) .

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

Figure 1A. Representative spinning disk confocal live-cell imaging time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), treated with control and KIF4A siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.B. Representative spinning disk confocal live-cell imaging time series images of S-trityl-L-cysteine (STLC)-treated U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red). Note that MT-flux is abrogated in presence of taxol (lower panel). Scale bars, 10 µm. Time, min:sec.C. Illustration of 405nm laser-photoactivated regions in monopolar spindles (blue circles, left). The effect of photoactivation and region selected for kymograph generation (dashed white rectangle, right). Scale bar, 10 µm. D. Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 30 sec. Quantification of MT-flux in bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments): - bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3)Quantification of MT-flux in monopolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments) monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3).


Figure 2A . Representative spinning disk confocal live - cell imaging time series images of U2OS PA - GFP - α - tubulin cells conditionally co - expressing KIF4A shRNA and RNAi - resistant mCherry - Kif4A variants induced using doxycycline . Chromosomes were stained using SiR - DNA . White arrowheads highlight poleward flux of the photoactivated regions . Scale bar , 10 µm . Time , min : sec . B . Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi - resistant mCherry - KIF4A variants . The anti - KIF4A antibody was used , together with anti - vinculin antibody as a loading control . C . Quantification of MT - flux upon shRNA - mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi - resistant mCherry - KIF4A variants . The bars in graph represent mean ± SD . N ( number of cells , number of independent experiments ) : uninduced shKIF4A ( 48 , 4 ) , shKIF4A ( 60 , 4 ) , uninduced KIF4A WT ( 36 , 5 ) , shKIF4A + KIF4A WT ( 43 , 5 ) , uninduced KIF4A K94A ( 42 , 7 ) , shKIF4A + KIF4A K94A ( 38 , 6 ) , uninduced KIF4A ΔZip1 ( 32 , 4 ) , shKIF4A + ΔZip1 ( 30 , 4 ) .

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

Figure 2A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec.B. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control.C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A + KIF4A K94A (38, 6), uninduced KIF4A ΔZip1 (32, 4), shKIF4A + ΔZip1 (30, 4).


Figure 3A . Representative spinning disk confocal time series of MT - flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes ( MUGs ) . U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , labeled for chromosomes with SiR - DNA ( grey ) are shown , with 5 mM caffeine used as a control . Scale bar , 10 µm . Time , min : sec . B . Quantification of the MT - flux rates from indicated conditions . MT - flux values with mean ± SD are plotted . N ( number of cells , number of independent experiments ) : Caffeine - only control ( 30 , 3 ) and MUGs ( 34 , 3 ) . C . Representative point - scanning confocal maximum - intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions , immunostained with antibodies against KIF4A and α - tubulin . DNA was counterstained with DAPI . KIF4A in cyan and α - tubulin in red in merged image . Scale bar , 10 µm .

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

Figure 3A. Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (grey) are shown, with 5 mM caffeine used as a control. Scale bar, 10 µm. Time, min:sec. B. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3).C. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 µm.


Figure 4A . Representative spinning disk confocal live - cell image series of MT - flux in U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) treated with indicated siRNAs . White arrowheads highlight poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec . B . Quantification of the impact of the MT - crosslinking proteins on MT - flux in U2OS PA - GFP / mCherry - α - tubulin cells transfected with respective siRNAs . Graph represent MT - flux values with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siPRC1 ( 45 , 3 ) , siNuMA ( 40 , 3 ) , siHSET ( 37 , 3 ) , siHSET + siNuMA ( 39 , 3 ) . C . Quantification of the impact of the motor activity of HSET on MT - flux in U2OS cells stably expressing mEOS - α - tubulin treated with control or HSET 3 ′ UTR siRNAs in presence or absence of the respective RNAi - resistant GFP - HSET constructs . Graph represent MT - flux values with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 23 , 3 ) , siHSET 3 ′ UTR ( 33 , 3 ) , siHSET 3 ′ UTR + HSET WT ( 29 , 5 ) , siHSET 3 ′ UTR + HSET N593K ( 30 , 4 ) . D . Representative kymographs of the photoactivated spindles displaying asynchronous flux motion ( split of the two red dashed lines towards individual poles ) upon RNAi - mediated depletion of MT - crosslinkers . E . Percentage of cells with asynchronous flux

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

Figure 4A. Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.B. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), siHSET + siNuMA (39, 3).C. Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), siHSET 3′UTR + HSET N593K (30, 4).D. Representative kymographs of the photoactivated spindles displaying asynchronous flux motion (split of the two red dashed lines towards individual poles) upon RNAi-mediated depletion of MT-crosslinkers. E. Percentage of cells with asynchronous flux movements


Figure 5A . Representative spinning disk confocal live - cell time series of MT - flux in U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) treated with indicated conditions . White arrowheads follow the poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec . B . Quantification of the impact of stable end - on KT - MT attachments on MT - flux in NDC80 - depleted cells under indicated conditions and in early prometaphase cells with and without the CENP - E inhibitor GSK923295 . MT - flux values are plotted with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siNDC80 ( 59 , 6 ) , siNDC80 + siKIF4A ( 41 , 3 ) , siNDC80 + STLC ( bipol . ) ( 28 , 3 ) , siNDC80 + siKIF15 ( 36 , 4 ) , siNDC80 + siCLASPs ( 33 , 3 ) , siNDC80 + siKIF2A ( 38 , 4 ) , siNDC80 + GSK923295 ( 38 , 3 ) , early prometaphase ( 23 , 3 ) , early prometaphase + GSK923295 ( 28 , 3 ) . C . Kymograph profiles ( left ) highlighting that the photoactivated regions in prometaphase ( upper panel ) split and move towards both poles , whereas metaphase spindles ( lower panel ) flux uniformly towards individual poles ( indicated by red dotted lines ) . Illustration of the photoactivated regions ( right ) in prometaphase and metaphase spindles , highlighting that interpolar MTs overlap over a broader region in early mitosis , becoming focused at the spindle equator in metaphase . Scale bar , 30 sec . D . Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase . N ( number of cells , number of independent experiments ) : prometaphase ( 23 , 3 ) , metaphase ( 22 , 3 ) .

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

Figure 5A. Representative spinning disk confocal live-cell time series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated conditions. White arrowheads follow the poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.B. Quantification of the impact of stable end-on KT-MT attachments on MT-flux in NDC80-depleted cells under indicated conditions and in early prometaphase cells with and without the CENP-E inhibitor GSK923295. MT-flux values are plotted with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siNDC80 (59, 6), siNDC80 + siKIF4A (41, 3), siNDC80 + STLC (bipol.) (28, 3), siNDC80 + siKIF15 (36, 4), siNDC80 + siCLASPs (33, 3), siNDC80 + siKIF2A (38, 4), siNDC80 + GSK923295 (38, 3), early prometaphase (23, 3), early prometaphase + GSK923295 (28, 3).C. Kymograph profiles (left) highlighting that the photoactivated regions in prometaphase (upper panel) split and move towards both poles, whereas metaphase spindles (lower panel) flux uniformly towards individual poles (indicated by red dotted lines). Illustration of the photoactivated regions (right) in prometaphase and metaphase spindles, highlighting that interpolar MTs overlap over a broader region in early mitosis, becoming focused at the spindle equator in metaphase. Scale bar, 30 sec. D. Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase. N (number of cells, number of independent experiments): prometaphase (23, 3), metaphase (22, 3).


Figure 6A . Representative maximum - intensity projected coherent - hybrid STED ( CH - STED ) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA ( DAPI ) , KTs ( CENP - C , magenta ) , α - tubulin ( red ) and CENP - E ( green ) . Scale bar , 10 µm . B . Model illustrating potential mechanisms for CENP - E mediated MT - flux forces either through its KT - or antiparallel MT - based localization and sliding . CENP - E localized at KTs laterally interacting with MTs ( white arrowheads in 1 , 2 ) , and CENP - E localized at interpolar MTs ( white arrows in 3 , 4 ) , in control and NDC80 - depleted mitotic cells . Scale bars , 2 µm . C . CH - STED analyses of HeLa cells stably expressing CENP - E - GFP illustrating bipolar spindles in prometaphase , metaphase and just after sister - chromatid separation at anaphase onset . Approximate x - y resolution is 70 nm . The expected / described localization of CENP - E - GFP ( green ) at KTs is shown . In addition , CENP - E - GFP was found associated with interpolar MTs ( ipMTs ) , including regions of overlapping anti - parallel MTs , and k - fibers . Chromosomes ( confocal mode only ) were revealed in the larger panels with DAPI ( cyan ) and MTs ( magenta ) were detected with an anti - α - tubulin antibody . Arrows indicate examples of clear MT bundles , including regions of overlapping anti - parallel MTs . Scale bar in all panels is 5 μm . D . Representative spinning disk confocal live - cell time series of HeLa cells stably expressing CENP - E - GFP . White arrowheads highlight CENP - E - GFP at KTs , white arrows highlight CENP - E - GFP on interpolar MTs . Scale bar , 10 µm . Time , hour : min .

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

Figure 6A. Representative maximum-intensity projected coherent-hybrid STED (CH-STED) images of mitotic spindles in U2OS cells treated with control and NDC80 siRNAs and stained for DNA (DAPI), KTs (CENP-C, magenta), α-tubulin (red) and CENP-E (green). Scale bar, 10 µm.B. Model illustrating potential mechanisms for CENP-E mediated MT-flux forces either through its KT- or antiparallel MT- based localization and sliding. CENP-E localized at KTs laterally interacting with MTs (white arrowheads in 1, 2), and CENP-E localized at interpolar MTs (white arrows in 3, 4), in control and NDC80-depleted mitotic cells. Scale bars, 2 µm.C. CH-STED analyses of HeLa cells stably expressing CENP-E-GFP illustrating bipolar spindles in prometaphase, metaphase and just after sister-chromatid separation at anaphase onset. Approximate x-y resolution is 70 nm. The expected/described localization of CENP-E-GFP (green) at KTs is shown. In addition, CENP-E-GFP was found associated with interpolar MTs (ipMTs), including regions of overlapping anti-parallel MTs, and k-fibers. Chromosomes (confocal mode only) were revealed in the larger panels with DAPI (cyan) and MTs (magenta) were detected with an anti-α-tubulin antibody. Arrows indicate examples of clear MT bundles, including regions of overlapping anti-parallel MTs. Scale bar in all panels is 5 μm.D. Representative spinning disk confocal live-cell time series of HeLa cells stably expressing CENP-E-GFP. White arrowheads highlight CENP-E-GFP at KTs, white arrows highlight CENP-E-GFP on interpolar MTs. Scale bar, 10 µm. Time, hour:min.


Figure 7A . Representative spinning disk confocal time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , with indicated treatments ( left ) . White arrowheads highlight poleward flux of the photoactivated regions . Scale bar , 10 µm . Time , min : sec . Right panels display corresponding kymographs of the photoactivated spindles used for quantification of the flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 2 µm ( horizontal ) and 30 sec ( vertical ) . B , C . Quantification of MT - flux in prometaphase ( B ) and metaphase ( C ) bipolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . siNDC80 , siNDC80 + GSK923295 , early prometaphase , early prometaphase + GSK923295 , siControl , and siKIF4A N ( number of cells , number of independent experiments ) : siNDC80 + KIF15 + STLC ( 33 , 5 ) , siNDC80 + KIF15 + STLC + GSK923295 ( 25 , 3 ) , early prometaphase + KIF15 + STLC + GSK923295 ( 11 , 2 ) , siKIF15 + STLC ( 29 , 3 ) , siKIF4A + siKIF15 + STLC ( 10 , 3 ) . D . Representative spinning disk confocal time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , with indicated treatments ( left ) and corresponding kymographs ( right ) as described in ( A ) . Scale bars , 2 µm ( horizontal ) and 30 sec ( vertical ) .

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

Figure 7A. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left). White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec. Right panels display corresponding kymographs of the photoactivated spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 2 µm (horizontal) and 30 sec (vertical).B, C. Quantification of MT-flux in prometaphase (B) and metaphase (C) bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. siNDC80, siNDC80 + GSK923295, early prometaphase, early prometaphase + GSK923295, siControl, and siKIF4A N (number of cells, number of independent experiments): siNDC80 + KIF15 + STLC (33, 5), siNDC80 + KIF15 + STLC + GSK923295 (25, 3), early prometaphase + KIF15 + STLC + GSK923295 (11, 2), siKIF15 + STLC (29, 3), siKIF4A + siKIF15 + STLC (10, 3).D. Representative spinning disk confocal time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), with indicated treatments (left) and corresponding kymographs (right) as described in (A). Scale bars, 2 µm (horizontal) and 30 sec (vertical).


Figure 8A - C . Graph depicting a direct correlation between MT - flux and mitotic spindle where mean spindle length is plotted over mean MT - flux rates for all indicated conditions ( A ) , mitotic conditions containing stable end - on attachments ( B ) , and NDC80 depleted conditions lacking stable end - on attachments ( C ) . Solid and dotted lines represent the linear regression and 95 % confidence interval , respectively . Pearson correlation coefficient ( r ) , coefficient of determination ( r2 ) and corresponding p values are indicated . The error bars over x and y axis represent the standard deviation of MT - flux and spindle length , respectively . D . Representative images from spinning disk confocal live - cell time series of U2OS PA - GFP - α - tubulin / mCherry - α - tubulin cells under indicated treatments , with SiR - DNA - labelled DNA . Microtubules are shown in red and DNA in cyan in the merged image . Scale bar : 10 µm . E . Quantification of spindle lengths at metaphase following indicated treatments . Graph represents the spindle length of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siMCAK ( 34 , 3 ) , siKIF4A + siKIF15 + STLC ( 46 , 6 ) , siKIF4A + siKIF15 + STLC + siMCAK ( 43 , 3 ) , siKIF4A + siKIF15 + STLC + siKIF2A ( 22 , 3 ) , siCLASPs ( 64 , 6 ) , siCLASPs + siMCAK ( 33 , 3 ) , siCLASPs + siKIF2A ( 32 , 3 ) .

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

Figure 8A-C. Graph depicting a direct correlation between MT-flux and mitotic spindle where mean spindle length is plotted over mean MT-flux rates for all indicated conditions (A), mitotic conditions containing stable end-on attachments (B), and NDC80 depleted conditions lacking stable end-on attachments (C). Solid and dotted lines represent the linear regression and 95% confidence interval, respectively. Pearson correlation coefficient (r), coefficient of determination (r2) and corresponding p values are indicated. The error bars over x and y axis represent the standard deviation of MT-flux and spindle length, respectively.D. Representative images from spinning disk confocal live-cell time series of U2OS PA-GFP-α-tubulin/mCherry-α-tubulin cells under indicated treatments, with SiR-DNA-labelled DNA. Microtubules are shown in red and DNA in cyan in the merged image. Scale bar: 10 µm.E. Quantification of spindle lengths at metaphase following indicated treatments. Graph represents the spindle length of individual cells with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siMCAK (34,3), siKIF4A + siKIF15 + STLC (46, 6), siKIF4A + siKIF15 + STLC + siMCAK (43, 3), siKIF4A + siKIF15 + STLC + siKIF2A (22, 3), siCLASPs (64, 6), siCLASPs + siMCAK (33, 3), siCLASPs + siKIF2A (32, 3).


Figure 3 ( G ) Tumour growth ( in mm3 ) of FBXW7 + / + and FBXW7 - / - xenografts in nude mice ( n = 10 animals per group ) . Treatment with either vehicle , paclitaxel ( 1 , 5mg / kg ) or tigecycline ( 50mg / kg ) started at day 6 post - tumour - injection , and was administered three times per week . Error bars indicate SEM . ( H ) Representative images of the xenografts defined in ( G ) at day 15 .

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

Figure 3(G) Tumour growth (in mm3) of FBXW7+/+ and FBXW7-/- xenografts in nude mice (n=10 animals per group). Treatment with either vehicle, paclitaxel (1,5mg/kg) or tigecycline (50mg/kg) started at day 6 post-tumour-injection, and was administered three times per week. Error bars indicate SEM. (H) Representative images of the xenografts defined in (G) at day 15.


Figure 5 ( D ) WB illustrating the levels of ATF4 in DLD - 1 cells treated as in ( C ) . GAPDH levels are shown as a loading control .

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

Figure 5(D) WB illustrating the levels of ATF4 in DLD-1 cells treated as in (C). GAPDH levels are shown as a loading control.


Figure 3Immunoblot analysis of phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) , phosphorylated p38 ( P - P38 ) , phosphorylated JNK1 / 2 ( P - JNK1 / 2 ) , phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , MEKK2 , TRAF2 , TRAF3 , TRAF6 , NEDD4L , and actin in HeLa cells transfected with Nedd4l siRNA ( siNedd4l ) or control RNA ( siNC ) ( A ) , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . Immunoblot analysis of phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) , phosphorylated p38 ( P - P38 ) , phosphorylated JNK1 / 2 ( P - JNK1 / 2 ) , phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , MEKK2 , TRAF2 , TRAF3 , TRAF6 , NEDD4L , and actin in Nedd4l + / + and Nedd4l - / - MEFs , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . Immunoblot analysis of phosphorylated p38 ( P - P38 ) , phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , MEKK2 , NEDD4L , and actin in Nedd4l + / + and Nedd4l - / - primary lung epithelial cells ( C ) , or Nedd4l + / + and Nedd4l + / - astrocytes ( D ) , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . ( E ) Immunoblot analysis of phosphorylated p65 subunit of NF - κB ( P - P65 ) , total P65 , phosphorylated p38 ( P - P38 ) , total P38 , MEKK2 , NEDD4L , and actin in HeLa cells transfected with Myc - tagged NEDD4L ， Myc - tagged NEDD4L - CA ( C942A ) , or Myc - tagged Null plasmids , treated with IL - 17 for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . ( F ) Immunoblot analysis of MEKK2 , TRAF2 , TRAF6 , NEDD4L , and actin in Nedd4l + / + , Nedd4l + / - , and Nedd4l - / - MEFs . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . ( G ) Immunoblot analysis of MEKK2 in lysates of Nedd4l + / + and Nedd4l - / - MEFs treated with CHX ( 50 μg / ml ) for indicated time . ( H ) The intensity values were measured by Image J software to calculate half - life of MEKK protein ( n = 3 biological replicates ) . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Densitometry of MEKK2 was relative to actin . All experiments were performed three times , and representative data are shown . Significant differences were tested using a two - tailed Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 . NS , no significance .

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

Figure 3Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in HeLa cells transfected with Nedd4l siRNA (siNedd4l) or control RNA (siNC) (A) , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.Immunoblot analysis of phosphorylated ERK1/2 (P-ERK1/2), phosphorylated p38 (P-P38), phosphorylated JNK1/2 (P-JNK1/2), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, TRAF2, TRAF3, TRAF6, NEDD4L, and actin in Nedd4l+/+ and Nedd4l-/- MEFs , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.Immunoblot analysis of phosphorylated p38 (P-P38), phosphorylated p65 subunit of NF-κB (P-P65), total P65, MEKK2, NEDD4L, and actin in Nedd4l+/+ and Nedd4l-/- primary lung epithelial cells (C), or Nedd4l+/+ and Nedd4l+/- astrocytes (D) , treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.(E) Immunoblot analysis of phosphorylated p65 subunit of NF-κB (P-P65), total P65, phosphorylated p38 (P-P38), total P38, MEKK2, NEDD4L, and actin in HeLa cells transfected with Myc-tagged NEDD4L，Myc- tagged NEDD4L-CA(C942A), or Myc- tagged Null plasmids, treated with IL-17 for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.(F)Immunoblot analysis of MEKK2, TRAF2, TRAF6, NEDD4L, and actin in Nedd4l+/+, Nedd4l+/-, and Nedd4l-/- MEFs. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown.(G)Immunoblot analysis of MEKK2 in lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with CHX (50 μg/ ml) for indicated time. (H)The intensity values were measured by Image J software to calculate half-life of MEKK protein ( n = 3 biological replicates). Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Densitometry of MEKK2 was relative to actin. All experiments were performed three times, and representative data are shown. Significant differences were tested using a two-tailed Student's t-test. *P<0.05, **P<0.01. NS, no significance.