Mammary gland whole ‐ mounts from control MLN4924 and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice at indicated ages . The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads ; premalignant lesions are marked by red arrowheads . Scale bar , 4 mm . Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice , as shown in ( B ) . Error bars represent SEM ( n = 6 ) . * P 0 . 05 ( Student ' s unpaired t ‐ test ) .

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

Mammary gland whole‐mounts from control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice at indicated ages. The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads; premalignant lesions are marked by red arrowheads. Scale bar, 4 mm. Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice, as shown in (B). Error bars represent SEM (n = 6). *P 0.05 (Student's unpaired t‐test).


Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice at hyperplasia , adenoma , and carcinoma stages . Scale bar , 100 μm . n = 8 .

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

Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice at hyperplasia, adenoma, and carcinoma stages. Scale bar, 100 μm. n = 8.


Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors ( top ) and metastases ( bottom ) from MLN4924 ; Shp2fl / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice . Scale bar , 100 μm . n = 8 .

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

Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors (top) and metastases (bottom) from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice. Scale bar, 100 μm. n = 8.


qRT - PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924 ; Shp2fl / fl ( n = 8 ) , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ( n = 6 ) , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ( n = 6 ) mice . Error bars represent upper and lower quartiles .

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

qRT-PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924;Shp2fl/fl (n = 8), MLN4924;MMTV‐Cre;Shp2+/fl (n = 6), and MLN4924;MMTV‐Cre;Shp2fl/fl (n = 6) mice. Error bars represent upper and lower quartiles.


Western blot analysis of Shp2protein levels in tumor tissues from MLN4924 ; Shp2fl / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice .

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

Western blot analysis of Shp2protein levels in tumor tissues from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice.


Immunohistochemistry analysis of EYFP ( top ) and SA ‐ β ‐ gal staining ( bottom ) on cryosections of mammary glandtumors from control MLN4924 ; Shp2fl / fl ; EYFP − / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . Note that SA ‐ β ‐ gal activity was present in the EYFP ‐ positive ( Shp2 ‐ negative ) areas ( right , arrows ) and was absent in the EYFP ‐ negative ( Shp2 ‐ positive ) areas ( right , arrowhead ) of mammary glandtumors of MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . Consecutive sections of EYFP and SA ‐ β ‐ gal staining for each group are shown . Scale bar : 100 μm . n = 5 .

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

Immunohistochemistry analysis of EYFP (top) and SA‐β‐gal staining (bottom) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Note that SA‐β‐gal activity was present in the EYFP‐positive (Shp2‐negative) areas (right, arrows) and was absent in the EYFP‐negative (Shp2‐positive) areas (right, arrowhead) of mammary glandtumors of MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Consecutive sections of EYFP and SA‐β‐gal staining for each group are shown. Scale bar: 100 μm. n = 5.


Immunofluorescence analysis of Ki67 , EYFP and CK8 ( pan ‐ epithelial marker ) ( top panel ) , p27 and EYFP ( middle panel ) , and p53 and EYFP ( bottom panel ) on cryosections of mammary glandtumors from control MLN4924 ; Shp2fl / fl ; EYFP − / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . DAPI was used for nuclear counterstain . Ki67 was absent in EYFP ‐ positive cells ( top right , arrows ) and was present in EYFP ‐ negative cells ( top right , arrowheads ) ; p27 was present in EYFP ‐ positive cells ( middle right , arrows ) and was absent in EYFP ‐ negative cells ( middle right , arrowhead ) ; and p53 was present in EYFP ‐ positive cells ( bottom right , arrows ) and was absent in EYFP ‐ negative cells ( bottom right , arrowhead ) . Scale bar : top panel , 100 μm ; middle and bottom panel , 50 μm . n = 5 .

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

Immunofluorescence analysis of Ki67, EYFP and CK8 (pan‐epithelial marker) (top panel), p27 and EYFP (middle panel), and p53 and EYFP (bottom panel) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. DAPI was used for nuclear counterstain. Ki67 was absent in EYFP‐positive cells (top right, arrows) and was present in EYFP‐negative cells (top right, arrowheads); p27 was present in EYFP‐positive cells (middle right, arrows) and was absent in EYFP‐negative cells (middle right, arrowhead); and p53 was present in EYFP‐positive cells (bottom right, arrows) and was absent in EYFP‐negative cells (bottom right, arrowhead). Scale bar: top panel, 100 μm; middle and bottom panel, 50 μm. n = 5.


qRT - PCR analysis of mRNA levels of Shp2 , Skp2 , Aurka , Dll1 , and Hey1 in EYFP + and EYFP − cells isolated by FACS from MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / fltumors . Error bars represent SEM ( n = 5 ) .

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

qRT-PCR analysis of mRNA levels of Shp2, Skp2, Aurka, Dll1, and Hey1 in EYFP+ and EYFP−cells isolated by FACS from MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/fltumors. Error bars represent SEM (n = 5).


Kaplan - Meier analysis of tumor formation in control ( blue , n = 8 ) and GS493 ‐ pretreated ( red , n = 8 ) MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice . The bar indicates the duration of treatment .

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

Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) MLN4924;MMTV‐Cre;Shp2fl/flmice. The bar indicates the duration of treatment.


Tumorgrowth curves for control ( blue , n = 5 ) and GS493 ‐ treated ( red , n = 5 ) MLN4924 mice . Note that after day 24 , all mice in the control group were sacrificed due to the tumor size . Error bars represent SEM ( n = 5 ) .

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

Tumorgrowth curves for control (blue, n = 5) and GS493‐treated (red, n = 5) MLN4924 mice. Note that after day 24, all mice in the control group were sacrificed due to the tumor size. Error bars represent SEM (n = 5).


( A ) SDS - PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT - B2 components , over - expressed in E . coli or insect cells , after the last purification step by SEC . IFT54 / 20 and IFT57 / 38 were purified as hetero - dimeric complexes whereas IFT80 , IFT172 and an N - terminal IFT172 ( 1 - 968 ) ( IFT172ΔC ) constructs were purified as single components . A cartoon representation of the predicted domain architecture of IFT - B2 components using HHPRED ( Söding et al , 2005 ) is shown at the center .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SDS-PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT-B2 components, over-expressed in E. coli or insect cells, after the last purification step by SEC. IFT54/20 and IFT57/38 were purified as hetero-dimeric complexes whereas IFT80, IFT172 and an N-terminal IFT172(1-968) (IFT172ΔC) constructs were purified as single components. A cartoon representation of the predicted domain architecture of IFT-B2 components using HHPRED (Söding et al, 2005) is shown at the center.


( B ) Pull - down analysis for the hexameric IFT - B2 complex . A mixture of IFT172ΔC , IFT80 , and the IFT57 / 38 complex ( ' input ' ) shows only weak non - specific binding to GSH - resin ( ' empty ' ) , but all proteins are pulled down by IFT54 / 20 - GST complex . Note that the IFT20 - GST and IFT38 proteins co - migrate on the gel and cannot be properly resolved . However , the presence of the IFT57 protein in the pull - down sample implies that its binding partner IFT38 is also there .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)Pull-down analysis for the hexameric IFT-B2 complex. A mixture of IFT172ΔC, IFT80, and the IFT57/38 complex ('input') shows only weak non-specific binding to GSH-resin ('empty'), but all proteins are pulled down by IFT54/20-GST complex. Note that the IFT20-GST and IFT38 proteins co-migrate on the gel and cannot be properly resolved. However, the presence of the IFT57 protein in the pull-down sample implies that its binding partner IFT38 is also there.


( C ) ( left ) SEC elution profiles of individual IFT - B2 components and of the assembled hexameric IFT - B2 complex . The elution peak of IFT - B2 is shifted compared to all of the individual components . Note that the peak heights were adjusted for better visualization of the result . ( right ) Coomassie stained SDS PAGE gel of the six main fractions of the IFT - B2 SEC elution peak demonstrating the presence of all six IFT - B2 subunits . Weak contaminating bands are marked with asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (left) SEC elution profiles of individual IFT-B2 components and of the assembled hexameric IFT-B2 complex. The elution peak of IFT-B2 is shifted compared to all of the individual components. Note that the peak heights were adjusted for better visualization of the result. (right) Coomassie stained SDS PAGE gel of the six main fractions of the IFT-B2 SEC elution peak demonstrating the presence of all six IFT-B2 subunits. Weak contaminating bands are marked with asterisks.


( A - C ) SEC elution profiles and corresponding SDS PAGE gels of IFT172ΔC , IFT57 / 38 , IFT80 , IFT54 / 20 as well as mixtures of IFT172ΔC / 57 / 38 , IFT80 / 57 / 38 and IFT172ΔC / 80 / 57 / 38 demonstrating that IFT57 / 38 associates with both IFT80 and IFT172ΔC . Only the central peak fraction from each SEC experiment is shown on the SDS PAGE gel . Note that the presence of both IFT172ΔC and IFT80 in a single peak in ( c ) ( peak 3 ) is due to the fact that these two proteins elute in the same position , and not because they physically interact ( see also Appendix Fig . S2B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC elution profiles and corresponding SDS PAGE gels of IFT172ΔC, IFT57/38, IFT80, IFT54/20 as well as mixtures of IFT172ΔC/57/38, IFT80/57/38 and IFT172ΔC/80/57/38 demonstrating that IFT57/38 associates with both IFT80 and IFT172ΔC. Only the central peak fraction from each SEC experiment is shown on the SDS PAGE gel. Note that the presence of both IFT172ΔC and IFT80 in a single peak in (c) (peak 3) is due to the fact that these two proteins elute in the same position, and not because they physically interact (see also Appendix Fig. S2B).


( D ) GSH resin pull - down of a mixture of IFT80 and IFT172ΔC ( input , lane 1 ) with GST - tagged CH - domains of IFT57 or IFT38 ( IFT57CH or IFT38CH ) or with the GST - tagged coiled - coil ( CC ) region of IFT57 mixed with His - tagged coiled - coil region of IFT38 . The SDS - PAGE gel shows interaction between IFT57CH - IFT172ΔC , IFT38CH - IFT80 and IFT57CC - IFT38CC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GSH resin pull-down of a mixture of IFT80 and IFT172ΔC (input, lane 1) with GST-tagged CH-domains of IFT57 or IFT38 (IFT57CH or IFT38CH) or with the GST-tagged coiled-coil (CC) region of IFT57 mixed with His-tagged coiled-coil region of IFT38. The SDS-PAGE gel shows interaction between IFT57CH-IFT172ΔC, IFT38CH-IFT80 and IFT57CC-IFT38CC.


( E ) Ni - NTA resin pull - down of untagged IFT54 / 20 with His - tagged IFT38 / 38 ( both subunits His - tagged ) . The two bands for IFT38 on the SDS PAGE gel correspond to His - tagged ( upper band ) and proteolytically cleaved untagged IFT38 ( lower band ) .

(E) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT38/38 (both subunits His-tagged). The two bands for IFT38 on the SDS PAGE gel correspond to His-tagged (upper band) and proteolytically cleaved untagged IFT38 (lower band).


( F ) Ni - NTA resin pull - down of untagged IFT54 / 20 with His - tagged IFT80 .

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

(F) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT80.


( A ) Ni2 + - NTA affinity pull - down of unpolymerised bovine αβ - tubulin with His - tagged IFT - B2 ( sub ) - complexes . A Coomassie stained SDS PAGE gel shows the IFT - B2 complexes used for pull - downs ( top ) and a western blot for anti - α - tubulin shows co - precipitated αβ - tubulin ( bottom ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with His-tagged IFT-B2 (sub)-complexes. A Coomassie stained SDS PAGE gel shows the IFT-B2 complexes used for pull-downs (top) and a western blot for anti-α-tubulin shows co-precipitated αβ-tubulin (bottom).


( F ) Ni2 + - NTA affinity pull - down of unpolymerised bovine αβ - tubulin with WT and mutant MmIFT54CH . ( top ) Coomassie stained SDS PAGE gel of input ( I ) and pulled down ( PD ) proteins and the corresponding western blot to visualize αβ - tubulin .

(F) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with WT and mutant MmIFT54CH. (top) Coomassie stained SDS PAGE gel of input (I) and pulled down (PD) proteins and the corresponding western blot to visualize αβ-tubulin.


( A ) Coomassie - stained SDS - PAGE gel showing GST - pulldown experiments between the IFT - B2 complex and an IFT88 / 52N subcomplex . The pre - assembled IFT - B2 complex ( lane 1 ) shows no non - specific interaction with empty GSH - beads ( lane 2 ) , but is efficiently pulled down by IFT88 / 52N - GST ( with an IFT52 ( 1 - 335 ) construct comprising the N - terminal domain as well as the IFT88 - binding region ) ( lane 3 ) . Both IFT88 and IFT52N are required for the interaction with IFT - B2 because neither the N - terminal IFT52 domain ( residues 1 - 275 ; lane 4 ) alone , nor an IFT88 / 52 complex lacking the N - terminal IFT52 domain ( with an IFT52 ( 281 - 335 ) construct containing only the IFT88 - binding region ) ( lane 5 ) show efficient pull - down of IFT - B2 . Contaminants from the purification of the complex shown in lane 5 are marked with asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie-stained SDS-PAGE gel showing GST-pulldown experiments between the IFT-B2 complex and an IFT88/52N subcomplex. The pre-assembled IFT-B2 complex (lane 1) shows no non-specific interaction with empty GSH-beads (lane 2), but is efficiently pulled down by IFT88/52N-GST (with an IFT52(1-335) construct comprising the N-terminal domain as well as the IFT88-binding region) (lane 3). Both IFT88 and IFT52N are required for the interaction with IFT-B2 because neither the N-terminal IFT52 domain (residues 1-275; lane 4) alone, nor an IFT88/52 complex lacking the N-terminal IFT52 domain (with an IFT52(281-335) construct containing only the IFT88-binding region) (lane 5) show efficient pull-down of IFT-B2. Contaminants from the purification of the complex shown in lane 5 are marked with asterisks.


( B ) Coomassie - stained SDS - PAGE gel showing a salt - stability test of the ( IFT88 / 52N ) - ( IFT - B2 ) interaction . Pre - assembled IFT - B2 complex was bound to IFT88 / 52N - GST immobilized on GSH - beads . The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl . After these washes the bound material was eluted and analyzed . IFT172ΔC is the only salt - labile component in this complex and is already washed off at a NaCl - concentration of 150 - 200 mM , whereas all the other IFT - B2 proteins remain associated even after washing with 1 M NaCl .

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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O

(B) Coomassie-stained SDS-PAGE gel showing a salt-stability test of the (IFT88/52N)-(IFT-B2) interaction. Pre-assembled IFT-B2 complex was bound to IFT88/52N-GST immobilized on GSH-beads. The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl. After these washes the bound material was eluted and analyzed. IFT172ΔC is the only salt-labile component in this complex and is already washed off at a NaCl-concentration of 150-200 mM, whereas all the other IFT-B2 proteins remain associated even after washing with 1 M NaCl.


( C ) Coomassie - stained SDS - PAGE gels showing GST - pulldown experiments between IFT88 / 52N - GST complex immobilized on GSH beads and various combinations of IFT - B2 proteins . The left gel shows the IFT - B2 input mixtures , the right gel shows the bound material after washing and elution from the beads . A pentameric IFT - B2 complex ( IFT80 / 57 / 54 / 38 / 20 ) is efficiently pulled down by IFT88 / 52N - GST , but not by empty beads ( compare lanes 1 and 2 ) . Omission of IFT54 / 20 ( lane 4 ) , or IFT80 ( lane 5 ) does not influence the binding of any of the other components , but a lack of IFT57 / 38 in the mixture abolishes detectable pull - downs of IFT54 / 20 and IFT80 ( lane 4 ) , indicating that IFT57 / 38 is the direct interaction partner of IFT88 / 52N . Indeed , IFT57 / 38 alone ( lane 7 ) , but not IFT54 / 20 ( lane 6 ) or IFT80 ( lane 8 ) , is sufficient to be pulled down in this assay . Contaminating bands from the IFT88 / 52N sample are marked with asterisks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie-stained SDS-PAGE gels showing GST-pulldown experiments between IFT88/52N-GST complex immobilized on GSH beads and various combinations of IFT-B2 proteins. The left gel shows the IFT-B2 input mixtures, the right gel shows the bound material after washing and elution from the beads. A pentameric IFT-B2 complex (IFT80/57/54/38/20) is efficiently pulled down by IFT88/52N-GST, but not by empty beads (compare lanes 1 and 2). Omission of IFT54/20 (lane 4), or IFT80 (lane 5) does not influence the binding of any of the other components, but a lack of IFT57/38 in the mixture abolishes detectable pull-downs of IFT54/20 and IFT80 (lane 4), indicating that IFT57/38 is the direct interaction partner of IFT88/52N. Indeed, IFT57/38 alone (lane 7), but not IFT54/20 (lane 6) or IFT80 (lane 8), is sufficient to be pulled down in this assay. Contaminating bands from the IFT88/52N sample are marked with asterisks.


( B ) Coomassie stained SDS PAGE gel of a pull - down of IFT - B2 using WT or mutant IFT88 / 521 - 335 - GST . Whereas single point mutations slightly reduced the amounts of pulled down IFT - B2 , the K130E / R204E double mutation reduced it to background levels ( compare lanes 2 and 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie stained SDS PAGE gel of a pull-down of IFT-B2 using WT or mutant IFT88/521-335-GST. Whereas single point mutations slightly reduced the amounts of pulled down IFT-B2, the K130E/R204E double mutation reduced it to background levels (compare lanes 2 and 6).


( A ) SEC - profiles of the IFT - B1 and IFT - B2 complexes as well as the 15 - subunit reconstituted IFT - B complex . IFT - B1 and IFT - B2 elute at similar volumes , but after mixing the resulting high - molecular weight complex is clearly shifted towards a smaller elution volume . The Coomassie - stained SDS - PAGE gel on the right shows the peak fractions of the individual runs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SEC-profiles of the IFT-B1 and IFT-B2 complexes as well as the 15-subunit reconstituted IFT-B complex. IFT-B1 and IFT-B2 elute at similar volumes, but after mixing the resulting high-molecular weight complex is clearly shifted towards a smaller elution volume. The Coomassie-stained SDS-PAGE gel on the right shows the peak fractions of the individual runs.


F . BAZ2A read coverage quantification at ± 1Kb from peak summit of BAZ2A - bound regions identified in PC3 cells and the corresponding reads for BAZ2A - BRD mutant ( Y1775F ) in PC3 cells . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Data information : Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BAZ2A read coverage quantification at ±1Kb from peak summit of BAZ2A-bound regions identified in PC3 cells and the corresponding reads for BAZ2A-BRD mutant (Y1775F) in PC3 cells. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


G . Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac . Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells ( BAZ2Awt and BAZ2A - BRD mutant ( Y1775F ) ) at ± 1Kb from BAZ2A peak summits . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Data information : Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Regions bound by BAZ2A in PC3 cells are enriched in H3K14ac. Read coverage quantification for H3K14ac levels at regions bound by BAZ2A in PC3 cells (BAZ2Awt and BAZ2A-BRD mutant (Y1775F)) at ±1Kb from BAZ2A peak summits. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Data information: Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


B . EP300 acetylates H3K14 in PC3 cells . Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A - 485 ( 10 μM ) . Histone H3 serves as protein loading control .

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B. EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A-485 (10 μM). Histone H3 serves as protein loading control.


F . qRT - PCR showing increased expression levels of genes with BAZ2A - bound promoter and downregulated in metastatic tumors ( AOX1 , MANB2 , DMPK , and DDB1 in PC3 cells treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 , * * * * < 0 . 0001 ) . Error bars represent SD .

F. qRT-PCR showing increased expression levels of genes with BAZ2A-bound promoter and downregulated in metastatic tumors (AOX1, MANB2, DMPK, and DDB1 in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, ****<0.0001). Error bars represent SD.


B . Inactive enhancers ( C2 ) are enriched in H3K14ac and BAZ2A levels . Read coverage at enhancer classes C1 and C2 for BAZ2A , H3K14ac , H3K27ac , H3K4me1 and H3K27me3 at ± 1Kb from annotated enhancer regions . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * * * < 0 . 001 ) . Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Inactive enhancers (C2) are enriched in H3K14ac and BAZ2A levels. Read coverage at enhancer classes C1 and C2 for BAZ2A, H3K14ac, H3K27ac, H3K4me1 and H3K27me3 at ±1Kb from annotated enhancer regions. Statistical significance (P-values) was calculated using two-tailed t-test (***< 0.001). Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range.


H . qRT - PCR showing increased expression levels of BAZ2A - bound C2 - enhancer genes in PC3 cells treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 , * * < 0 . 001 ) . Error bars represent SD .

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

H. qRT-PCR showing increased expression levels of BAZ2A-bound C2-enhancer genes in PC3 cells treated with siRNA-EP300. Data were from three independent experiments. Values were normalized to GAPDH mRNA. Statistical significance (P-values) was calculated using two-tailed t-test (*<0.05, **<0.001). Error bars represent SD.


C . qRT - PCR of genes differentially expressed in PC3 - CSCs compared to PC3 cells showing the upregulation of CSC markers ( ALDH1A2 , LGR5 , CD24 ) and downregulation of genes related to developmental processes ( HOXA1 , HOXA4 , SOX7 ) . Data are from two independent experiments . mRNA levels were normalized to GAPDH .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR of genes differentially expressed in PC3-CSCs compared to PC3 cells showing the upregulation of CSC markers (ALDH1A2, LGR5, CD24) and downregulation of genes related to developmental processes (HOXA1, HOXA4, SOX7). Data are from two independent experiments. mRNA levels were normalized to GAPDH.


D . BAZ2A is required for the dedifferentiation of PC3 into PC3 - CSCs . Left panel : Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A . Right panel : BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA - BAZ2A were measured by qRT - PCR and normalized to GAPDH mRNA from 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BAZ2A is required for the dedifferentiation of PC3 into PC3-CSCs. Left panel: Representative images from two independent experiments showing the impairment of PC3 cells to form tumorspheres upon stable depletion of BAZ2A. Right panel: BAZ2A mRNA levels in three independent PC3 cell lines stably expressing shRNA-BAZ2A were measured by qRT-PCR and normalized to GAPDH mRNA from 2 independent experiments.


E . Left panel : qRT - PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A - BRD mutant ( BAZ2AY1775F ) . Values were normalized to MRPS7 mRNA . Data are from 3 independent experiments . Right panel : Quantification of tumorspheres from 3 independent experiments . Statistical significance ( P - values ) was calculated using two - tailed t - test ( * < 0 . 05 ; * * < 0 . 01 ) . Error bars represent SD .

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

E. Left panel: qRT-PCR showing BAZ2A mRNA levels in PC3 cells depleted of endogenous BAZ2A with siRNA specifically targeting human BAZ2A sequences and expressing mouse BAZ2Awt and BAZ2A-BRD mutant (BAZ2AY1775F). Values were normalized to MRPS7 mRNA. Data are from 3 independent experiments. Right panel: Quantification of tumorspheres from 3 independent experiments. Statistical significance (P-values) was calculated using two-tailed t-test (* <0.05; **< 0.01). Error bars represent SD.


B . Cell proliferation curves of PC3 cells treated with siRNA - BAZ2A . Error bars represent the standard deviation from three independent experiments . C . Cell proliferation curves of PC3 cells treated with 5μM BAZ2 - ICR and GSK2801 . Error bars represent the standard deviation from three independent experiments .

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B. Cell proliferation curves of PC3 cells treated with siRNA-BAZ2A. Error bars represent the standard deviation from three independent experiments. C. Cell proliferation curves of PC3 cells treated with 5μM BAZ2-ICR and GSK2801. Error bars represent the standard deviation from three independent experiments.


D . Representative images of tumorspheres generated from PC3 cells treated with BAZ2 - ICR ( 1μM ) or GSK2801 ( 1μM ) .

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D. Representative images of tumorspheres generated from PC3 cells treated with BAZ2-ICR (1μM) or GSK2801 (1μM).


E . Half - maximal effective concentration ( EC50 ) of BAZ2 - ICR . Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2 - ICR concentration . The amount of tumorspheres was assessed by measurement of cell viability . Error bars represent SD . Values are from three independent experiments .

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E. Half-maximal effective concentration (EC50) of BAZ2-ICR. Measurements were performed 5 days after treatment of PC3 cells and initiation of tumorspheres using the indicated BAZ2-ICR concentration. The amount of tumorspheres was assessed by measurement of cell viability. Error bars represent SD. Values are from three independent experiments.


A . Representative bright field images of organoids derived from mouse prostate cells treated with DMSO , BAZ2 - ICR ( 5μM ) , GSK2801 ( 5μM ) or GSK126 ( 5μM ) inhibitors followed by transduction with shRNA - Control or shRNA - Pten .

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

A. Representative bright field images of organoids derived from mouse prostate cells treated with DMSO, BAZ2-ICR (5μM), GSK2801 (5μM) or GSK126 (5μM) inhibitors followed by transduction with shRNA-Control or shRNA-Pten.


C . Representative images of Ki67 - immunostained mouse prostate organoid sections expressing shRNA - control and shRNA - Pten and treated with DMSO or BAZ2 - ICR ( 5μM ) . D . Quantification of Ki67 + cells in mouse prostate organoids shown in C . * * < 0 . 01 , * * * * P < 0 . 0001 , two - tailed Student ' s t test ; ns , not significant . The median values are represented by a line .

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C. Representative images of Ki67-immunostained mouse prostate organoid sections expressing shRNA-control and shRNA-Pten and treated with DMSO or BAZ2-ICR (5μM). D. Quantification of Ki67+ cells in mouse prostate organoids shown in C. **< 0.01, ****P < 0.0001, two-tailed Student's t test; ns, not significant. The median values are represented by a line.


E . Quantification of mouse prostate organoids per field expressing shRNA - control and shRNA - Pten and treated with DMSO , GSK126 ( 5μM ) , BAZ2 - ICR ( 5μM ) , or GSK2801 ( 5μM ) . Values were normalized to shRNA - control samples . Average values of two independent experiments .

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E. Quantification of mouse prostate organoids per field expressing shRNA-control and shRNA-Pten and treated with DMSO, GSK126 (5μM), BAZ2-ICR (5μM), or GSK2801 (5μM). Values were normalized to shRNA-control samples. Average values of two independent experiments.


A , The indicated cells co - transfected with p5HRE - Luc and either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( empty vector : EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay . B , U2OS cells co - transfected with p5HRE - Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, The indicated cells co-transfected with p5HRE-Luc and either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (empty vector: EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay. B, U2OS cells co-transfected with p5HRE-Luc and the indicated siRNA were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.


C , HCT116 p53 - / - cells co - transfected with p5HRE - Luc , either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) , and either pcDNA3 / p53 ( at two concentrations : + and + + ) or pcDNA3 ( EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies .

C, HCT116 p53-/- cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 (at two concentrations: + and ++) or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay and Western blotting using the indicated antibodies.


D , HCT116 p53 + / + cells co - transfected with p5HRE - Luc , either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) , and either pcDNA3 / p53 R175H , R248W , R273H , or pcDNA3 ( EV ) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HCT116 p53+/+ cells co-transfected with p5HRE-Luc, either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV), and either pcDNA3/p53 R175H, R248W, R273H, or pcDNA3 (EV) were cultured under the indicated oxygen conditions for 24 hours for the luciferase assay.


The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) either pcDNA3 / p53 ( p53 ) or pcDNA3 ( EV ) ( C ) with the indicated siRNA were cultured under < 0 . 1 % O2 conditions for 24 hours and subjected to qRT - PCR for the indicated

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

The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) either pcDNA3/p53 (p53) or pcDNA3 (EV) (C) with the indicated siRNA were cultured under < 0.1% O2 conditions for 24 hours and subjected to qRT-PCR for the indicated mRNA


The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) to the transwell invasion

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

The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) to the transwell invasion assay


The indicated cells transiently transfected with either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) with the indicated siRNA or to the transwell invasion

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

The indicated cells transiently transfected with either pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) with the indicated siRNA or to the transwell invasion assay


G , The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( left ) and the representative images of lungs ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O 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, The number of metastatic colonies in lungs in the pulmonary metastasis model using the indicated cells stably transfected with ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (left) and the representative images of lungs (right).


The indicated cells stably transfected with the ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( H ) were cultured under < 0 . 1 % O2 ( H ) for the indicated periods and subjected to the in vitro cell proliferation assay .

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

The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) or its empty vector (EV) (H) were cultured under < 0.1% O2 (H) for the indicated periods and subjected to the in vitro cell proliferation assay.


The indicated cells stably transfected with the ZBTB2 expression vector ( ZBTB2 ) transiently transfected with siScr or siZBTB2 ( I ) the indicated oxygen conditions ( I ) for the indicated periods and subjected to the in vitro cell proliferation assay .

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

The indicated cells stably transfected with the ZBTB2 expression vector (ZBTB2) transiently transfected with siScr or siZBTB2 (I) the indicated oxygen conditions (I) for the indicated periods and subjected to the in vitro cell proliferation assay.


J , K , Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed .

J,K, Growth of the indicated tumor xenografts with or without ZBTB2 overexpression was analyzed.


A , Tissue microarrays of human lung adenocarcinoma ( 243 samples ) stained with anti - ZBTB2 antibodies . High magnification images are shown in the left lower side of each image . Scale bar , 500 and 100 μm in the low and high magnification images , 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

A, Tissue microarrays of human lung adenocarcinoma (243 samples) stained with anti-ZBTB2 antibodies. High magnification images are shown in the left lower side of each image. Scale bar, 500 and 100 μm in the low and high magnification images, respectively.


B , Fisher ' s exact test for percentage of CA9 - positive tumors in the four indicated groups . Percentages and absolute numbers of each group are indicated in the graph .

O O O O O O O O O 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, Fisher's exact test for percentage of CA9-positive tumors in the four indicated groups. Percentages and absolute numbers of each group are indicated in the graph.


C , Kaplan - Meier analysis of the disease - free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status .

O O O O 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, Kaplan-Meier analysis of the disease-free survival of the lung cancer patients stratified by the expression levels of ZBTB2 and p53 status.


D - F , TCGA - based Kaplan - Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX ( D ) , ZMAT3 ( E ) , and CEACAM1 ( 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

D-F, TCGA-based Kaplan-Meier analysis of overall survival of lung adenocarcinoma patients stratified by the expression levels of ZBTB2 and those of BAX (D), ZMAT3 (E), and CEACAM1 (F).


A , The indicated cells transiently transfected with pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( EV ) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies .

A, The indicated cells transiently transfected with pEF6/ZBTB2 (ZBTB2) or pEF6/myc-His B (EV) were cultured under the indicated oxygen conditions for 24 hours and subjected to Western blotting using the indicated antibodies.


The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A and N803A ( E ) were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated with the indicated siRNA The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


The indicated cells transiently transfected with pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A and N803A ( E ) were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated ( B , D , E , F ) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with pcDNA6/Gal4 DBD-HIF-1α TAD P564A and N803A (E) were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated (B,D,E,F) The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A were additionally co - transfected with either pEF6 / myc - His B ( EV ) , pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 1 - 23 ] ( del [ 1 - 23 ] ) , pEF6 / ZBTB2 del [ BTB / POZ ] ( del [ BTB ] ) , pEF6 / ZBTB2 del [ ZF1 ] ( del [ ZF1 ] ) , pEF6 / ZBTB2 del [ ZF2 ] ( del [ ZF2 ] ) , pEF6 / ZBTB2 del [ ZF3 ] ( del [ ZF3 ] ) , or pEF6 / ZBTB2 del [ ZF4 ] - ( del [ ZF4 ] ) , as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay .

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

The indicated cells transiently transfected with both pG5H1bLuc and either pcDNA6/Gal4 DBD-HIF-1α TAD P564A were additionally co-transfected with either pEF6/myc-His B (EV), pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[1-23] (del[1-23]), pEF6/ZBTB2 del[BTB/POZ] (del[BTB]), pEF6/ZBTB2 del[ZF1] (del[ZF1]), pEF6/ZBTB2 del[ZF2] (del[ZF2]), pEF6/ZBTB2 del[ZF3] (del[ZF3]), or pEF6/ZBTB2 del[ZF4]- (del[ZF4]), as indicated The cells were then cultured under the indicated oxygen conditions for 24 hours and subjected to the luciferase assay.


H , After transient transfection with the expression vectors for ZBTB2 - V5 , ZBTB2 - myc , or their empty vector ( - ) , ZBTB2 - myc protein was immunoprecipitated using the anti - myc antibody and co - precipitated ZBTB2 - V5 was detected using the anti - V5 antibody ( upper ) . One - tenth of the whole cell lysate ( WCL ) was subjected to immunoblotting with the indicated antibodies ( lower ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, After transient transfection with the expression vectors for ZBTB2-V5, ZBTB2-myc, or their empty vector (-), ZBTB2-myc protein was immunoprecipitated using the anti-myc antibody and co-precipitated ZBTB2-V5 was detected using the anti-V5 antibody (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).


I , J , The indicated cells transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT or pcDNA4 / LgBiT and either pcDNA4 / ZBTB2 - SmBiT or pcDNA4 / SmBiT , as indicated , were cultured under < 0 . 1 % oxygen ( I ) or the indicated oxygen conditions ( J ) and subjected to the split luciferase complementation assay .

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

I,J, The indicated cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT or pcDNA4/LgBiT and either pcDNA4/ZBTB2-SmBiT or pcDNA4/SmBiT, as indicated, were cultured under < 0.1% oxygen (I) or the indicated oxygen conditions (J) and subjected to the split luciferase complementation assay.


K , HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 ( ZF2 or ZF3 - deletion ) were cultured under < 0 . 1 % oxygen conditions , and subjected to immunoprecipitation with anti - myc antibody . Coprecipitated DNA was subjected to the qRT - PCR experiment using primers against HRE regions of the STC1 promoter .

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

K, HeLa cells overexpressing wildtype or the indicated mutant of ZBTB2 (ZF2 or ZF3-deletion) were cultured under < 0.1% oxygen conditions, and subjected to immunoprecipitation with anti-myc antibody. Coprecipitated DNA was subjected to the qRT-PCR experiment using primers against HRE regions of the STC1 promoter.


L , The same experiment as I was conducted using the indicated combination of pcDNA4 / ZBTB2 - LgBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - LgBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - LgBiT ( ΔZF3 ) , and pcDNA4 / ZBTB2 - SmBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - SmBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - SmBiT ( ΔZF3 ) .

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

L, The same experiment as I was conducted using the indicated combination of pcDNA4/ZBTB2-LgBiT (WT), pcDNA4/ZBTB2ΔZF2-LgBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-LgBiT (ΔZF3), and pcDNA4/ZBTB2-SmBiT (WT), pcDNA4/ZBTB2ΔZF2-SmBiT (ΔZF2), or pcDNA4/ZBTB2ΔZF3-SmBiT (ΔZF3).


B , HCT116 p53 - / - cells co - transfected with pG5H1bLuc , pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A , and either pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 del [ 8 - 11 ] ( ZBTB2 del [ 8 - 11 ] ) , pEF6 / ZBTB2 del [ 51 - 54 ] ( ZBTB2 del [ 51 - 54 ] ) , or pEF6 / myc - His B ( EV ) , as indicated , were cultured under < 0 . 1 % oxygen conditions for 24 hours and subjected to the luciferase assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HCT116 p53-/- cells co-transfected with pG5H1bLuc, pcDNA6/Gal4 DBD-HIF-1α TAD P564A, and either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 del[8-11] (ZBTB2 del[8-11]), pEF6/ZBTB2 del[51-54] (ZBTB2 del[51-54]), or pEF6/myc-His B (EV), as indicated, were cultured under < 0.1% oxygen conditions for 24 hours and subjected to the luciferase assay.


C , After transient transfection with or without the expression vectors for ZBTB2 - V5 , ZBTB2 4A - V5 , ZBTB2 - myc , ZBTB2 4A - myc , or their empty vector , as indicated , the myc - fused proteins were immunoprecipitated using an anti - myc antibody and co - precipitated ZBTB2 - V5 or ZBTB2 4A - V5 was detected ( upper ) . One - tenth of the whole cell lysate ( WCL ) was subjected to immunoblotting with the indicated antibodies ( lower ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, After transient transfection with or without the expression vectors for ZBTB2-V5, ZBTB2 4A-V5, ZBTB2-myc, ZBTB2 4A-myc, or their empty vector, as indicated, the myc-fused proteins were immunoprecipitated using an anti-myc antibody and co-precipitated ZBTB2-V5 or ZBTB2 4A-V5 was detected (upper). One-tenth of the whole cell lysate (WCL) was subjected to immunoblotting with the indicated antibodies (lower).


D , HCT116 p53 - / - ( left ) cells transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - LgBiT 4A ( 4A ) , or pcDNA4 / LgBiT ( - ) and either pcDNA4 / ZBTB2 - SmBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - SmBiT ( 4A ) , or pcDNA4 / SmBiT ( - ) , as indicated , were cultured under < 0 . 1 % oxygen and subjected to the split luciferase complementation assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HCT116 p53-/- (left) cells transiently transfected with or without either pcDNA4/ZBTB2-LgBiT (ZBTB2), pcDNA4/ZBTB2 4A-LgBiT 4A (4A), or pcDNA4/LgBiT (-) and either pcDNA4/ZBTB2-SmBiT (ZBTB2), pcDNA4/ZBTB2 4A-SmBiT (4A), or pcDNA4/SmBiT (-), as indicated, were cultured under < 0.1% oxygen and subjected to the split luciferase complementation assay.


G , The same kind of luciferase assay to quantify the transactivation activity as B was carried out using either pEF6 / ZBTB2 ( ZBTB2 ) , pEF6 / ZBTB2 4A ( 4A ) , or pEF6 / myc - His B ( EV ) .

O O O O O O O O O O O O O O O O O O O O O O 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, The same kind of luciferase assay to quantify the transactivation activity as B was carried out using either pEF6/ZBTB2 (ZBTB2), pEF6/ZBTB2 4A (4A), or pEF6/myc-His B (EV).


H , Growth of the indicated tumor xenografts was analyzed .

H, Growth of the indicated tumor xenografts was analyzed.


( B ) Effect of ND - 011992 on the intracellular ATP level in M . bovis BCG . The bacteria were treated with DMSO ( blue triangle ) , 100 nM Q203 alone ( green triangle ) , 20 μM ND - 011992 alone ( brown square ) , and a dose - range of ND - 011992 in the presence of Q203 ( red squares ) for 15 hours before quantification of intracellular ATP levels .

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

(B) Effect of ND-011992 on the intracellular ATP level in M. bovis BCG. The bacteria were treated with DMSO (blue triangle), 100 nM Q203 alone (green triangle), 20 μM ND-011992 alone (brown square), and a dose-range of ND-011992 in the presence of Q203 (red squares) for 15 hours before quantification of intracellular ATP levels.


( C ) Oxygen consumption assay in M . bovis BCG using methylene blue as an oxygen sensor . ( D ) Oxygen consumption assay in M . bovis BCG using the Seahorse XFe96 Extracellular flux analyser . 12 μM ND - 011992 was injected alone ( green triangles ) , 100 nM Q203 ( blue circles ) , or in combination with Q203 ( red squares ) . For each condition , OCR readings were normalised to the last basal OCR reading before drug injection . OCR : Oxygen Consumption Rate . The dotted lines indicate the timepoints of drug injection .

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

(C) Oxygen consumption assay in M. bovis BCG using methylene blue as an oxygen sensor. (D) Oxygen consumption assay in M. bovis BCG using the Seahorse XFe96 Extracellular flux analyser. 12 μM ND-011992 was injected alone (green triangles), 100 nM Q203 (blue circles), or in combination with Q203 (red squares). For each condition, OCR readings were normalised to the last basal OCR reading before drug injection. OCR: Oxygen Consumption Rate. The dotted lines indicate the timepoints of drug injection.


( E ) Dose - dependent inhibition of M . bovis BCG OCR by ND - 011992 ( in the presence of 100 nM Q203 ) measured on a Seahorse XFe96 analyser platform . For each condition , OCR readings were normalised to the last basal OCR reading before drug injection .

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 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

(E) Dose-dependent inhibition of M. bovis BCG OCR by ND-011992 (in the presence of 100 nM Q203) measured on a Seahorse XFe96 analyser platform. For each condition, OCR readings were normalised to the last basal OCR reading before drug injection.


( F ) Effect of ND - 011992 on the OCR of M . smegmatis IMVs using the Oroboros O2k fluorespirometer . IMVs OCR from the parental strain ( green triangles ) , ∆ cydAB knockout ( blue circles ) , and ∆ cydAB complemented with M . tuberculosis CydABDC + ( red squares ) energized with NADH were determined . Q203 was used at 1 μM . 100 % OCR was defined as the OCR of the untreated samples for each strain .

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

(F) Effect of ND-011992 on the OCR of M. smegmatis IMVs using the Oroboros O2k fluorespirometer. IMVs OCR from the parental strain (green triangles), ∆cydAB knockout (blue circles), and ∆cydAB complemented with M. tuberculosis CydABDC+ (red squares) energized with NADH were determined. Q203 was used at 1 μM. 100% OCR was defined as the OCR of the untreated samples for each strain.


( A ) Differential gene expression analysis of H37Rv treated with Q203 , ND - 011992 , or combination ( H37Rv - Combo ) , and ∆ cydAB treated with Q203 compared to untreated controls . The log2 - fold differences in gene expression of various conditions relative to the untreated control is indicated using a sliding scale where higher expression is reflected in red and lower expression is reflected in blue , with a midpoint signifying no difference in white .

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

(A) Differential gene expression analysis of H37Rv treated with Q203, ND-011992, or combination (H37Rv-Combo), and ∆cydAB treated with Q203 compared to untreated controls. The log2-fold differences in gene expression of various conditions relative to the untreated control is indicated using a sliding scale where higher expression is reflected in red and lower expression is reflected in blue, with a midpoint signifying no difference in white.


( C ) Absence of the Cyt - bcc : aa3 sensitized H37Rv to ND - 011992 . M . tuberculosis H37Rv wildtype ( Blue circles ) , ∆ ctaE - qcrCAB ( red squares ) and complemented strain ( green triangles ) were treated with ND - 011992 . Growth inhibitory potency ( MIC50 ) was recorded after 2 to 3 weeks of incubation .

(C) Absence of the Cyt-bcc:aa3 sensitized H37Rv to ND-011992. M. tuberculosis H37Rv wildtype (Blue circles), ∆ctaE-qcrCAB (red squares) and complemented strain (green triangles) were treated with ND-011992. Growth inhibitory potency (MIC50) was recorded after 2 to 3 weeks of incubation.


( D ) The reduced minus oxidized difference spectrum of M . smegmatis ΔqcrCAB inverted membrane vesicles ( IMVs ) . The green line represents the spectrum without treatment , while the blue line represents the spectrum of the IMVs in the presence of 10 μM ND - 011992 .

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

(D) The reduced minus oxidized difference spectrum of M. smegmatis ΔqcrCAB inverted membrane vesicles (IMVs). The green line represents the spectrum without treatment, while the blue line represents the spectrum of the IMVs in the presence of 10 μM ND-011992.


( A ) Combined potency of ND - 011992 + Q203 against the drug - resistant clinical isolate M . tuberculosis 123 - 20 - 0047 .

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

(A) Combined potency of ND-011992 + Q203 against the drug-resistant clinical isolate M. tuberculosis 123-20-0047.


( C ) Growth inhibition assay in M . bovis BCG . 106 bacteria were plated onto 7H10 agar supplemented with DMSO , 25 nM Q203 , 3 µM ND - 011992 , or 25 nM Q203 + 3 µM ND - 011992 .

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

(C) Growth inhibition assay in M. bovis BCG. 106 bacteria were plated onto 7H10 agar supplemented with DMSO, 25 nM Q203, 3 µM ND-011992, or 25 nM Q203 + 3 µM ND-011992.


( D ) Fluctuation analysis in M . tuberculosis H37Rv . M . tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 ( red ) . Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND - 011992 or 2 µg / mL rifampicin ( blue ) . The corresponding values of the median frequency of resistance is indicated in the graph .

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

(D) Fluctuation analysis in M. tuberculosis H37Rv. M. tuberculosis ΔcydAB was plated on 7H10 containing 100 nM Q203 (red). Parental H37Rv was plated on 7H10 with 100 nM Q203 and 6 µM ND-011992 or 2 µg/mL rifampicin (blue). The corresponding values of the median frequency of resistance is indicated in the graph.


( A ) Bactericidal activity of ND - 011992 and ND - 011992 - Q203 combination in replicating M . tuberculosis H37Rv . Bedaquiline ( BDQ ) was used at 500 nM , and Q203 at 100 nM . Inoc . : starting inoculum ; DMSO : vehicle control . Bacteria viability was determined by enumerating colony - forming units ( CFU ) on nutrient - agar plates after 15 days of incubation . Dotted line represents 90 % reduction in CFU / mL compared to the initial inoculum at time 0 . * P < 0 . 05 ; * * P < 0 . 01 unpaired Student ' s t - test , one - tailed ; n = 3 ; comparing Q203 alone vs Q203 + ND - 011992 . Exact P - values ( from left to right ) : 0 . 0346 , 0 . 0143 , 0 . 00016 , 0 . 000052 . ( B ) Bactericidal activity of ND - 011992 and ND - 011992 - Q203 combination in nutrient - starved M . tuberculosis H37Rv . Bedaquiline ( BDQ ) was used at 500 nM , isoniazid was used at 1 μM , Q203 was used at 100 nM , and ND - 011992 was used at 3 , 6 , 12 , and 30 μM . Bacteria viability was determined by enumerating colony - forming units ( CFU ) after 15 days of incubation . Dotted line represents 90 % reduction in CFU / mL compared to the initial inoculum at time 0 . * * P < 0 . 01 unpaired Student ' s t - test , one - tailed ; n = 3 ; comparing Q203 alone vs Q203 + ND - 011992 . Exact P - values ( from left to right ) : 0 . 0098 , 0 . 000027 , 0 . 000067 .

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

(A) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in replicating M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, and Q203 at 100 nM. Inoc.: starting inoculum; DMSO: vehicle control. Bacteria viability was determined by enumerating colony-forming units (CFU) on nutrient-agar plates after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0. *P < 0.05; **P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0346, 0.0143, 0.00016, 0.000052. (B) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in nutrient-starved M. tuberculosis H37Rv. Bedaquiline (BDQ) was used at 500 nM, isoniazid was used at 1 μM, Q203 was used at 100 nM, and ND-011992 was used at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony-forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0. **P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0098, 0.000027, 0.000067.


( C ) Bactericidal activity of ND - 011992 and ND - 011992 - Q203 combination in M . tuberculosis H37Rv under hypoxia . BDQ was used at 500 nM , isoniazid at 1 μM , metronidazole ( MTZ ) at 200 μM , Q203 at 100 nM , and ND - 011992 at 3 , 6 , 12 , and 30 μM . Bacteria viability was determined by enumerating colony - forming units ( CFU ) after 15 days of incubation . Dotted line represents 90 % reduction in CFU / mL compared to the initial inoculum at time 0 . * * P < 0 . 01 unpaired Student ' s t - test , one - tailed ; n = 3 ; comparing Q203 alone vs Q203 + ND - 011992 . Exact P - values ( from left to right ) : 0 . 0092 , 0 . 00010 , 0 . 00079 .

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(C) Bactericidal activity of ND-011992 and ND-011992-Q203 combination in M. tuberculosis H37Rv under hypoxia. BDQ was used at 500 nM, isoniazid at 1 μM, metronidazole (MTZ) at 200 μM, Q203 at 100 nM, and ND-011992 at 3, 6, 12, and 30 μM. Bacteria viability was determined by enumerating colony-forming units (CFU) after 15 days of incubation. Dotted line represents 90% reduction in CFU/mL compared to the initial inoculum at time 0.**P < 0.01 unpaired Student's t-test, one-tailed; n = 3; comparing Q203 alone vs Q203 + ND-011992. Exact P-values (from left to right): 0.0092, 0.00010, 0.00079.


( D ) Efficacy of ND - 011992 - Q203 combination treatment in a mouse model of acute tuberculosis . Bacterial load ( CFU ) were enumerated in the lungs of mice before treatment , and after 5 days of treatment with either the vehicle control ( brown squares ) , Q203 at 5 mg / kg ( red squares ) , ND - 011992 at 25 mg / kg ( green triangles ) , or ND - 011992 at 25mg / kg with Q203 at 5 mg / kg ( orange diamonds ) . * * P = 0 . 0006 unpaired Student ' s t - test , two - tailed ; n = 4 ; comparing Q203 alone vs Q203 + ND - 011992 .

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(D) Efficacy of ND-011992-Q203 combination treatment in a mouse model of acute tuberculosis. Bacterial load (CFU) were enumerated in the lungs of mice before treatment, and after 5 days of treatment with either the vehicle control (brown squares), Q203 at 5 mg/kg (red squares), ND-011992 at 25 mg/kg (green triangles), or ND-011992 at 25mg/kg with Q203 at 5 mg/kg (orange diamonds). **P = 0.0006 unpaired Student's t-test, two-tailed; n = 4; comparing Q203 alone vs Q203 + ND-011992.


Quantification of IL - 1β concentration ( pg / ml ) in the supernatant of primary macrophage cultures from COVID - 19 patients ( n = 44 ; red bars ) or SC - naïve individuals ( n = 24 ; blue bars ) stimulated with LPS or S - protein ( 0 . 1 µg / ml ) . To activate IL - 1β secretion nigericin was added for 2h . For statistical analysis , two - way ANOVA with tukey post hoc test was used . TNF - α ( pg / ml ) secreted from macrophages derived from SC - naïve and COVID - 19 patients as described above . Cells were stimulated with LPS ( SC - naïve / COVID - 19 n = 4 ) , LPS with nigericin ( SC - naïve n = 4 ; COVID - 19 n = 6 ) , S - protein ( SC - naïve n = 5 ; COVID - 19 n = 4 ) , S - protein with nigericin ( SC - naïve n = 3 ; COVID - 19 n = 4 ) or left unstimulated ( SC - naïve n = 3 ; COVID - 19 n = 4 ) . Significances shown in the figure are always in comparison to the unstimulated control .

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Quantification of IL-1β concentration (pg/ml) in the supernatant of primary macrophage cultures from COVID-19 patients (n = 44; red bars) or SC-naïve individuals (n = 24; blue bars) stimulated with LPS or S-protein (0.1 µg/ml). To activate IL-1β secretion nigericin was added for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used. TNF-α (pg/ml) secreted from macrophages derived from SC-naïve and COVID-19 patients as described above. Cells were stimulated with LPS (SC-naïve/COVID-19 n = 4), LPS with nigericin (SC-naïve n = 4; COVID-19 n = 6), S-protein (SC-naïve n = 5; COVID-19 n = 4), S-protein with nigericin (SC-naïve n = 3; COVID-19 n = 4) or left unstimulated (SC-naïve n = 3; COVID-19 n = 4). Significances shown in the figure are always in comparison to the unstimulated control.


Representative confocal microscope pictures of macrophages from SC - naïve ( top ) and COVID - 19 patients ( bottom ) . Nuclei are stained with DAPI ( blue ) and ASC speck are labeled by immunofluorescence staining ( green ) . Macrophages were stimulated with LPS ( left ) and S - Protein ( right ) for 4h followed by stimulation with nigericin in both cases . Pictures were taken with a 60x objective using the same microscope settings .

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Representative confocal microscope pictures of macrophages from SC-naïve (top) and COVID-19 patients (bottom). Nuclei are stained with DAPI (blue) and ASC speck are labeled by immunofluorescence staining (green). Macrophages were stimulated with LPS (left) and S-Protein (right) for 4h followed by stimulation with nigericin in both cases. Pictures were taken with a 60x objective using the same microscope settings.


Subgroup analysis of IL - 1β secretion of macrophages after stimulation with S - protein and nigericin from COVID - 19 patients with severe ( red / black ; n = 23 ) or mild ( white / red ; n = 21 ) disease . Student ' s t - test with Welsh corrections was used to calculate statistical differences . Graph shows mean ± SEM .

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Subgroup analysis of IL-1β secretion of macrophages after stimulation with S-protein and nigericin from COVID-19 patients with severe (red/black; n=23) or mild (white/red; n=21) disease. Student's t-test with Welsh corrections was used to calculate statistical differences. Graph shows mean ± SEM.


CD14 + cells of two convalescent individuals were isolated sequentially at d18 , d26 and d56 after confirmation of SARS CoV2 infection by PCR . Following M - CSF differentiation , macrophages were stimulated with LPS ( red triangle ) or S - Protein ( red circles ) and IL - 1β concentration ( pg / ml ) was quantified upon incubation with nigericin . Blue dashed line indicates mean IL - 1β level of SC - naïve individuals stimulated with S - protein and nigericin .

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CD14+ cells of two convalescent individuals were isolated sequentially at d18, d26 and d56 after confirmation of SARS CoV2 infection by PCR. Following M-CSF differentiation, macrophages were stimulated with LPS (red triangle) or S-Protein (red circles) and IL-1β concentration (pg/ml) was quantified upon incubation with nigericin. Blue dashed line indicates mean IL-1β level of SC-naïve individuals stimulated with S-protein and nigericin.


Volcano plot showing DEGs in S - protein stimulated macrophages from convalescent COVID - 19 patients ( SC - conv ) compared to unstimulated cells . Negative log10 adjusted p - values are plotted against the log2 fold - change . Select genes are labeled . P - values are calculated using Wald test and p - adjusted values using the FDR / Benjamini - Hochberg approach .

Volcano plot showing DEGs in S-protein stimulated macrophages from convalescent COVID-19 patients (SC-conv) compared to unstimulated cells. Negative log10 adjusted p-values are plotted against the log2 fold-change. Select genes are labeled. P-values are calculated using Wald test and p-adjusted values using the FDR/Benjamini-Hochberg approach.


IL - 1β concentration ( pg / ml ) in supernatants of COVID - 19 patient derived macrophages pretreated with DMSO ( n = 44 ) , KINK - 1 ( n = 11 ) and MMG - 11 ( n = 19 ) and subsequent stimulation with LPS and nigericin . For statistical analysis , one - way ANOVA was used .

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IL-1β concentration (pg/ml) in supernatants of COVID-19 patient derived macrophages pretreated with DMSO (n = 44), KINK-1 (n = 11) and MMG-11 (n = 19) and subsequent stimulation with LPS and nigericin. For statistical analysis, one-way ANOVA was used.


IL - 1β concentration ( pg / ml ) in supernatants from macrophages of SC naïve ( blue ; n = 7 ) and COVID - 19 patients ( red ; n = 11 ) . Macrophages were pre - treated with DMSO ( SC - naïve n = 7 ; COVID - 19 n = 11 ) , KINK - 1 ( SC - naïve n = 6 ; COVID - 19 n = 7 ) and MMG - 11 ( SC - naïve n = 4 ; COVID - 19 n = 4 ) for 2h and then stimulated with zymosan ( 4h ) and nigericin ( 2h ) as indicated . For statistical analysis , two - way ANOVA with tukey post hoc test was used .

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IL-1β concentration (pg/ml) in supernatants from macrophages of SC naïve (blue; n = 7) and COVID-19 patients (red; n = 11). Macrophages were pre-treated with DMSO (SC-naïve n = 7; COVID-19 n = 11), KINK-1 (SC-naïve n = 6; COVID-19 n = 7) and MMG-11 (SC-naïve n = 4; COVID-19 n = 4) for 2h and then stimulated with zymosan (4h) and nigericin (2h) as indicated. For statistical analysis, two-way ANOVA with tukey post hoc test was used.


IL - 1β concentration ( pg / ml ) in supernatants from COVID - 19 patients which were stimulated for 2h with a blocking anti - TLR2 monoclonal antibody in different concentrations as indicated . Subsequently , macrophages were stimulated with LPS ( 1 ng / ml : n = 6 ; 10 and 100 ng / ml : n = 7 ) and S - Protein ( 1 ng / ml : n = 12 ; 10 and 100 ng / ml : n = 13 ) for 4h and both groups for 2h with nigericin . For statistical analysis , one - way ANOVA was

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IL-1β concentration (pg/ml) in supernatants from COVID-19 patients which were stimulated for 2h with a blocking anti-TLR2 monoclonal antibody in different concentrations as indicated. Subsequently, macrophages were stimulated with LPS (1 ng/ml: n = 6; 10 and 100 ng/ml: n = 7) and S-Protein (1 ng/ml: n = 12; 10 and 100 ng/ml: n = 13) for 4h and both groups for 2h with nigericin. For statistical analysis, one-way ANOVA was used


Volcano plot showing DEGs in unstimulated COVID - 19 patient - derived macrophages ( SC - conv ) compared to macrophages from SARS - CoV - 2 naïve individuals ( SC - naïve ) . Negative log10 padj values are plotted against the log2 fold - change . Selected genes of interest are labeled .

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Volcano plot showing DEGs in unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to macrophages from SARS-CoV-2 naïve individuals (SC-naïve). Negative log10 padj values are plotted against the log2 fold-change. Selected genes of interest are labeled.


IL - 1β concentration ( pg / ml ) in supernatants of SC - naïve individuals ( n = 7 ) which were pre - treated for 24h with FCCP ( 1µg / ml ) and H2O2 ( 10µg / ml ) . Macrophages were then incubated with LPS or S - Protein ( 4h ) and nigericin ( 2h ) as indicated in the figure . Control cells were left unstimulated or stimulated with LPS or S - protein without pre - treatment with FCSP or H2O2 ( n = 6 ) . For statistical analysis , Brown - Forsythe and Welsh ANOVA test was used .

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IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.


Representative gene loci ( TLR2 ( top ) and S100A7A / 8 / 9 / 12 ( bottom ) H3K27ac peaks ) comparing SC - naïve ( blue ) and SC - conv ( red ) monocytes . Red peaks reach higher values than blue peaks ( representative examples of four samples for each condition ) .

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Representative gene loci (TLR2 (top) and S100A7A/8/9/12 (bottom) H3K27ac peaks) comparing SC-naïve (blue) and SC-conv (red) monocytes. Red peaks reach higher values than blue peaks (representative examples of four samples for each condition).


Quantification of IL - 1β concentrations ( pg / ml ) in the supernatant of primary macrophage cultures from patients with active / untreated tuberculosis ( n = 6 ; red / black bars ) or healthy / SARS - CoV - 2 naïve individuals ( n = 3 ; blue bars ) or the same patients after 6 months of anti - tuberculous treatment ; n = 6 ; white / red bars ) stimulated with LPS or S - protein ( 0 . 1 µg / ml ) for 4h and subsequent incubation with nigericin for 2h . For statistical analysis , two - way ANOVA with tukey post hoc test was used .

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Quantification of IL-1β concentrations (pg/ml) in the supernatant of primary macrophage cultures from patients with active/untreated tuberculosis (n = 6; red/black bars) or healthy/SARS-CoV-2 naïve individuals (n = 3; blue bars) or the same patients after 6 months of anti-tuberculous treatment; n = 6; white/red bars) stimulated with LPS or S-protein (0.1 µg/ml) for 4h and subsequent incubation with nigericin for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.


G The entry efficiency of SARS - CoV - 2 and RshSTT182 pseudoviruses in HeLa - hACE2 cells . Green fluorescence indicates the entry of the pseudoviruses into the Hela - hACE2 cells . Unaltered HeLa cells were used as a negative control . H Statistics for transduction of pseudoviruses . Data represents the results of five replicates in one representative assay . All data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

G The entry efficiency of SARS-CoV-2 and RshSTT182 pseudoviruses in HeLa-hACE2 cells. Green fluorescence indicates the entry of the pseudoviruses into the Hela-hACE2 cells. Unaltered HeLa cells were used as a negative control. H Statistics for transduction of pseudoviruses. Data represents the results of five replicates in one representative assay. All data are presented as mean ± SD. Pseudovirus infection assays were performed at least twice.


A Flow cytometry characterization of binding between bat - ACE2 orthologs and the SARS - CoV - 2 or RshSTT182 / 200 RBDs . BHK cells expressing eGFP - fused ACE2s were stained with the indicated His - tagged proteins . The SARS - CoV - 2 NTD was used as a negative control .

A Flow cytometry characterization of binding between bat-ACE2 orthologs and the SARS-CoV-2 or RshSTT182/200 RBDs. BHK cells expressing eGFP-fused ACE2s were stained with the indicated His-tagged proteins. The SARS-CoV-2 NTD was used as a negative control.


A Flow cytometry characterization of binding between ACE2s and the RshSTT182 / 200 or SARS - CoV - 2 RBDs . Cells stained with the RshSTT182 / 200 RBD , the SARS - CoV - 2 RBD and the SARS - CoV - 2 NTD proteins are shown in orange , marine and red , respectively . The SARS - CoV - 2 NTD was used as a negative control .

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A Flow cytometry characterization of binding between ACE2s and the RshSTT182/200 or SARS-CoV-2 RBDs. Cells stained with the RshSTT182/200 RBD, the SARS-CoV-2 RBD and the SARS-CoV-2 NTD proteins are shown in orange, marine and red, respectively. The SARS-CoV-2 NTD was used as a negative control.


D The entry efficiency of SARS - CoV - 2 and RshSTT182 pseudoviruses into HeLa - hACE2 cells . Green fluorescence indicates the entry of the pseudoviruses into the Hela - hACE2 cells . Unaltered HeLa cells were used as a negative control . E Statistics for transduction of pseudoviruses . Data represents the results of five replicates in one representative assay . All data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

D The entry efficiency of SARS-CoV-2 and RshSTT182 pseudoviruses into HeLa-hACE2 cells. Green fluorescence indicates the entry of the pseudoviruses into the Hela-hACE2 cells. Unaltered HeLa cells were used as a negative control. E Statistics for transduction of pseudoviruses. Data represents the results of five replicates in one representative assay. All data are presented as mean ± SD. Pseudovirus infection assays were performed at least twice.


B The neutralization curve of serum samples in COVID - 19 convalescents and vaccinees against RshSTT182 and SARS - CoV - 2 are shown . Bar represents the SD of two replicates .

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B The neutralization curve of serum samples in COVID-19 convalescents and vaccinees against RshSTT182 and SARS-CoV-2 are shown. Bar represents the SD of two replicates.


C . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . Apc1 - loop500 WT or its derivatives ( ∆ 11 or 3A ) were incubated with the 35S - labelled Flag - B56γ in interphase extract ( Inter ) or anaphase extract ( Ana ) supplemented with CycB ∆ 167 at 23 ˚ C for 1 hr . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or coomassie brilliant blue ( CBB ) staining . The bar graph is quantification of bound B56γ . The intensities of MBP control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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C. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. Apc1-loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S-labelled Flag-B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23˚C for 1 hr. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


D . Cdk - dependent in vitro kinase assay of Apc1 - loop500 . MBP fused WT or 3A Apc1 - loop500 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 23 ˚ C for 10 min , separated by SDS - PAGE and detected by autoradiography .

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D. Cdk-dependent in vitro kinase assay of Apc1-loop500. MBP fused WT or 3A Apc1-loop500 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 23˚C for 10 min, separated by SDS-PAGE and detected by autoradiography.


E . Cdk can promote Apc1 - loop500 and B56 interaction . MBP fused WT or 3A Apc1 - loop500 fragment was incubated in the presence or absence of Cdk2 - cyclin A at 30 ˚ C for 60 min . MBP - fused peptides ( - / + kinase ) were isolated and incubated with purified Flag - B56γ at 23 ˚ C for 30 min . The bound proteins were recovered by amylose beads and analysed by SDS - PAGE and immunoblotting with indicated antibodies . F . Quantification of E . Bound B56γ to WT Apc1 - loop500 control ( - Cdk ) was arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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E. Cdk can promote Apc1-loop500 and B56 interaction. MBP fused WT or 3A Apc1-loop500 fragment was incubated in the presence or absence of Cdk2-cyclin A at 30˚C for 60 min. MBP-fused peptides (-/+ kinase) were isolated and incubated with purified Flag-B56γ at 23˚C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS-PAGE and immunoblotting with indicated antibodies. F. Quantification of E. Bound B56γ to WT Apc1-loop500 control (-Cdk) was arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant wild - type ( WT ) or B56 binding site mutant APC / C ( 1 - L557A / V560A ) was incubated with APC / C - depleted ( ∆ APC ) interphase extract ( Inter ) or ∆ APC anaphase extract supplemented with CycB ∆ 167 ( Ana ) at 23 ˚ C for indicated times . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . pApc1 ( pS314 / pS318 ) is a phospho - site specific antibody that binds only when both S314 and S318 are phosphorylated . B . Quantification of A . The intensities of WT control in interphase were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild-type (WT) or B56 binding site mutant APC/C (1-L557A/V560A) was incubated with APC/C-depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23˚C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho-site specific antibody that binds only when both S314 and S318 are phosphorylated. B. Quantification of A. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


C . B56 binding site mutant APC / C ( 1 - L557A / V560A ) is less active in ubiquitylation assay than WT APC / C . The purified recombinant WT APC / C or B56 binding motif mutant APC / C ( 1 - L557A / V560A ) was incubated with ∆ APC anaphase extract . The recovered APC / C - Cdc20 complex was subjected to ubiquitylation assay using 35S - labelled cyclin B as a substrate . Samples were taken at indicated time points and analysed by SDS - PAGE and autoradiography . D . Quantification of C . Error bars , s . e . m . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. D. Quantification of C. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdc20 binding to APC / C loop domain mutants in interphase extract . The purified recombinant WT or Apc1 mutant APC / Cs ( 1 - ∆ L300 , 1 - ∆ L300 / ∆ 11 or 1 - ∆ L300 / L557A / V560A ) were incubated with ∆ APC interphase extract at 23 ˚ C for 1 hr . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments . B . ( left panel ) Cdc20 binding to APC / C loop domain mutants in anaphase extracts . Same as A , but ∆ APC anaphase extract was used and incubated at 23 ˚ C for 50 min in anaphase extract . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1-∆L300, 1-∆L300/∆11 or 1-∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23˚C for 1 hr. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. B. (left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as A, but ∆APC anaphase extract was used and incubated at 23˚C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdk - dependent in vitro kinase assay of Apc1 - loop300 . 3xFlag - tagged WT or 7T Apc1 - loop300 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 30 ˚ C for 30 min , and separated by SDS - PAGE and detected by autoradiography . ( right panel ) 32P - phosphorylated WT or 7T Apc1 - loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS - PAGE followed by autoradiography . B . Quantification of removal of radioactivity in A . Error bars , s . e . m . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography. B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments.