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

G, 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.


( A ) miR - 155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB / c mice 6 weeks

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

(A) 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 .

O O O O O O O O O O O O O O O O O O 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) 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 .

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

(C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E 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.


( 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 .

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

(A-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 .

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G 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.


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 ) The colocalization of BCG with lysosome was detected by confocal microscopy . The percentage of co - localization of BCG with CD63 - positive ( B )

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

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

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.


( 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 ) .

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

(A-D) 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 )

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

(A-D) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E 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.


( 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 ) .

(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 ) .

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

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

(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.


( 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 .

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

(A) 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 .

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

(C) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 )

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

(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 )

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

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 ) .

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

(C 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( 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 .

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

(A) 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 .

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 )

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

(C-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-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).


( 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 .

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

(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 ) .

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

D) 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.


A . 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 .

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

A. Representative 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 .

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

B. 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 .

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

C. 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 )

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

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

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).


A . 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 .

A. 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 .

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

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

C. 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).


A . 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 ) .

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

A. Representative 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

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.


A . 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 .

A. 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

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

C. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. 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


A . 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 .

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

A. Representative 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 ) .

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

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

C. 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).


A . 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 .

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

A. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

C. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. 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.


A . 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 ) .

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

A. Representative 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 ) .

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

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

D. 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).


A - 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 .

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

A-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 .

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

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

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).


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

(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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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 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 .

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

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 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 .

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

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 .

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

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 .

(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 .

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

(F)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 O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G)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.


( A ) Immunoblot analysis of NEDD4L , MEKK2 , and MEKK3 immunoprecipitated with NEDD4L - specific antibody from lysates of HeLa cells pre - treated with 20μM MG - 132 for 6 h and then treated with IL - 17 for the indicated time . Data information : All experiments were performed three times , and representative data are shown .

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

(A)Immunoblot analysis of NEDD4L, MEKK2, and MEKK3 immunoprecipitated with NEDD4L-specific antibody from lysates of HeLa cells pre-treated with 20μM MG-132 for 6 h and then treated with IL-17 for the indicated time. Data information: All experiments were performed three times, and representative data are shown.


( B ) Immunoblot analysis of NEDD4L precipitated with GST - tagged Null or GST - tagged MEKK2 recombinant protein . Data information : All experiments were performed three times , and representative data are shown .

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

(B)Immunoblot analysis of NEDD4L precipitated with GST-tagged Null or GST-tagged MEKK2 recombinant protein. Data information: All experiments were performed three times, and representative data are shown.


( C ) Immunoblot of HA - tagged MEKK2 co - immunoprecipitated with anti - Myc antibody from lysates of HEK293T cells co - transfected with plasmids expressing HA - tagged MEKK2 and Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : All experiments were performed three times , and representative data are shown .

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

(C)Immunoblot of HA-tagged MEKK2 co-immunoprecipitated with anti-Myc antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.


( D ) Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l + / + , and Neddl4 - / - treated with DMSO , 20μM MG - 132 , or 0 . 2 μM bafilomycin A1 ( Baf A1 ) for 6 h . Densitometry of MEKK2 was relative to actin . Data information : All experiments were performed three times , and representative data are shown .

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

(D)Immunoblot analysis of MEKK2 and NEDD4L in Nedd4l+/+, and Neddl4- /- treated with DMSO, 20μM MG-132, or 0.2 μM bafilomycin A1 (Baf A1) for 6 h. Densitometry of MEKK2 was relative to actin. Data information: All experiments were performed three times, and representative data are shown.


( E ) Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro . Data information : All experiments were performed three times , and representative data are shown .

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

(E)Immunoblot analysis of MEKK2 incubated with NEDD4L or TRAF6 recombinant protein and the full complement of ubiquitination reaction components in vitro. Data information: All experiments were performed three times, and representative data are shown.


( F ) Immunoblot analysis of K48 - , K63 - linked , and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2 - specific antibody from lysates of Nedd4l + / + and Nedd4l - / - MEFs treated with MG - 132 . Data information : All experiments were performed three times , and representative data are shown .

(F)Immunoblot analysis of K48-, K63-linked, and total ubiquitination of MEKK2 following precipitation of MEKK2 with MEKK2-specific antibody from lysates of Nedd4l+/+ and Nedd4l-/- MEFs treated with MG-132. Data information: All experiments were performed three times, and representative data are shown.


( G ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following precipitation of MEKK2 with Flag - tagged specific antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 and Myc - tagged wild - type NEDD4L or mutant NEDD4L with C2 ( ∆ C2 ) , WW ( ∆ WW ) or HECT ( ∆ HECT ) domain deleted . Data information : All experiments were performed three times , and representative data are shown .

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

(G)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following precipitation of MEKK2 with Flag-tagged specific antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2 and Myc-tagged wild-type NEDD4L or mutant NEDD4L with C2 (∆C2), WW (∆WW) or HECT (∆HECT) domain deleted. Data information: All experiments were performed three times, and representative data are shown.


( H ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , Myc - tagged NEDD4L , and HA - tagged wild - type or K6O , K11O , K27O , K29O , K33O , K48O , K63O mutant Ub . ( I ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , Myc - tagged NEDD4L , and HA - tagged wild - type or K6R , K11R , K27R , K29R , K33R , K48R , K63R mutant Ub . Data information : All experiments were performed three times , and representative data are shown .

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

(H)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6O, K11O, K27O, K29O, K33O, K48O, K63O mutant Ub. (I)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, Myc-tagged NEDD4L, and HA-tagged wild-type or K6R, K11R, K27R, K29R, K33R, K48R, K63R mutant Ub. Data information: All experiments were performed three times, and representative data are shown.


( J ) Immunoblot analysis of total ubiquitination of Flag - tagged MEKK2 following immunoprecipitating MEKK2 with anti - Flag antibody from lysates of HEK293T cells co - transfected with plasmids expressing Flag - tagged MEKK2 , increased dosage of Myc - tagged NEDD4L , and HA - tagged wild - type , K27O , or K63O mutant Ub . Data information : All experiments were performed three times , and representative data are shown .

(J)Immunoblot analysis of total ubiquitination of Flag-tagged MEKK2 following immunoprecipitating MEKK2 with anti-Flag antibody from lysates of HEK293T cells co-transfected with plasmids expressing Flag-tagged MEKK2, increased dosage of Myc-tagged NEDD4L, and HA-tagged wild-type, K27O, or K63O mutant Ub. Data information: All experiments were performed three times, and representative data are shown.


( A , Immunoblot analysis of phosphorylated p38 ( p - p38 ) , phosphorylated NF - κB p65 ( P - P65 ) , MEKK2 and actin in HeLa cells ( A ) transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 ( HeLa cells 50 ng / ml , for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

(A, Immunoblot analysis of phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in HeLa cells (A) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


B ) Immunoblot analysis of phosphorylated p38 ( p - p38 ) , phosphorylated NF - κB p65 ( P - P65 ) , MEKK2 and actin in MEFs ( B ) transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 MEFs 100ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

B) Immunoblot analysis of phosphorylated p38 (p-p38), phosphorylated NF-κB p65 (P-P65), MEKK2 and actin in MEFs (B) transfected with Mekk2 siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 MEFs 100ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


( C , Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells ( C ) transfected with siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 ( HeLa cells 50 ng / ml , for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

(C, Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (C) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 (HeLa cells 50 ng/ml, for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


D ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in MEFs ( D ) transfected with siRNA ( siMekk2 ) or control RNA ( siNC ) and stimulated with IL - 17 MEFs 100ng / ml ) for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

D) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in MEFs (D) transfected with siRNA (siMekk2) or control RNA (siNC) and stimulated with IL-17 MEFs 100ng/ml) for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


( E ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( P - P65 ) , NEDD4L , MEKK2 and actin in HeLa cells transfected with control RNA ( siNC ) , Nedd4l siRNA ( siNedd4l ) , Mekk2 siRNA ( siMekk2 ) or Nedd4l siRNA plus Mekk2 siRNA ( siNedd4l + siMekk2 ) , and stimulated with IL - 17 ( 50 ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

(E) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), NEDD4L, MEKK2 and actin in HeLa cells transfected with control RNA (siNC), Nedd4l siRNA (siNedd4l), Mekk2 siRNA(siMekk2) or Nedd4l siRNA plus Mekk2 siRNA (siNedd4l+ siMekk2), and stimulated with IL-17(50 ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


( F ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( p - p65 ) , NEDD4L , MEKK2 and actin in Nedd4l + / + and Nedd4l - / - MEFs transfected with Mekk2 siRNA ( siMekk2 ) or control RNA ( siNc ) , and stimulated with IL - 17 ( 100 ng / ml ) for indicated time . Data information : Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . All experiments were performed two times , and representative data are shown .

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

(F) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (p-p65), NEDD4L, MEKK2 and actin in Nedd4l+/+ and Nedd4l-/-MEFs transfected with Mekk2 siRNA (siMekk2) or control RNA (siNc), and stimulated with IL-17 (100 ng/ml) for indicated time. Data information: Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. All experiments were performed two times, and representative data are shown.


( G , Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells ( G ) treated as described in ( E ) ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

(G, Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells (G) treated as described in (E) (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


H ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in MEFs ( H ) treated as described in ( F ) ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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

H) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in MEFs (H) treated as described in (F) (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


( A ) Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL - 17 ( 50 ng / ml ) for indicated time , and then incubated with or without calf intestinal alkaline phosphatasetime ( CIAP ) for 30 mins . Data information : All experiments were performed two times , and representative data are shown .

(A)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells stimulated with IL-17 (50 ng/ml) for indicated time, and then incubated with or without calf intestinal alkaline phosphatasetime (CIAP) for 30 mins. Data information: All experiments were performed two times, and representative data are shown.


( B ) Realtime PCR analysis of Il - 6 , Cxcl2 , and Ccl20 mRNA expression in HeLa cells transfected with HA - tagged MEKK2 S520A or control vector , and stimulated with IL - 17 ( 50 ng / ml ) for indicated time ( n = 3 technical replicates ) . Data information : Data are shown as the mean ± s . e . m . of technical replicates . All experiments were performed two times , and representative data are shown .

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(B) Realtime PCR analysis of Il-6, Cxcl2, and Ccl20 mRNA expression in HeLa cells transfected with HA-tagged MEKK2 S520A or control vector, and stimulated with IL-17 (50 ng/ml) for indicated time (n = 3 technical replicates). Data information: Data are shown as the mean ± s.e.m. of technical replicates. All experiments were performed two times, and representative data are shown.


( C ) Immunoblot analysis of phosphorylated p38 , phosphorylated NF - κB p65 ( P - P65 ) , HA , and actin in HeLa cells treated as in ( B ) . Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins , respectively . Data information : All experiments were performed two times , and representative data are shown .

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(C) Immunoblot analysis of phosphorylated p38, phosphorylated NF-κB p65 (P-P65), HA, and actin in HeLa cells treated as in (B). Numbers between two blots indicate densitometry of phosphorylated proteins relative to that of total proteins, respectively. Data information: All experiments were performed two times, and representative data are shown.


( D ) Immunoblot analysis of MEKK2 and TAK1 co - immunoprecipitated with anti - TAK1 antibody from lysates of HeLa cells treated with 50ng / ml IL - 17 for indicated time . Data information : All experiments were performed two times , and representative data are shown .

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(D)Immunoblot analysis of MEKK2 and TAK1 co-immunoprecipitated with anti-TAK1 antibody from lysates of HeLa cells treated with 50ng/ml IL-17 for indicated time. Data information: All experiments were performed two times, and representative data are shown.


( E ) Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1 - specific ( siTak1 ) or control RNA ( siNC ) , and then stimulated with IL - 17 ( 50 ng / ml ) for indicated time . Data information : All experiments were performed two times , and representative data are shown .

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(E)Immunoblot analysis of Ser520 phosphorylation of MEKK2 immunoprecipitated from HeLa cells transfected with Tak1-specific (siTak1) or control RNA (siNC), and then stimulated with IL-17 (50 ng/ml) for indicated time. Data information: All experiments were performed two times, and representative data are shown.


( F ) Immunoblot analysis of Myc - tagged NEDD4L co - immunoprecipitated with anti - HA antibody from lysates of HEK293T cells co - transfected with plasmids expressing Myc - tagged NEDD4L and HA - tagged wild - type MEKK2 or mutant MEKK2 ( S520A ) . Data information : All experiments were performed two times , and representative data are shown .

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(F)Immunoblot analysis of Myc-tagged NEDD4L co-immunoprecipitated with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing Myc-tagged NEDD4L and HA-tagged wild-type MEKK2 or mutant MEKK2 (S520A). Data information: All experiments were performed two times, and representative data are shown.


( G ) Immunoblot analysis of total ubiquitination of HA - tagged MEKK2 and HA - tagged MEKK2 ( S520A ) following precipitation of MEKK2 with anti - HA antibody from lysates of HEK293T cells co - transfected with plasmids expressing HA - tagged MEKK2 or HA - tagged MEKK2 ( S520A ) together with Myc - tagged NEDD4L and Flag - tagged Ub . Data information : All experiments were performed two times , and representative data are shown .

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(G)Immunoblot analysis of total ubiquitination of HA-tagged MEKK2 and HA-tagged MEKK2(S520A) following precipitation of MEKK2 with anti-HA antibody from lysates of HEK293T cells co-transfected with plasmids expressing HA-tagged MEKK2 or HA-tagged MEKK2(S520A) together with Myc-tagged NEDD4L and Flag-tagged Ub. Data information: All experiments were performed two times, and representative data are shown.


( H ) Immunoblot analysis of HA - tagged MEKK2 , HA - tagged MEKK2 ( S520A ) , Myc - tagged NEDD4L , and actin in HEK293T cells transfected with the indicated plasmids with anti - HA , anti - Myc , or anti - actin antibody . Data information : All experiments were performed two times , and representative data are shown .

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(H)Immunoblot analysis of HA-tagged MEKK2, HA-tagged MEKK2 (S520A), Myc-tagged NEDD4L, and actin in HEK293T cells transfected with the indicated plasmids with anti-HA, anti-Myc, or anti-actin antibody. Data information: All experiments were performed two times, and representative data are shown.


B , phase contrast images of the morphological changes in MDR cells , residual MDR cells and regrown cells for Du145TXR or MCF - 7ADR . Red circles mark magnified areas ( bottom ) . Scale bars , 50 μm ( low magnification images ) .

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B, phase contrast images of the morphological changes in MDR cells, residual MDR cells and regrown cells for Du145TXR or MCF-7ADR. Red circles mark magnified areas (bottom). Scale bars, 50 μm (low magnification images).


C - D , cell viability of residual MDR cells ( RMCs ) and MDR cells ( MCs ) of ( C ) Du145TXR or ( D ) MCF - 7ADR cells using the same combination treatment for 24 hours on the indicated days . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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C-D, cell viability of residual MDR cells (RMCs) and MDR cells (MCs) of (C) Du145TXR or (D) MCF-7ADR cells using the same combination treatment for 24 hours on the indicated days. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


F - G , drug tolerant rate of ( F ) Du145 / Du145TXR ( 20 nM taxol / 20 nM taxol plus 50 μM verapamil ) or ( G ) MCF - 7 / MCF - 7ADR ( 200 nM adriamycin / 200 nM adriamycin plus 50 μM verapamil ) cells treated with drugs on the indicated days . Drug tolerant rate defined by comparing cell viability followed treatment , RMCs VS . MCs or CCs VS . PeCs , > 1 representing drug tolerant . Induction Time indicates the time of entering DTP state ; Sustaining Time indicates the time of maintaining DTP state . Blue colors represent control groups ; Red colors represent MDR cancer cells . Mean with ± SD .

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F-G, drug tolerant rate of (F) Du145/Du145TXR (20 nM taxol/20 nM taxol plus 50 μM verapamil) or (G) MCF-7/MCF-7ADR (200 nM adriamycin/200 nM adriamycin plus 50 μM verapamil) cells treated with drugs on the indicated days. Drug tolerant rate defined by comparing cell viability followed treatment, RMCs VS. MCs or CCs VS. PeCs, > 1 representing drug tolerant. Induction Time indicates the time of entering DTP state; Sustaining Time indicates the time of maintaining DTP state. Blue colors represent control groups; Red colors represent MDR cancer cells. Mean with ± SD.


D , heat map based on scoring rank indicated consistently expressed genes identified by RNA - seq of MCs VS . MPCs in Du145TXR and MCF - 7ADR .

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D, heat map based on scoring rank indicated consistently expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR and MCF-7ADR.


F , immunoblotting of NPC1L1 in MCs , MPCs and RCs of Du145TXR or MCF - 7ADR . G , immunoblotting of NPC1L1 in Du145TXR or MCF - 7ADR cells transfected with siNPC1L1 or siScramble .

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F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR. G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.


H - I , colony formation assay and quantification of ( H ) Du145TXR or ( I ) MCF - 7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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H-I, colony formation assay and quantification of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


A - C , flow cytometric analysis of ROS levels in Du145TXR or MCF - 7ADR cells treated with indicated agents for 12 and 36 hours . ( A ) Representative images and quantification of ROS in ( B ) Du145TXR or ( C ) MCF - 7ADR cells are shown . One - way ANOVA was used to analyze statistical differences . Mean with ± SD Data information : Results are representative of three independent experiments .

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A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments.


D - E , cell viability of ( D ) Du145TXR or ( E ) MCF - 7ADR treated with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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D-E, cell viability of (D) Du145TXR or (E) MCF-7ADR treated with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


J - K , cell viability of ( J ) Du145TXR or ( K ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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J-K, cell viability of (J) Du145TXR or (K) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments.


C - D , endocytosis analysis of ( C ) Du145TXR or ( D ) MCF - 7ADR cells treated with indicated agents for 8 hours , followed by staining with 250 μg / mL FITC - DEX for 4 hours . Scale bars , 10 μm . Data information : Results are representative of three independent experiments .

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C-D, endocytosis analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments.


E - F , phase contrast images of the morphological changes in ( E ) Du145TXR and ( F ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours . Scale bars , 10 or 25 μm . The red arrows indicate vacuoles . Data information : Results are representative of three independent experiments .

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E-F, phase contrast images of the morphological changes in (E) Du145TXR and (F) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours. Scale bars, 10 or 25 μm. The red arrows indicate vacuoles. Data information: Results are representative of three independent experiments.


I - J , colony formation assay and quantification of ( I ) Du145TXR or ( J ) MCF - 7ADR cells treated with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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I-J, colony formation assay and quantification of (I) Du145TXR or (J) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


C , immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours . Data information : Results are representative of three independent experiments .

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C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments.


F - G , relative luciferase activities of wild - type ( WT ) or mutant ( MUT ) NPC1L1 promoter ( - 205 ~ - 215 , # 1 ) reporter constructs in ( F ) Du145TXR or ( G ) MCF - 7ADR cells treated with indicated agents . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


J - K , colony formation assay and quantification of ( J ) Du145TXR or ( K ) MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


immunohistochemical staining of Ki67 in indicated groups . ( D ) Representative images are shown . Scale bar , 50 μm . , immunohistochemical staining of cleaved - caspase 3 ( CC3 ) in indicated groups . ( F ) Representative images are shown . Scale bar , 50 μm .

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immunohistochemical staining of Ki67 in indicated groups. (D) Representative images are shown. Scale bar, 50 μm. , immunohistochemical staining of cleaved-caspase 3 (CC3) in indicated groups. (F) Representative images are shown. Scale bar, 50 μm.


( I ) Corresponding images and isolated tumors are shown . The red dotted line represents cured tumors .

(I) Corresponding images and isolated tumors are shown. The red dotted line represents cured tumors.


A Lysates of HeLa cells stably expressing GFP alone or GFP - FXR1 were subjected to immunoprecipitation using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( shown a mean value , * P < 0 . 05 , * * P < 0 . 01 ; N = 3 ) .

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A Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (shown a mean value, *P < 0.05, **P < 0.01; N = 3).


B Lysates of HeLa cells stably expressing GFP alone or 3xGFP - Nup85 were immunoprecipitated using GFP - Trap beads ( GFP - IP ) , analysed by Western blot and quantified ( SE , short exposure , LE , long exposure ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) .

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B Lysates of HeLa cells stably expressing GFP alone or 3xGFP-Nup85 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (SE, short exposure, LE, long exposure) (shown a mean value, *P < 0.05; N = 3).


C Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot . The arrow points to the heavy chain of IgG ( IgG HC ) ( shown a mean value , * P < 0 . 05 ; N = 3 ) .

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C Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot. The arrow points to the heavy chain of IgG (IgG HC) (shown a mean value, *P < 0.05; N = 3).


D HeLa cells were treated with indicated siRNAs , synchronized by double thymidine block , and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor ( LBR ) to label the NE , and FXR1 .

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D HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1.


E HeLa cells stably expressing GFP - FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414 , which labels FG - Nups . The magnified framed regions are shown in the corresponding numbered panels . The arrowheads indicate NE and cytoplasmic localization of GFP - FXR1 .

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E HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1.