f5 ( a ) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE - 125132 . Scale bar , 15 μm . ( b ) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE - 125132 . ( c ) IL - 8 enzyme - linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . ( d ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 or WYE - 125132 . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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f5(a) immunofluorescence microscopy images of U2OS cells stained for LAMP1 and the Golgi marker RCAS1 after 24 h glutamine deprivation with or without treatment with 1 μM BrefA or 100 nM WYE-125132. Scale bar, 15 μm.(b) Cytokine array analysis of conditioned medium from U2OS cells subjected to glutamine deprivation in the presence of 1 μM BrefA or 100 nM WYE-125132.(c) IL-8 enzyme-linked immunosorbent assay in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132.(d) RT-PCR analysis of IL-8 mRNA expression in U2OS cells after 24 h glutamine deprivation with or without CCI-779 or WYE-125132. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


f6 ( a ) Immunoblot analysis of autophagy , ER stress - and mTOR - related proteins , and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI - 779 . ( b ) RT - PCR analysis of IL - 8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK ( JNK # 1 and JNK # 2 ) and subjected to 24 h glutamine deprivation . ( c ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) of conditioned media from the experiment described in b . ( d ) Immunoblot analysis of JNK protein expression in JNK siRNA - treated cells . e ) RT - PCR analysis of IL - 8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 ( JNKi ) . ( f ) IL - 8 ELISA of conditioned medium from the experiment described in e . ( g ) IL - 8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 ( IRE - A and IRE - B ) and subjected to glutamine deprivation for 24 h . ( h ) RT - PCR analysis of IL - 8 mRNA expression from experiment described in g . ( i ) RT - PCR analysis of CHOP mRNA expression from experiment described in g . ( j ) RT - PCR analysis of IRE1 mRNA expression from experiment described in g . ( k ) Immunoblot analysis of experiment described in g showing phosphorylation of JNK . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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

f6(a) Immunoblot analysis of autophagy, ER stress- and mTOR-related proteins, and JNK in U2OS cells after 24 h glutamine deprivation with or without CCI-779.(b) RT-PCR analysis of IL-8 mRNA expression in U2OS cells transfected with two different siRNAs targeting JNK (JNK #1 and JNK #2) and subjected to 24 h glutamine deprivation.(c) IL-8 enzyme-linked immunosorbent assay (ELISA) of conditioned media from the experiment described in b.(d) Immunoblot analysis of JNK protein expression in JNK siRNA-treated cells.e) RT-PCR analysis of IL-8 expression in U2OS cells after 24 h glutamine deprivation with or without the JNK inhibitor SP600125 (JNKi).(f) IL-8 ELISA of conditioned medium from the experiment described in e.(g) IL-8 ELISA of U2OS cells transfected with two different siRNAs targeting IRE1 (IRE-A and IRE-B) and subjected to glutamine deprivation for 24 h.(h) RT-PCR analysis of IL-8 mRNA expression from experiment described in g. (i) RT-PCR analysis of CHOP mRNA expression from experiment described in g. (j) RT-PCR analysis of IRE1 mRNA expression from experiment described in g.(k) Immunoblot analysis of experiment described in g showing phosphorylation of JNK. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


f7 ( a ) IL - 8 enzyme - linked immunosorbent assay ( ELISA ) on U2OS cells starved of glutamine or treated with BPTES ( 10 μM ) for 24 h . ( b ) RT - PCR analysis of IL - 8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG . ( c ) Metabolite analysis of U2OS cells starved of glutamine , treated with BPTES or glutamine - starved co - treated with DMαKG . ( d ) IL - 8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co - treated with DMαKG . ( e ) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co - treated with DMαKG . Error bars in all figures represent s . d . of three biological replicates . OD , optical density .

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f7(a) IL-8 enzyme-linked immunosorbent assay (ELISA) on U2OS cells starved of glutamine or treated with BPTES (10 μM) for 24 h.(b) RT-PCR analysis of IL-8 and CHOP mRNA expression in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG.(c) Metabolite analysis of U2OS cells starved of glutamine, treated with BPTES or glutamine-starved co-treated with DMαKG.(d) IL-8 ELISA of U2OS cells starved of glutamine or treated with BPTES for 24 h and co-treated with DMαKG.(e) Immunoblot analysis of pS6K and pJNK in U2OS cells starved of glutamine for 24 h and co-treated with DMαKG. Error bars in all figures represent s.d. of three biological replicates. OD, optical density.


Figure 1 ( E ) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay . Interaction between bls - CRBN and FLAG - GST - protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology . ( F ) In vitro binding assay for thalidomide , pomalidomide and lenalidomide . Interaction between bls - CRBN and FLAG - GST - PLZF in the presence of DMSO ( 3 . 125 , 6 . 25 , 12 . 5 , 25 , 50 , 100 or 200 µM ) thalidomide , pomalidomide or lenalidomide was analysed using the AlphaScreen technology . Data information : All relative AlphaScreen ( AS ) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) .

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

Figure 1(E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology.(F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).


Figure 2 ( E ) In vitro binding and ubiquitination assay of AGIA - PLZF . Empty vector , AGIA - PLZF or FLAG - CRBN expressing HEK293T cells were lysed and the lysates were mixed . The first immunoprecipitation with anti - AGIA or anti - FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide . The purified AGIA - PLZF or CRL4FLAG - CRBN complex , including AGIA - PLZF and FLAG - CRBN , was incubated with recombinant E1 , E2 and HA - ubiquitin in the presence of DMSO or 200 µM lenalidomide . The second immunoprecipitation was performed using anti - AGIA antibody . Ubiquitination of PLZF was analysed by immunoblot .

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Figure 2(E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.


Figure 3 ( B ) In vitro binding assay using truncated PLZF . Thalidomide - dependent interaction between bls - CRBN and FLAG - GST - PLZF - full length ( FL ) or truncated FLAG - GST - PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology . Data information : All relative AlphaScreen ( AS ) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * * * * P < 0 .

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Figure 3(B) In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 µM thalidomide using the AlphaScreen technology. Data information: All relative AlphaScreen (AS) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, ****P < 0.0001


Figure 4 ( D ) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . ( E ) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP - 1 cells treated with DMSO , thalidomide or 5 - hydroxythalidomide for 24 h . Data information : Data in bar graphs in ( D ) and ( E ) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100 . Error bars donated ± standard deviation ( n = 3 ) and P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ) .

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Figure 4(D) Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. (E) Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide or 5-hydroxythalidomide for 24 h. Data information: Data in bar graphs in (D) and (E) were calculated as relative PLZF band intensity with PLZF band intensity of DMSO as 100. Error bars donated ± standard deviation (n = 3) and P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01 and ***P < 0.001).


Figure 5 ( A , B ) Immunoblot analysis of AGIA - MmPLZF ( A ) or - MmSALL4 ( B ) in FLAG - MmCRBN - WT , - MmCRBN - I391V or - MmCRBN - V380E / I391V expressing CRBN - / - HEK293T cells treated with DMSO or thalidomide ( Tha ) for 16 h . Data information : Data in bar graphs were calculated as relative Mm ) PLZF or SALL4 band intensity with Mm ) PLZF or SALL4 , respectively . The band intensity of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ) . ( C , D ) Immunoblot analysis of AGIA - GgPLZF ( C ) or - GgSALL4 ( D ) in FLAG - GgCRBN - WT or - GgCRBN - I390V expressing CRBN - / - HEK293T cells treated with DMSO or thalidomide ( Tha ) for 16 h . ( E , F ) Immunoblot analysis of AGIA - Mm PLZF ( E ) or - MmSALL4 ( F ) in FLAG - MmCRBN - WT expressing CRBN - / - HEK293T cells treated with indicated concentration of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . ( G , H ) Immunoblot analysis of AGIA - GgPLZF ( G ) or - GgSALL4 ( H ) in FLAG - GgCRBN - WT expressing CRBN - / - HEK293T cells treated with indicated concentration of DMSO , thalidomide ( Tha ) or 5 - hydroxythalidomide ( 5 - HT ) for 16 h . Data information : Data in bar graphs were calculated as relative ( Gg or Mm ) PLZF or SALL4 band intensity with ( Gg or Mm ) PLZF or SALL4 , respectively . The band intensity of DMSO was 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ) .

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

Figure 5(A, B) Immunoblot analysis of AGIA-MmPLZF (A) or -MmSALL4 (B) in FLAG-MmCRBN-WT, -MmCRBN-I391V or -MmCRBN-V380E/I391V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. Data information: Data in bar graphs were calculated as relative Mm) PLZF or SALL4 band intensity with Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).(C, D) Immunoblot analysis of AGIA-GgPLZF (C) or -GgSALL4 (D) in FLAG-GgCRBN-WT or -GgCRBN-I390V expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) for 16 h. (E, F) Immunoblot analysis of AGIA-Mm PLZF (E) or -MmSALL4 (F) in FLAG-MmCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. (G, H) Immunoblot analysis of AGIA-GgPLZF (G) or -GgSALL4 (H) in FLAG-GgCRBN-WT expressing CRBN-/- HEK293T cells treated with indicated concentration of DMSO, thalidomide (Tha) or 5-hydroxythalidomide (5-HT) for 16 h. Data information: Data in bar graphs were calculated as relative (Gg or Mm) PLZF or SALL4 band intensity with (Gg or Mm) PLZF or SALL4, respectively. The band intensity of DMSO was 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).


Figure 6 ( E ) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl ( n = 10 ) or shPLZF ( n = 9 ) were analysed by Victoria blue staining . h ; humerus , r ; radius , u ; ulna , fe ; femur , fi ; fibula , t ; tibia . Data in bar graphs were calculated as relative bone length with bone length of shControl as 100 . Error bars denote ± standard deviation ( forelimb and hindlimb shControl ( n = 3 ) , forelimb shPLZF ( n = 5 ) or hindlimb shPLZF ( n = 6 ) ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) .

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Figure 6(E) Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl (n = 3), forelimb shPLZF (n = 5) or hindlimb shPLZF (n = 6)). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01 and ****P < 0.0001).


Figure 7 ( F ) Photographs show E4 chicken embryos treated with HBC ( control , n = 10 , C1 , C3 , C5 and C6 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 , T1 , T2 , T6 and T9 ) or 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 , H1 , H2 , H8 and H9 ) corresponding to immunoblot analysis in Fig 7G . Red arrows show treated regions . Data information Data in bar graphs in ( F ) were calculated as relative limb bud length with left limb bud length as one . Error bars denote ± standard deviation ( F ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * * P < 0 . 01 , and * * * P < 0 . 001 ) . ( G ) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud . Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC ( n = 10 ) , 1 µg / µl ( 3 . 9 mM ) thalidomide ( n = 11 ) and 1 µg / µl ( 3 . 6 mM ) 5 - hydroxythalidomide ( 5 - HT ) ( n = 10 ) . Data information : Data in bar graphs in ( G ) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4 , respectively . The band intensity of HBC was 100 . Error bars denote ± standard deviation ( ( E ) , n = 3 or ( G ) , n = 4 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( NS = Not Significant , * P < 0 . 05 , and * * P < 0 . 01 ) .

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Figure 7(F) Photographs show E4 chicken embryos treated with HBC (control, n = 10, C1, C3, C5 and C6), 1 µg/µl (3.9 mM) thalidomide (n = 11, T1, T2, T6 and T9) or 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n = 10, H1, H2, H8 and H9) corresponding to immunoblot analysis in Fig 7G. Red arrows show treated regions. Data information Data in bar graphs in (F) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation (F), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001).(G) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC (n =10), 1 µg/µl (3.9 mM) thalidomide (n =11) and 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n =10). Data information: Data in bar graphs in (G) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 or (G), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01).


Figure 8E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS - GgPLZF was treated with HBC or 1 µg / µl ( 3 . 9 mM ) thalidomide , and the chicken forelimb bud phenotypes were observed in bright field ( BF ) or fluorescence of enhanced GFP ( EGFP ) . Fgf10 / Fgf8 expression was analysed by section by in situ hybridisation . ( Q ) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in ( Q ) were calculated as relative limb bud length with limb bud length of control embryo as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * * P < 0 . 01 ) . ( R ) Quantitative analysis of Fgf10 / Fgf8 expression Data in bar graphs in ( R ) were calculated as relative Fgf10 / Fgf8 mRNA expressions with Fgf10 / Fgf8 mRNA expressions of control embryo as 100 . Error bars denote ± standard deviation ( n = 3 ) . P - values were calculated by one - way ANOVA with Tukey ' s post - hoc test ( * P < 0 . 05 and * * P < 0 . 01 ) .

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Figure 8E3 chicken forelimb bud electroporated with empty pCAGGS or pCAGGS-GgPLZF was treated with HBC or 1 µg/µl (3.9 mM) thalidomide, and the chicken forelimb bud phenotypes were observed in bright field (BF) or fluorescence of enhanced GFP (EGFP). Fgf10/Fgf8 expression was analysed by section by in situ hybridisation. (Q) Quantitative analysis of phenotypes of chicken limb bud Data in bar graphs in (Q) were calculated as relative limb bud length with limb bud length of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01).(R) Quantitative analysis of Fgf10/Fgf8 expression Data in bar graphs in (R) were calculated as relative Fgf10/Fgf8 mRNA expressions with Fgf10/Fgf8 mRNA expressions of control embryo as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (*P < 0.05 and **P < 0.01).


Figure 1A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells . MIC10 KO cells show a drastic reduction of MIC13 , MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged . MIC60 KO cells show a drastic reduction of protein levels of all MICOS components . B Representative electron micrographs of WT , MIC10 KO and MIC60 KO HAP1 cells . Scale bar 500 nm . C Quantification of CJs per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) . D Quantification of cristae per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) . E Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 034 for basal ( using one sample t - test ) and * P = 0 . 018 for maximum respiration ( using unpaired Student ' s t - test ) . F Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 01 for basal ( using one sample t - test ) , * * * P = 0 . 0004 for maximum respiration and * P = 0 . 012 for non - mitochondrial respiration ( using unpaired Student ' s t - test ) .

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Figure 1A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction of MIC13, MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction of protein levels of all MICOS components.B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm.C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test).F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).


Figure 2A Representative STED super - resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies ( top panel ) and TOMM70 ( middle panel ) . Bottom panel shows merged images . Arrowheads show colocalization of MIC60 and TOMM70 punctae . Scale bar 500 nm . B Quantification of median interpunctae distance ( IPD ) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Median IPD from n = 10 - 16 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . ns , not significant , unpaired Student ' s t - test .

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Figure 2A Representative STED super-resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies (top panel) and TOMM70 (middle panel). Bottom panel shows merged images. Arrowheads show colocalization of MIC60 and TOMM70 punctae. Scale bar 500 nm.B Quantification of median interpunctae distance (IPD) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Median IPD from n = 10-16 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ns, not significant, unpaired Student's t-test.


Figure 3FRAP curves ( curve fitted ) of WT Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown . C FRAP curves ( curve fitted ) of MIC13 KO Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown . D Histogram showing average percentage of mobile fractions , obtained from curve fits of all mitochondria expressing GFP - tagged versions of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) in WT and MIC13 KO Hela cells ( data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments , 6 - 10 mitochondria from each experiment ) .

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Figure 3FRAP curves (curve fitted) of WT Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.C FRAP curves (curve fitted) of MIC13 KO Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP-tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO Hela cells (data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments, 6-10 mitochondria from each experiment).


Figure 4A - D Representative single particle tracks of cells expressing MIC60 - ( A ) and MIC10 - SNAP ( B ) in WT Hela cells and MIC60 - ( C ) and MIC10 - SNAP ( D ) in MIC13 KO Hela cells , stained with silicone - rhodamine dye , and imaged at a rate of 33 ms / frame . Single tracks were colour - coded according to temporal appearance . Scale Bar 5 µm . Insets in each image show zoomed - in images of few representative tracks . E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients ( insD ) in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP stained with silicone - rhodamine . Number of tracks analysed ( obtained from 2 independent experiments ) : n = 2541 , 2540 , 3560 and 1441 in WT Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , MIC13 KO Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , respectively . ( * * * * P ≤ 0 . 0001 for all possible comparisons , unpaired Student ' s t - test ) . F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10 - SNAP . Number of corresponding subtracks analysed ( obtained from 2 independent experiments ) : n = 2561 , 2443 , 3402 , 1360 , 561 and 1110 . Data are mean ± SEM . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired Student ' s t - test .

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Figure 4A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks.E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test).F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10-SNAP. Number of corresponding subtracks analysed (obtained from 2 independent experiments): n = 2561, 2443, 3402, 1360, 561 and 1110. Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student's t-test.


Figure 5A , C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT ( A ) and MIC13 KO ( C ) Hela cells expressing MIC10 - SNAP stained with silicone - rhodamine . Box in ( A and C ) mark selection shown as a zoom in panel ( B and D ) respectively . Scale bar 500 nm . E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10 - SNAP ( 3 independent experiments , 5 - 8 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * P = 0 . 026 ( merging and splitting events in WT cells ) and * * * * P < 0 . 0001 ( merging and splitting events of WT vs . MIC13 KO ) , unpaired Student ' s t - test .

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Figure 5A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm.E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10-SNAP (3 independent experiments, 5-8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. *P = 0.026 (merging and splitting events in WT cells) and ****P < 0.0001 (merging and splitting events of WT vs. MIC13 KO), unpaired Student's t-test.


Figure 6A Representative live - cell STED super - resolution images ( t = 0 s ) , showing WT Hela cells expressing MIC13 - SNAP , from a time - series of images acquired at a time interval of 2 . 5 s stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( B ) . Scale bar 500 nm . C Representative live - cell STED super - resolution images ( t = 0 s ) , showing WT Hela cells expressing MIC13 - SNAP , from a time - series of images acquired at a time interval of 1 . 3 s stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( D ) . Scale bar 500 nm . D Time - lapse image series of a mitochondrion expressing MIC13 - SNAP imaged at a time interval of 1 . 3 s / frame . Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13 - SNAP . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm . E First image from ( D ) was used for showing the intensity profiles along the length of three cristae ( numbered 1 - 3 and marked with white arrows ) in ( F ) . F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM . Magenta boxes depict intensity maxima at IBM .

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Figure 6A Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 2.5 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm.C Representative live-cell STED super-resolution images (t=0 s), showing WT Hela cells expressing MIC13-SNAP, from a time-series of images acquired at a time interval of 1.3 s stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing MIC13-SNAP imaged at a time interval of 1.3 s/ frame. Green and magenta asterisks show cycles of cristae merging and splitting marked by MIC13-SNAP. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E First image from (D) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities at the edges of the line scans representing the IBM. Magenta boxes depict intensity maxima at IBM.


Figure 7A Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells expressing ATP5I - SNAP stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( B ) . Scale bar 500 nm . B Time - lapse image series of a mitochondrion expressing ATP5I - SNAP in WT Hela cells ( 2 . 5 s / frame ) . Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Green ` Y ´ represent Y - type imminent mergence event . Scale bar 500 nm . C Representative live - cell STED super - resolution images ( t = 0 s ) showing MIC13 KO Hela cells expressing ATP5I - SNAP stained with silicone - rhodamine . Box marks selection shown as a zoom in panel ( D ) . Scale bar 500 nm . D Time - lapse image series of a mitochondrion expressing ATP5I - SNAP in MIC13 KO Hela cells ( 2 . 5 s / frame ) . Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I - SNAP respectively . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm . E First image from ( B ) was used for showing the intensity profiles along the length of three cristae ( numbered 1 - 3 and marked with white arrows ) in ( F ) . F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM . The represented IBM area is superimposed from intensities of MIC13 - SNAP from Figure 6F . Magenta box depicts intensity maxima at cristae membrane apart from IBM . G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I - SNAP ( 3 independent experiments , 3 - 7 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * * * * P < 0 . 0001 , unpaired Student ' s t - test .

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Figure 7A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (B). Scale bar 500 nm. B Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in WT Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Green `Y´ represent Y- type imminent mergence event . Scale bar 500 nm. C Representative live-cell STED super-resolution images (t=0 s) showing MIC13 KO Hela cells expressing ATP5I-SNAP stained with silicone-rhodamine. Box marks selection shown as a zoom in panel (D). Scale bar 500 nm. D Time-lapse image series of a mitochondrion expressing ATP5I-SNAP in MIC13 KO Hela cells (2.5 s/frame). Green and magenta asterisks show merging and splitting events of cristae marked by ATP5I-SNAP respectively. Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E First image from (B) was used for showing the intensity profiles along the length of three cristae (numbered 1-3 and marked with white arrows) in (F). F Intensity profiles of lines drawn across the length of cristae show increased intensities in the middle of the line scans representing the CM. The represented IBM area is superimposed from intensities of MIC13-SNAP from Figure 6F. Magenta box depicts intensity maxima at cristae membrane apart from IBM. G Blind quantification of cristae merging and splitting events in WT and MIC13 KO Hela cells expressing ATP5I-SNAP (3 independent experiments, 3-7 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. ****P < 0.0001, unpaired Student's t-test.


Figure 8A Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells stained with TMRM . Box marks selection shown as a zoom in panel ( B ) ( red hot LUT ) . Scale bar 500 nm . B Time - lapse image series of two mitochondria stained with TMRM ( 1 . 5 s / frame ) . Green asterisks show merging events and redistribution of TMRM intensity from a high - intensity cristae to a low - intensity cristae ( two such instances are shown at 3 and 7 . 4 s ) . Green arrows pointing inward connected by solid line at 1 . 5 and 5 . 9 s indicate imminent merging events . Scale bar 500 nm . C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT Hela cells stained with TMRM . Box marks selection shown as a zoom in panel ( D ) ( red hot LUT ) . Scale bar 500 nm . D Time - lapse image series of two mitochondria stained with TMRM ( 1 . 5 s / frame ) . Green and magenta asterisks show cristae merging and splitting cycles ( shown at 5 . 9 s and 7 . 4 s ) . Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm . E Representative time - lapse images of WT Hela cells stained with NAO , and imaged using Airyscan microscope , indicate cristae merging and splitting dynamics . Yellow and magenta asterisks show merging and splitting events of cristae respectively . Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events , respectively . Scale bar 500 nm .

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Figure 8A Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (B) (red hot LUT). Scale bar 500 nm. B Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green asterisks show merging events and redistribution of TMRM intensity from a high-intensity cristae to a low-intensity cristae (two such instances are shown at 3 and 7.4 s). Green arrows pointing inward connected by solid line at 1.5 and 5.9 s indicate imminent merging events. Scale bar 500 nm. C Representative live-cell STED super-resolution images (t=0 s) showing WT Hela cells stained with TMRM. Box marks selection shown as a zoom in panel (D) (red hot LUT). Scale bar 500 nm. D Time-lapse image series of two mitochondria stained with TMRM (1.5 s/frame). Green and magenta asterisks show cristae merging and splitting cycles (shown at 5.9 s and 7.4 s). Green arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm. E Representative time-lapse images of WT Hela cells stained with NAO, and imaged using Airyscan microscope, indicate cristae merging and splitting dynamics. Yellow and magenta asterisks show merging and splitting events of cristae respectively. Yellow arrows pointing inward connected by solid line and magenta arrows pointing outward connected by dotted line show sites of imminent merging and splitting events, respectively. Scale bar 500 nm.


Figure 9A Mitochondrion in WT Hela cells expressing ATP5I - PAGFP was photoactivated , in the area shown by a blue box , at 0 s . Inset of white box at 0 . 98 s clearly shows fluorescent crista ( red arrow ) , absent at 0 . 38 s , 0 . 59 s & 0 . 78 s ( red arrowhead showing the area enclosed by white dotted lines ) , confirming merging of non - fluorescent cristae vesicle with the IBM . Threshold images of insets are shown above the whole images . Scale bar 500 nm . B Dual - colour STED super - resolution images of fused mitochondrion in WT Hela cells ( Left Panel ) showing colocalization of cristae marked with ATP5I - GFP ( using anti - GFP antibody ) and ATP5I - SNAP ( stained with silicone - rhodamine ) . ( Right Panel ) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I - GFP ( using anti - GFP antibody ) or ATP5I - SNAP ( stained with silicone - rhodamine ) . Arrowheads show merged cristae while arrows show cristae that maintain their individual identity . Scale bar 500 nm .

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Figure 9A Mitochondrion in WT Hela cells expressing ATP5I-PAGFP was photoactivated, in the area shown by a blue box, at 0 s. Inset of white box at 0.98 s clearly shows fluorescent crista (red arrow), absent at 0.38 s, 0.59 s & 0.78 s (red arrowhead showing the area enclosed by white dotted lines), confirming merging of non-fluorescent cristae vesicle with the IBM. Threshold images of insets are shown above the whole images. Scale bar 500 nm.B Dual-colour STED super-resolution images of fused mitochondrion in WT Hela cells (Left Panel) showing colocalization of cristae marked with ATP5I-GFP (using anti-GFP antibody) and ATP5I-SNAP (stained with silicone-rhodamine). (Right Panel) Fused mitochondrion in MIC13 KO Hela cells showing cristae marked with either ATP5I-GFP (using anti-GFP antibody) or ATP5I-SNAP (stained with silicone-rhodamine). Arrowheads show merged cristae while arrows show cristae that maintain their individual identity. Scale bar 500 nm.


Figure 3 ( Left panel ) A10 cells were transfected with Flag - FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . ( Bottom right panel ) A line was drawn through a representative cell to indicate relative intensity of RGB signals . ( Top right panel ) An orthogonal projection was generated by a Z - stack ( 240 nm interval ) image set using the ZEN program ( Zeiss ) depicting the region surrounding the nucleus in the XY , XZ , and YZ planesB . ( i ) HEK 293T cells were transfected with Flag - FMRP as in A , and endogenous β - catenin protein complexes were immunoprecipitated with β - catenin antibody . Co - immunoprecipitated Flag - FMRP protein was detected by Western blot analysis with the Flag antibody . Non - programmed rabbit IgG was used as a control . ( ii ) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β - catenin or FMRP antibody . The corresponding protein was detected in the precipitated immunocomplex by Western blot . Non - programmed rabbit IgG was used as a control ( i ) Purified 6x - His - FMRP and various regions of β - catenin proteins fused to GST were incubated with glutathione - agarose beads . Purified GST or GST - fusion proteins were visualized by Western blotting using the GST antibody , and sizes of corresponding GST - fusion proteins were confirmed . Pulled - down FMRP proteins by GST fused β - catenin were detected by immunoblotting with the FMRP antibody . ( ii ) Interaction between FMRP and β - catenin was assessed in the presence or absence of RNA . HEK 293T cells were subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody , in the presence or absence of RNase A ( 10 µg / mL ) . The corresponding protein was detected in the precipitated immunocomplex by Western blot , and non - programmed rabbit IgG was used as a control . RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A ( Top panel ) A10 cells were transfected with GBP - Lifeact and either EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red ) ( Top panel ) A10 cells were transfected wit EYFP - β - catenin , Flag - FMRP , or mCherry - β - catenin . The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag ( red ) , as indicated . ( Bottom panel ) A10 cells were transfected with GBP - Lifeact similar to above , but with both EYFP - β - catenin and Flag - FMRP , and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag ( red ) E . A10 cells were transfected as in ( D ) with GBP - Lamin

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

Figure 3(Left panel) A10 cells were transfected with Flag-FMRP for 3 hr then cultured for 24 hr prior to fixation and staining for β-catenin (green), Flag (FMRP, red), and nucleus (blue). (Bottom right panel) A line was drawn through a representative cell to indicate relative intensity of RGB signals. (Top right panel) An orthogonal projection was generated by a Z-stack (240 nm interval) image set using the ZEN program (Zeiss) depicting the region surrounding the nucleus in the XY, XZ, and YZ planesB. (i) HEK 293T cells were transfected with Flag-FMRP as in A, and endogenous β-catenin protein complexes were immunoprecipitated with β-catenin antibody. Co-immunoprecipitated Flag-FMRP protein was detected by Western blot analysis with the Flag antibody. Non-programmed rabbit IgG was used as a control. (ii) Primary VSMCs were harvested and subjected to an endogenous coIP analysis with either β-catenin or FMRP antibody. The corresponding protein was detected in the precipitated immunocomplex by Western blot. Non-programmed rabbit IgG was used as a control(i) Purified 6x-His-FMRP and various regions of β-catenin proteins fused to GST were incubated with glutathione-agarose beads. Purified GST or GST-fusion proteins were visualized by Western blotting using the GST antibody, and sizes of corresponding GST-fusion proteins were confirmed. Pulled-down FMRP proteins by GST fused β-catenin were detected by immunoblotting with the FMRP antibody. (ii) Interaction between FMRP and β-catenin was assessed in the presence or absence of RNA. HEK 293T cells were subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody, in the presence or absence of RNase A (10 µg/mL). The corresponding protein was detected in the precipitated immunocomplex by Western blot, and non-programmed rabbit IgG was used as a control. RNA was extracted from parallel cultured cells and total RNA content was analyzed by agarose gel electrophoresis in the presence or absence of RNase A(Top panel) A10 cells were transfected with GBP-Lifeact and either EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red)(Top panel) A10 cells were transfected wit EYFP-β-catenin, Flag-FMRP, or mCherry-β-catenin. The cells were fixed and subjected to either fluorescence microscopy or immunofluorescent staining for Flag (red), as indicated. (Bottom panel) A10 cells were transfected with GBP-Lifeact similar to above, but with both EYFP-β-catenin and Flag-FMRP, and the cells were subjected to fluorescent microscopy and immunofluorescent staining for Flag (red) E. A10 cells were transfected as in (D) with GBP-Lamin B1


Figure 4A . Co - sedimentation of protein / RNA complexes on a 10 - 50 % sucrose gradient . Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients ; peaks corresponding to the 40S and 60S subunits , 80S monosome , and polysomes were detected by UV absorbance at 254 nm , and indicated proteins in these fractions were detected by Western blot . Corresponding total cell lysate was used as input ( i ) Precipitated proteins in A10 cell lysates with m7GTP - agarose beads were identified by Western blot analysis . eIF4E and tubulin were shown as positive and negative controls , respectively . Total lysates were used as input . ( ii ) A10 cell lysates were incubated with GTP - agarose beads , and none of the proteins tested were precipitated with the beads . Total lysates were used as input control ( i ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to m7GTP - agarose pull - down as in ( C ) ( ii ) HEK 293T cells were transfected with empty vector or Flag - FMRP , and lysates were subjected to GTP - agarose pull - down as in ( i ) . E . HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP - agarose pull - downs as in ( C ) F . HEK 293T cells lysates were subjected to m7GTP - agarose pull - downs as in ( C ) in the presence or absence of RNase A ( 10 µg / mL ) . RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis .

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Figure 4A. Co-sedimentation of protein/RNA complexes on a 10-50% sucrose gradient. Cell lysates generated from the cells transfected with either siRNA targeting fmr1 or its scrambled control were ultracentrifuged in sucrose gradients; peaks corresponding to the 40S and 60S subunits, 80S monosome, and polysomes were detected by UV absorbance at 254 nm, and indicated proteins in these fractions were detected by Western blot. Corresponding total cell lysate was used as input(i) Precipitated proteins in A10 cell lysates with m7GTP-agarose beads were identified by Western blot analysis. eIF4E and tubulin were shown as positive and negative controls, respectively. Total lysates were used as input. (ii) A10 cell lysates were incubated with GTP-agarose beads, and none of the proteins tested were precipitated with the beads. Total lysates were used as input control(i) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to m7GTP-agarose pull-down as in (C)(ii) HEK 293T cells were transfected with empty vector or Flag-FMRP, and lysates were subjected to GTP-agarose pull-down as in (i). E. HEK 293T cells were transfected with either siRNAs targeting fmr1 or scrambled control and were subjected to m7GTP-agarose pull-downs as in (C)F. HEK 293T cells lysates were subjected to m7GTP-agarose pull-downs as in (C) in the presence or absence of RNase A (10 µg/mL). RNA was extracted from parallel lysates and RNA content was analyzed by agarose gel electrophoresis.


Figure 5Primary VSMCs ( left panel ) , A10 ( middle panel ) , and HEK 293T cells ( right panel ) were transfected with siRNAs targeting ctnnb1 ( β - catenin ) or scrambled RNA . After 36 hr , the cells were pulsed with 0 . 5 µM puromycin for 15 min and then harvested . The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody . β - catenin blots indicated efficacy of siRNA and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation . A one - way ANOVA and Tukey post - hoc test was performed , with p - values indicated above the error barsA10 cells were transfected with Flag - FMRP then treated with either cycloheximide or its solvent ( DMSO ) for 4 hr prior to fixation . β - catenin ( green ) , Flag ( FMRP , red ) were visualized by immunofluorescence . Nucleus ( blue ) was identified by DNA staining with Hoechst 33342 . Arrows indicate a loss of β - catenin signal from the perinuclear region that was seen in the control following cycloheximide treatmentA10 cells were transfected with Flag - FMRP as above and then pre - treated with rapamycin or solvent ( DMSO ) for 1 hr in serum - free media . Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β - catenin antibody . Non - programmed rabbit IgG was used as a control . Existence of Flag - FMRP in the immunocomplex was detected by Flag antibody and β - catenin by GFP antibodyA10 cells were transfected with Flag - FMRP followed by rapamycin treatment similar to ( D ) . Cells were fixed and stained for β - catenin ( green ) , Flag ( FMRP , red ) , and nucleus ( blue ) . A loss of β - catenin signal from the perinuclear region that was seen in the control is indicated by the arrows .

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Figure 5Primary VSMCs (left panel), A10 (middle panel), and HEK 293T cells (right panel) were transfected with siRNAs targeting ctnnb1 (β-catenin) or scrambled RNA. After 36 hr, the cells were pulsed with 0.5 µM puromycin for 15 min and then harvested. The puromycin incorporated peptides were detected by Western blot analysis with puromycin antibody. β-catenin blots indicated efficacy of siRNA and actin was used as a loading control. The average puromycin/actin ratio (n=3) is shown in the graphs to the right of each cell type and the error bars indicate standard deviation. A one-way ANOVA and Tukey post-hoc test was performed, with p-values indicated above the error barsA10 cells were transfected with Flag-FMRP then treated with either cycloheximide or its solvent (DMSO) for 4 hr prior to fixation. β-catenin (green), Flag (FMRP, red) were visualized by immunofluorescence. Nucleus (blue) was identified by DNA staining with Hoechst 33342. Arrows indicate a loss of β-catenin signal from the perinuclear region that was seen in the control following cycloheximide treatmentA10 cells were transfected with Flag-FMRP as above and then pre-treated with rapamycin or solvent (DMSO) for 1 hr in serum-free media. Serum was added for 2 hr before the cells were harvested and subjected to coIP analysis using β-catenin antibody. Non-programmed rabbit IgG was used as a control. Existence of Flag-FMRP in the immunocomplex was detected by Flag antibody and β-catenin by GFP antibodyA10 cells were transfected with Flag-FMRP followed by rapamycin treatment similar to (D). Cells were fixed and stained for β-catenin (green), Flag (FMRP, red), and nucleus (blue). A loss of β-catenin signal from the perinuclear region that was seen in the control is indicated by the arrows.


Figure 6 ( Top panel ) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were treated with 0 . 5 µM puromycin for 15 min and harvested for Western blot analysis . Total β - catenin levels are indicated , and actin was used as a loading control . The average puromycin / actin ratio ( n = 3 ) was graphed . Error bars indicate standard deviation . An independent samples t - test was performed ( 2 - tailed ) , with the p - value indicated above the error bar . ( Bottom panel ) Cells were treated similarly to above and fixed and stained for β - catenin ( green ) and nucleus ( blue ) The experiment in ( A ) was repeated using A10 cells ( i ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . The cells were harvested and subjected to an endogenous coIP analysis using either β - catenin or FMRP antibody . The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis . Non - programed rabbit IgG was used as a control . Efficacy of Wnt - 3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β - catenin ( S33 / 37 / T41 ) by Wnt - 3a . ( ii ) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt - 3a ( 100 ng / mL ) or its solvent ( 0 . 1 % BSA in PBS ) for 4 hr . Cell lysates were then subjected to m7GTP - agarose pull - down analysis . eIF4E and tubulin were used as positive and negative controls , respectively , and phospho - β - catenin ( S33 / 37 / T41 ) Western blots were used as controls for Wnt - 3a stimulation .

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Figure 6(Top panel) Primary VSMCs were serum deprived overnight prior to stimulation with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were treated with 0.5 µM puromycin for 15 min and harvested for Western blot analysis. Total β-catenin levels are indicated, and actin was used as a loading control. The average puromycin/actin ratio (n=3) was graphed. Error bars indicate standard deviation. An independent samples t-test was performed (2-tailed), with the p-value indicated above the error bar. (Bottom panel) Cells were treated similarly to above and fixed and stained for β-catenin (green) and nucleus (blue) The experiment in (A) was repeated using A10 cells(i) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. The cells were harvested and subjected to an endogenous coIP analysis using either β-catenin or FMRP antibody. The corresponding proteins in the precipitated immunocomplex were detected by Western blot analysis. Non-programed rabbit IgG was used as a control. Efficacy of Wnt-3a treatment was assessed by inhibition of GSK3 mediated phosphorylation of β-catenin (S33/37/T41) by Wnt-3a. (ii) HEK 293T cells were serum deprived for 1 hr and then stimulated with Wnt-3a (100 ng/mL) or its solvent (0.1% BSA in PBS) for 4 hr. Cell lysates were then subjected to m7GTP-agarose pull-down analysis. eIF4E and tubulin were used as positive and negative controls, respectively, and phospho-β-catenin (S33/37/T41) Western blots were used as controls for Wnt-3a stimulation.


Figure 1Representative images of seed fluorescence segregation in 420 / + + in wild type ( Col ) and hcr2 . Scatterplots to the right show red ( dsRed ) and green ( eGFP ) fluorescence values in 420 / + + Col ( top ) and hcr2 ( bottom ) . Scale bars : 2 mm . End - point RT - PCR analysis of HSBP in Col , hsbp - 3 , hsbp - 2 and hsbp - 1 . Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp - 3 , respectively . Image J was used to measure relative PCR band intensity for hsbp - 3 ( 53 % ) , hsbp - 2 ( 9 % ) and hsbp - 1 ( 70 % ) . TUB2 was used as an internal control . As in E , but showing immunoblot analysis of HSBP . hsbp - 3 , hsbp - 2 and hsbp - 1 accumulate about 58 % , 17 % and 77 % of HSBP levels , respectively . Coomassie - stained membrane was used as a loading control .

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Figure 1Representative images of seed fluorescence segregation in 420/++ in wild type (Col) and hcr2. Scatterplots to the right show red (dsRed) and green (eGFP) fluorescence values in 420/++ Col (top) and hcr2 (bottom). Scale bars: 2 mm.End-point RT-PCR analysis of HSBP in Col, hsbp-3, hsbp-2 and hsbp-1. Hash and asterisk indicate aberrant long and short splicing variants of HSBP in hsbp-3, respectively. Image J was used to measure relative PCR band intensity for hsbp-3 (53%), hsbp-2 (9%) and hsbp-1 (70%). TUB2 was used as an internal control. As in E, but showing immunoblot analysis of HSBP. hsbp-3, hsbp-2 and hsbp-1 accumulate about 58%, 17% and 77% of HSBP levels, respectively. Coomassie-stained membrane was used as a loading control.


Figure 6Representative images of ASY1 ( green ) and RAD51 ( red ) immunostaining in Col and hsbp - 3 . Nuclei spreads were stained with DAPI ( blue ) . Scale bars : 10 μm . Quantification of RAD51 foci numbers per cell in Col ( blue ) and hsbp - 3 ( red ) . n = 20 cells of biological replicates . Representative images of MLH1 ( red ) immunostaining in Col , hsbp - 3 , hsbp - 2 , Col / Ler , and meiMIGS - HSBP Col / Ler . Nuclear DNA was stained with DAPI . Scale bar : 5 μm . Quantification of the number of MLH1 foci per cell shown in ( G ) . n ≥ 32 cells of biological replicates . Representative images of HEI10 ( red ) and ASY1 ( green ) immunostaining in Col and hsbp - 3 . Scale bar : 2 μm . Quantification of the number of HEI10 foci per cell shown in ( I ) . n ≥ 21 cells of biological replicates . Immunoblot analysis of HEI10 and HEI10 - Myc in Col , hsbp - 3 , HEI10 - myc and HEI10 - myc plants . Experiments were performed at least three times .

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Figure 6Representative images of ASY1 (green) and RAD51 (red) immunostaining in Col and hsbp-3. Nuclei spreads were stained with DAPI (blue). Scale bars: 10 μm. Quantification of RAD51 foci numbers per cell in Col (blue) and hsbp-3 (red). n = 20 cells of biological replicates.Representative images of MLH1 (red) immunostaining in Col, hsbp-3, hsbp-2, Col/Ler, and meiMIGS-HSBP Col/Ler. Nuclear DNA was stained with DAPI. Scale bar: 5 μm. Quantification of the number of MLH1 foci per cell shown in (G). n ≥ 32 cells of biological replicates.Representative images of HEI10 (red) and ASY1 (green) immunostaining in Col and hsbp-3. Scale bar: 2 μm.Quantification of the number of HEI10 foci per cell shown in (I). n ≥ 21 cells of biological replicates.Immunoblot analysis of HEI10 and HEI10-Myc in Col, hsbp-3, HEI10-myc and HEI10-myc plants. Experiments were performed at least three times.


Figure 2 ( A - D ) The serial dilutions of post - second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2 - Line19F - FFL virions . The serum samples were serially diluted , and the detection of antibodies were measured by optical density ( OD ) at 490 nm . End - point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control ( PBS immunized animal ) .

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Figure 2(A-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal).


Figure 3 ( A ) Lung RSV titers ( PFU / gram of lung tissue ) were determined in individual lungs ( n = 5 mice per group ) collected at 5 days post - RSV infection . Results were determined and presented as box and whisker plots , where boxes extends from 25th to 75th percentile , whiskers show minimum to maximum value and central band represents the median value for the group .

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Figure 3(A) Lung RSV titers (PFU/gram of lung tissue) were determined in individual lungs (n = 5 mice per group) collected at 5 days post-RSV infection. Results were determined and presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.


Figure 4 ( A ) Serum samples collected from individual mouse ( M 1 - 5 ) immunized with F 110 - 136 peptide were tested for antibody binding against F - p27 ( 110 - 136 ) peptide or F 1 - 34 peptide ( control ) in ELISA . ( B ) A549 cells were infected with RSV ( MOI = 0 . 1 ) for 16 h , and fixed . Cells were treated with mock control rabbit sera ( left panels ) , or rabbit anti - sera against F ( center panels ) , or against F - p27 ( 110 - 136 ) peptide ( right panels ) , followed by Alexa 594 conjugated anti - Rabbit IgG ( red ) . Nuclei are stained with DAPI ( blue ) . Scale bar = 10 µm . The number of cells positive for RSV - F ( middle panel ) and RSV - p27 ( right panel ) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the ' merge ' panel . The experiments were performed twice and variation between the two independent expeiremnts was < 6 % .

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Figure 4(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA.(B) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.


Figure 5Mice lungs were collected from PBS - ( mock ) vaccinated BALB / c mice at day 5 post - RSV infection , fixed , paraffin embedded , and slides were processed for imaging . Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera ( left panels ) or rabbit anti - sera against RSV F protein ( center panels ) , or against RSV F - p27 ( 110 - 136 ) peptide ( right panels ) . Scale bar = 100 µm . The percentage of cells positive for RSV - F ( middle panel ) and RSV - p27 ( right panel ) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown . The experiments were performed twice and variation between the two independent expeiremnts was < 9 % .

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Figure 5Mice lungs were collected from PBS-(mock) vaccinated BALB/c mice at day 5 post-RSV infection, fixed, paraffin embedded, and slides were processed for imaging. Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera (left panels) or rabbit anti-sera against RSV F protein (center panels), or against RSV F-p27 (110-136) peptide (right panels). Scale bar = 100 µm. The percentage of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown. The experiments were performed twice and variation between the two independent expeiremnts was <9%.


Figure 6 ( A ) Lung tissues from individual mice ( n = 5 ) at day 5 post - RSV challenge ( or uninfected control ) were used to evaluate CD4 / CD8 T cells separately for airway and distal lungs . Lung slides were fixed and stained with DAPI to visualize nuclei ( blue ) and either anti - CD4 ( green ) or anti - CD8 ( red ) T cells . The number of CD4 / CD8 T cells were determined in each lung airway and distal region . Individual CD4 + / CD8 + T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice . Results are presented as box and whisker plots , where boxes extends from 25th to 75th percentile , whiskers show minimum to maximum value and central band represents the median value for the group .

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Figure 6(A) Lung tissues from individual mice (n=5) at day 5 post-RSV challenge (or uninfected control) were used to evaluate CD4/CD8 T cells separately for airway and distal lungs. Lung slides were fixed and stained with DAPI to visualize nuclei (blue) and either anti-CD4 (green) or anti-CD8 (red) T cells. The number of CD4/CD8 T cells were determined in each lung airway and distal region. Individual CD4+/CD8+ T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.


Figure 1A Western blot analysis of YTHDC1 in HEK293T and MCF - 7 knockdown samples . B Volcano plot of genes with significant switching to lengthened ( red ) or shortened ( blue ) 3 ' UTRs according to test of linear trend alternative to independence in HEK293T ( left panel ) and MCF - 7 cells ( right panel ) . Embedded bar plots show the number of significant genes . The X axis shows the Pearson correlation r from the linear trend alternative test , and the y axis shows the FDR . E The qRT - PCR validation of APA switching . Eight genes with lengthened and shortened 3 ' UTRs in HEK293T and in MCF - 7 cells were chosen . PRPF4B , CTNNBIP1 , PHB and RCL1 were selected from HEK293T cells , and rest genes are MCF - 7 cells . KD ( Extended / Common ) / Ctr ( Extended / Common ) < 1 indicates a shorter 3 ' UTR in the knockdown sample , and vice versa . Data are presented as mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , the p values were obtained from unpaired two - tailed Student ' s t - test .

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Figure 1A Western blot analysis of YTHDC1 in HEK293T and MCF-7 knockdown samples.B Volcano plot of genes with significant switching to lengthened (red) or shortened (blue) 3' UTRs according to test of linear trend alternative to independence in HEK293T (left panel) and MCF-7 cells (right panel). Embedded bar plots show the number of significant genes. The X axis shows the Pearson correlation r from the linear trend alternative test, and the y axis shows the FDR.E The qRT-PCR validation of APA switching. Eight genes with lengthened and shortened 3' UTRs in HEK293T and in MCF-7 cells were chosen. PRPF4B, CTNNBIP1, PHB and RCL1 were selected from HEK293T cells, and rest genes are MCF-7 cells. KD (Extended/Common)/Ctr (Extended/Common) <1 indicates a shorter 3' UTR in the knockdown sample, and vice versa. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, the p values were obtained from unpaired two-tailed Student's t-test.


Figure 2A Higher proportion of target genes in the list of genes with shortened 3 ' UTRs after knockdown of YTHDC1 . The p values were obtained by Fisher ' s exact test . * p = 1 . 227 × 10 - 3 , * * p = 3 . 725 × 10 - 4 , NA : no significance . C Peak density distribution of YTHDC1 binding sites near different poly ( A ) sites . Genes with shortened 3 ' UTR shows a higher density of YTHDC1 binding peaks near proximal APA sites compared to background genes . The density of YTHDC1 binding site peak were calculated as the number of peak in a 10 - nt interval divided by the total number of mRNAs that contained this position . E Scatter plot of protein and mRNA ratios of dual luciferase report assay with λN - BoxB system . λN , YTHDC1 and λN - YTHDC1 were co - transfected with a bicistronic vector . Two batches of experiments with three biological repeats were performed . Both luciferase activity and mRNA levels were measured . To test the effect of YTHDC1 binding on the proximal APA site , a linear model with the co - transfected genes and batch as independent variables was fitted . Data are presented as mean ± SEM of three biological replicates . * * p < 0 . 01 with t - test from linear model . F Overexpression of YTHDC1 - WT and YTHDC1 - Mut ( W377A , W428A ) revealed by Western blot . G Scatter plot of qRT - PCR for APA validation . Mutation of the m6A binding site of YTHDC1 loses its inhibitory effect on proximal APA sites . Extended / Common ratios of ten genes were obtained by qRT - PCR in HEK293T cells overexpressing YTHDC1 - WT and YTHDC1 - Mut . The ratios were normalized to those of control cells . Data are presented as mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , ns : no significance . the p values were obtained from unpaired two - tailed Student ' s t - test .

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Figure 2A Higher proportion of target genes in the list of genes with shortened 3'UTRs after knockdown of YTHDC1. The p values were obtained by Fisher's exact test. *p=1.227×10-3, **p=3.725×10-4, NA: no significance.C Peak density distribution of YTHDC1 binding sites near different poly(A) sites. Genes with shortened 3' UTR shows a higher density of YTHDC1 binding peaks near proximal APA sites compared to background genes. The density of YTHDC1 binding site peak were calculated as the number of peak in a 10-nt interval divided by the total number of mRNAs that contained this position.E Scatter plot of protein and mRNA ratios of dual luciferase report assay with λN-BoxB system. λN, YTHDC1 and λN-YTHDC1 were co-transfected with a bicistronic vector. Two batches of experiments with three biological repeats were performed. Both luciferase activity and mRNA levels were measured. To test the effect of YTHDC1 binding on the proximal APA site, a linear model with the co-transfected genes and batch as independent variables was fitted. Data are presented as mean ± SEM of three biological replicates. ** p<0.01 with t-test from linear model.F Overexpression of YTHDC1-WT and YTHDC1-Mut (W377A, W428A) revealed by Western blot.G Scatter plot of qRT-PCR for APA validation. Mutation of the m6A binding site of YTHDC1 loses its inhibitory effect on proximal APA sites. Extended/Common ratios of ten genes were obtained by qRT-PCR in HEK293T cells overexpressing YTHDC1-WT and YTHDC1-Mut. The ratios were normalized to those of control cells. Data are presented as mean ± SEM of three biological replicates. *p<0.05, **p<0.01, ns: no significance. the p values were obtained from unpaired two-tailed Student's t-test.


Figure 3A - C The Co - IP results of endogenous WTAP , METTL3 and METTL14 with YTHDC1 . The endogenous WTAP and YTHDC1 can be pulled down reciprocally by each other in the presence of RNase A . D The Co - IP results of Myc - CBLL1 , Myc - RBM15 , Myc - ZC3H13 and Myc - VIRMA with Endogenous YTHDC1 . None of them can pull down Endogenous YTHDC1 in the presence of RNase A . E . Endogenous YTHDC1 interacts with RPB3 in a RNA independent way .

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Figure 3A-C The Co-IP results of endogenous WTAP, METTL3 and METTL14 with YTHDC1. The endogenous WTAP and YTHDC1 can be pulled down reciprocally by each other in the presence of RNase A.D The Co-IP results of Myc-CBLL1, Myc-RBM15, Myc-ZC3H13 and Myc-VIRMA with Endogenous YTHDC1. None of them can pull down Endogenous YTHDC1 in the presence of RNase A.E. Endogenous YTHDC1 interacts with RPB3 in a RNA independent way.


Figure 4A Co - IP assay using Myc - tagged 3 ' end processing factors to pull down endogenous YTHDC1 without the addition of RNase A . FIP1L1 , PABPC1 and PABPC4 can pull down endogenous YTHDC1 . B Co - IP assay using Myc - FIP1L1 , Myc - PABPC1 and Myc - PABPC4 to pull down endogenous YTHDC1 in the presence of RNase A . C Co - IP assay with antibodies against endogenous YTHDC1 and FIP1L1 . D Coomassie brilliant blue staining of purified different epitope - tagged YTHDC1 and FIP1L1 . E An in vitro pull - down assay using purified FLAG - YTHDC1 and Myc - FIP1L1 showed that FLAG - YTHDC1 directly interacts with Myc - FIP1L1 . F Endogenous YTHDC1 and FIP1L1 were stained with antibodies labeled with Alexa Fluor 488 and Alexa Fluor 568 fluorophores , respectively . Immunofluorescence results show that YTHDC1 and FIP1L1 have a high spatial association in the nucleus . Scale bars 15µm . G Scan graph analysis and nuclear staining density quantification of YTHDC1 and FIP1L1 , indicating high colocalization in the nucleus . H Forty cells were randomly selected , and colocalization was assessed by calculating the average overlap coefficient , Pearson ' s correlation and colocalization rate according to Leica TCS SP8 microscope software . Data are presented as mean ± SD of 40 biological replicates .

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Figure 4A Co-IP assay using Myc-tagged 3' end processing factors to pull down endogenous YTHDC1 without the addition of RNase A. FIP1L1, PABPC1 and PABPC4 can pull down endogenous YTHDC1.B Co-IP assay using Myc-FIP1L1, Myc-PABPC1 and Myc-PABPC4 to pull down endogenous YTHDC1 in the presence of RNase A.C Co-IP assay with antibodies against endogenous YTHDC1 and FIP1L1.D Coomassie brilliant blue staining of purified different epitope-tagged YTHDC1 and FIP1L1.E An in vitro pull-down assay using purified FLAG-YTHDC1 and Myc-FIP1L1 showed that FLAG-YTHDC1 directly interacts with Myc-FIP1L1.F Endogenous YTHDC1 and FIP1L1 were stained with antibodies labeled with Alexa Fluor 488 and Alexa Fluor 568 fluorophores, respectively. Immunofluorescence results show that YTHDC1 and FIP1L1 have a high spatial association in the nucleus. Scale bars 15µm.G Scan graph analysis and nuclear staining density quantification of YTHDC1 and FIP1L1, indicating high colocalization in the nucleus. H Forty cells were randomly selected, and colocalization was assessed by calculating the average overlap coefficient, Pearson's correlation and colocalization rate according to Leica TCS SP8 microscope software. Data are presented as mean ± SD of 40 biological replicates.


Figure 5B Co - IP assays using truncated FIP1L1 proteins revealed that the proline - rich domain of FIP1L1 plays an important role in interacting with YTHDC1 . C Mutation of FIP1L1 nearly abrogates the interaction between YTHDC1 and FIP1L1 compared with wild type . D - G Co - IP assays using different domains of YTHDC1 revealed that the N - terminus ( 1 - 344 aa ) and YTH domain ( 364 - 507 aa ) of YTHDC1 interact with FIP1L1 .

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Figure 5B Co-IP assays using truncated FIP1L1 proteins revealed that the proline-rich domain of FIP1L1 plays an important role in interacting with YTHDC1.C Mutation of FIP1L1 nearly abrogates the interaction between YTHDC1 and FIP1L1 compared with wild type.D-G Co-IP assays using different domains of YTHDC1 revealed that the N-terminus (1-344 aa) and YTH domain (364-507 aa) of YTHDC1 interact with FIP1L1.