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

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(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 ) . two bands for IFT38 on SDS PAGE gel correspond to His - tagged ( upper band ) proteolytically untagged IFT38 ( lower band ) .

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(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 .

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(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 IFT - B2 complexes used for pull - downs ( top ) a western blot for anti - α - tubulin shows co - precipitated αβ - tubulin ( bottom ) .

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(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 mutant MmIFT54CH . ( top ) Coomassie stained SDS PAGE gel of input ( I ) pulled down ( PD ) corresponding western blot to visualize αβ - tubulin .

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(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 IFT - B2 an IFT88 / 52N subcomplex . pre - assembled IFT - B2 ( 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 N - terminal domain as well as IFT88 - binding region ) ( lane 3 ) . Both IFT88 IFT52N are required for interaction with IFT - B2 because neither N - terminal IFT52 domain ( residues 1 - 275 ; lane 4 ) alone , nor an IFT88 / 52 lacking N - terminal IFT52 domain ( with an IFT52 ( 281 - 335 ) construct containing only IFT88 - binding region ) ( lane 5 ) show efficient pull - down of IFT - B2 . Contaminants from purification of 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_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 B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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(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 immobilized on GSH beads various combinations of IFT - B2 . left gel shows IFT - B2 input mixtures , right gel shows bound material after washing elution from beads . A pentameric IFT - B2 ( IFT80 / 57 / 54 / 38 / 20 ) is efficiently pulled down by IFT88 / 52N - GST , but not by empty beads ( compare lanes 1 2 ) . Omission of IFT54 / 20 ( lane 4 ) , or IFT80 ( lane 5 ) does not influence binding of any of other components , but a lack of IFT57 / 38 in mixture abolishes detectable pull - downs of IFT54 / 20 IFT80 ( lane 4 ) , indicating that IFT57 / 38 is 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 . Contaminating bands from IFT88 / 52N sample are marked with asterisks .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 amounts of pulled down IFT - B2 , K130E / R204E double mutation reduced it to background ( compare lanes 2 6 ) .

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

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

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

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . Western blot showing H3K14ac in PC3 treated for three days without with selective catalytic EP300 inhibitor A - 485 ( 10 μM ) . Histone H3 serves as 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 of genes with BAZ2A - bound promoter downregulated in metastatic tumors ( AOX1 , MANB2 , DMPK , DDB1 in PC3 treated with siRNA - EP300 . Data were from three independent experiments . Values were normalized to GAPDH mRNA . Statistical significance ( P - values ) was calculated using two - t - ( * < 0 . 05 , * * * * < 0 . 0001 ) . Error bars represent SD .

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

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

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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 showing upregulation of CSC markers ( ALDH1A2 , LGR5 , CD24 ) downregulation of genes related to developmental processes ( HOXA1 , HOXA4 , SOX7 ) . Data are from two independent experiments . mRNA were normalized to GAPDH .

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

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

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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 . proliferation curves of PC3 treated with siRNA - BAZ2A . Error bars represent standard deviation from three independent experiments . C . proliferation curves of PC3 treated with 5μM BAZ2 - ICR GSK2801 . Error bars represent 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 of tumorspheres generated from PC3 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 ( EC50 ) of BAZ2 - ICR . Measurements were performed 5 days after treatment of PC3 initiation of tumorspheres using indicated BAZ2 - ICR . amount of tumorspheres was assessed by measurement of 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 of organoids derived from mouse prostate treated with DMSO , BAZ2 - ICR ( 5μM ) , GSK2801 ( 5μM ) or GSK126 ( 5μM ) inhibitors followed by transduction with shRNA - Control or shRNA - Pten .

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

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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 . of mouse prostate organoids per field expressing shRNA - control shRNA - Pten 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 , indicated co - transfected with p5HRE - Luc either pEF6 / ZBTB2 ( ZBTB2 ) or pEF6 / myc - His B ( empty vector : EV ) were cultured under indicated oxygen conditions for 24 hours for luciferase . B , U2OS co - transfected with p5HRE - Luc indicated siRNA were cultured under indicated oxygen conditions for 24 hours for luciferase .

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

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

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

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.


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

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

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


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

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

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


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

O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O 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 , number of metastatic colonies in lungs in pulmonary metastasis model using indicated stably transfected with ZBTB2 expression vector ( ZBTB2 ) or its empty vector ( EV ) ( left ) representative of lungs ( right ) .

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 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).


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

O O O O B-CONTROLLED_VAR 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

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.


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

O O O O B-CONTROLLED_VAR 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

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 indicated xenografts with or without ZBTB2 overexpression was analyzed .

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

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 are shown in left lower side of each image . Scale bar , 500 100 μm in low high magnification , respectively .

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

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 for of CA9 - positive tumors in four indicated groups . Percentages absolute numbers of each group are indicated in graph .

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

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 of disease - free survival of lung cancer stratified by expression of ZBTB2 p53 status .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 - Kaplan - Meier of overall survival of lung adenocarcinoma stratified by expression of ZBTB2 those of BAX ( D ) , ZMAT3 ( E ) , 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

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

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

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.


indicated transiently transfected with both pG5H1bLuc either pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A 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 indicated siRNA were then cultured under indicated oxygen conditions for 24 hours subjected to luciferase .

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

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.


indicated transiently transfected with both pG5H1bLuc 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 indicated siRNA were then cultured under indicated oxygen conditions for 24 hours subjected to luciferase .

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

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.


indicated transiently transfected with pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A 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 ) were then cultured under indicated oxygen conditions for 24 hours subjected to luciferase .

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

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.


indicated transiently transfected with both pG5H1bLuc 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 were then cultured under indicated oxygen conditions for 24 hours subjected to luciferase .

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

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

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

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

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 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

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

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 , same experiment as I was conducted using indicated combination of pcDNA4 / ZBTB2 - LgBiT ( WT ) , pcDNA4 / ZBTB2ΔZF2 - LgBiT ( ΔZF2 ) , or pcDNA4 / ZBTB2ΔZF3 - LgBiT ( ΔZF3 ) , 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

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 - / - co - transfected with pG5H1bLuc , pcDNA6 / Gal4 DBD - HIF - 1α TAD P564A , 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 subjected to luciferase .

O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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 expression vectors for ZBTB2 - V5 , ZBTB2 4A - V5 , ZBTB2 - myc , ZBTB2 4A - myc , or their empty vector , as indicated , myc - fused were immunoprecipitated using an anti - myc antibody co - precipitated ZBTB2 - V5 or ZBTB2 4A - V5 was detected ( upper ) . One - tenth of whole lysate ( WCL ) was subjected to immunoblotting with indicated antibodies ( lower ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 B-MEASURED_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 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 ) transiently transfected with or without either pcDNA4 / ZBTB2 - LgBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - LgBiT 4A ( 4A ) , or pcDNA4 / LgBiT ( - ) either pcDNA4 / ZBTB2 - SmBiT ( ZBTB2 ) , pcDNA4 / ZBTB2 4A - SmBiT ( 4A ) , or pcDNA4 / SmBiT ( - ) , as indicated , were cultured under < 0 . 1 % oxygen subjected to split luciferase complementation .

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 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 B-CONTROLLED_VAR 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 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 , same kind of luciferase to quantify 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 B-CONTROLLED_VAR 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

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 indicated xenografts was analyzed .

H, Growth of the indicated tumor xenografts was analyzed.


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 in M . bovis BCG using methylene blue as an oxygen sensor . ( D ) Oxygen consumption in M . bovis BCG using 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 last basal OCR reading before drug injection . OCR : Oxygen Consumption Rate . dotted lines indicate timepoints of drug injection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 presence of 100 nM Q203 ) measured on a Seahorse XFe96 analyser platform . For each condition , OCR readings were normalised to last basal OCR reading before drug injection .

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

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

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

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

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(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 drug - resistant clinical isolate M . tuberculosis 123 - 20 - 0047 .

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


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

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(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 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 6 µM ND - 011992 or 2 µg / mL rifampicin ( blue ) . corresponding values of median frequency of resistance is indicated in graph .

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

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(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 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 , ND - 011992 at 3 , 6 , 12 , 30 μM . viability was determined by enumerating colony - forming units ( CFU ) after 15 days of incubation . Dotted represents 90 % reduction in CFU / mL compared to initial inoculum at time 0 . * * P < 0 . 01 unpaired Student ' s t - , one - ; 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 . load ( CFU ) were enumerated in lungs of mice before treatment , after 5 days of treatment with either 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 - , two - ; 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.


of IL - 1β ( pg / ml ) in supernatant of macrophage cultures from COVID - 19 ( n = 44 ; red bars ) or SC - naïve individuals ( n = 24 ; blue bars ) stimulated with LPS or S - ( 0 . 1 µg / ml ) . To activate IL - 1β secretion nigericin was added for 2h . For statistical , two - way ANOVA with tukey post hoc was used . TNF - α ( pg / ml ) secreted from macrophages derived from SC - naïve COVID - 19 as described above . were stimulated with LPS ( SC - naïve / COVID - 19 n = 4 ) , LPS with nigericin ( SC - naïve n = 4 ; COVID - 19 n = 6 ) , S - ( SC - naïve n = 5 ; COVID - 19 n = 4 ) , S - with nigericin ( SC - naïve n = 3 ; COVID - 19 n = 4 ) or left unstimulated ( SC - naïve n = 3 ; COVID - 19 n = 4 ) . Significances shown in figure are always in comparison to 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 ) COVID - 19 ( bottom ) . Nuclei are stained with DAPI ( blue ) ASC speck are labeled by immunofluorescence ( green ) . Macrophages were stimulated with LPS ( left ) S - ( right ) for 4h followed by stimulation with nigericin in both cases . Pictures were taken with a 60x objective using 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 of IL - 1β secretion of macrophages after stimulation with S - nigericin from COVID - 19 with severe ( red / black ; n = 23 ) or mild ( / red ; n = 21 ) disease . Student ' s t - with Welsh corrections was used to calculate statistical differences . Graph shows mean ± .

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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 + of two convalescent individuals were isolated sequentially at d18 , d26 d56 after confirmation of SARS CoV2 infection by PCR . Following M - CSF differentiation , macrophages were stimulated with LPS ( red triangle ) or S - ( red circles ) IL - 1β ( pg / ml ) was quantified upon incubation with nigericin . Blue dashed indicates mean IL - 1β level of SC - naïve individuals stimulated with S - 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 - stimulated macrophages from convalescent COVID - 19 ( SC - conv ) compared to unstimulated . Negative log10 adjusted p - values are plotted against log2 fold - change . Select genes are labeled . P - values are calculated using Wald p - adjusted values using FDR / Benjamini - Hochberg approach .

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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 ( pg / ml ) in supernatants of COVID - 19 derived macrophages pretreated with DMSO ( n = 44 ) , KINK - 1 ( n = 11 ) MMG - 11 ( n = 19 ) subsequent stimulation with LPS nigericin . For statistical , 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 ( pg / ml ) in supernatants from macrophages of SC naïve ( blue ; n = 7 ) COVID - 19 ( 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 ) MMG - 11 ( SC - naïve n = 4 ; COVID - 19 n = 4 ) for 2h then stimulated with zymosan ( 4h ) nigericin ( 2h ) as indicated . For statistical , two - way ANOVA with tukey post hoc 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 ( pg / ml ) in supernatants from COVID - 19 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 ; 10 100 ng / ml : n = 7 ) S - ( 1 ng / ml : n = 12 ; 10 100 ng / ml : n = 13 ) for 4h both groups for 2h with nigericin . For statistical , 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 - derived macrophages ( SC - conv ) compared to macrophages from SARS - CoV - 2 naïve individuals ( SC - naïve ) . Negative log10 padj values are plotted against 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 ( pg / ml ) in supernatants of SC - naïve individuals ( n = 7 ) which were pre - treated for 24h with FCCP ( 1µg / ml ) H2O2 ( 10µg / ml ) . Macrophages were then incubated with LPS or S - ( 4h ) nigericin ( 2h ) as indicated in figure . Control were left unstimulated or stimulated with LPS or S - without pre - treatment with FCSP or H2O2 ( n = 6 ) . For statistical , Brown - Forsythe Welsh ANOVA 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 ) S100A7A / 8 / 9 / 12 ( bottom ) H3K27ac peaks ) comparing SC - naïve ( blue ) 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).


of IL - 1β concentrations ( pg / ml ) in supernatant of macrophage cultures from with active / untreated tuberculosis ( n = 6 ; red / black bars ) or healthy / SARS - CoV - 2 naïve individuals ( n = 3 ; blue bars ) or same after 6 months of anti - tuberculous treatment ; n = 6 ; / red bars ) stimulated with LPS or S - ( 0 . 1 µg / ml ) for 4h subsequent incubation with nigericin for 2h . For statistical , two - way ANOVA with tukey post hoc 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 entry efficiency of SARS - CoV - 2 RshSTT182 pseudoviruses in HeLa - hACE2 . Green indicates entry of pseudoviruses into Hela - hACE2 . Unaltered HeLa were used as a negative control . H Statistics for transduction of pseudoviruses . Data represents results of five replicates in one representative . data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

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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 SARS - CoV - 2 or RshSTT182 / 200 RBDs . BHK expressing eGFP - fused ACE2s were stained with indicated His - tagged . SARS - CoV - 2 NTD was used as a negative control .

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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 RshSTT182 / 200 or SARS - CoV - 2 RBDs . stained with RshSTT182 / 200 RBD , SARS - CoV - 2 RBD SARS - CoV - 2 NTD are shown in orange , marine red , respectively . 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 entry efficiency of SARS - CoV - 2 RshSTT182 pseudoviruses into HeLa - hACE2 . Green indicates entry of pseudoviruses into Hela - hACE2 . Unaltered HeLa were used as a negative control . E Statistics for transduction of pseudoviruses . Data represents results of five replicates in one representative . 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 neutralization curve of serum samples in COVID - 19 convalescents vaccinees against RshSTT182 SARS - CoV - 2 are shown . Bar represents 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 using MBP fused Apc1 - loop500 fragments B56γ . Apc1 - loop500 WT or its derivatives ( ∆ 11 or 3A ) were incubated with 35S - labelled Flag - B56γ in interphase extract ( Inter ) or anaphase extract ( Ana ) supplemented with CycB ∆ 167 at 23 ˚ C for 1 hr . bound were recovered by amylose beads , separated by SDS - PAGE detected by autoradiography or coomassie brilliant blue ( CBB ) . bar graph is of bound B56γ . 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 of Apc1 - loop500 . MBP fused WT or 3A Apc1 - loop500 fragment was incubated with Cdk2 - cyclin A in presence of [ γ - 32P ] - ATP at 23 ˚ C for 10 min , separated by SDS - PAGE 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 B56 interaction . MBP fused WT or 3A Apc1 - loop500 fragment was incubated in presence or absence of Cdk2 - cyclin A at 30 ˚ C for 60 min . MBP - fused peptides ( - / + kinase ) were isolated incubated with purified Flag - B56γ at 23 ˚ C for 30 min . bound were recovered by amylose beads analysed by SDS - PAGE immunoblotting with indicated antibodies . F . 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 in Xenopus egg extracts . 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 . APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads bound were analysed by SDS - PAGE immunoblotting with indicated antibodies . pApc1 ( pS314 / pS318 ) is a phospho - site specific antibody that binds only when both S314 S318 are phosphorylated . B . of A . 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 than WT APC / C . purified recombinant WT APC / C or B56 binding motif mutant APC / C ( 1 - L557A / V560A ) was incubated with ∆ APC anaphase extract . recovered APC / C - Cdc20 was subjected to ubiquitylation using 35S - labelled cyclin B as a substrate . Samples were taken at indicated time points analysed by SDS - PAGE autoradiography . D . of C . Error bars , s . e . m . from three independent experiments .

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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 . 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 . APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads bound were analysed by SDS - PAGE immunoblotting with indicated antibodies . ( right panel ) of bound Cdc20 . 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 incubated at 23 ˚ C for 50 min in anaphase extract . ( right panel ) of bound Cdc20 . intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

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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.


C . Cdc20 - 5A efficiently binds to B56 binding site mutant APC / C . purified recombinant WT APC / C or Apc1 - loop500 mutant APC / C ( 1 - L557A / V560A ) was incubated with WT Cdc20 or non - phosphorylatable Cdc20 mutant ( 5A ) in ∆ APC ∆ Cdc20 anaphase extract at 23 ˚ C for 55 min . APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads bound were analysed by SDS - PAGE immunoblotting with indicated antibodies . D . of C . intensities of WT APC / C control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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C. Cdc20-5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1-loop500 mutant APC/C (1-L557A/V560A) was incubated with WT Cdc20 or non-phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23˚C for 55 min. 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. D. Quantification of C. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


E . APC / C Cdc20 immunoprecipitated from CSF extract were incubated in presence of a range of concentrations or absence of PP2A - B56γ at 23 ˚ C . Samples were taken at indicated time points analysed by SDS - PAGE immunoblotting with antibodies , including phospho - site specific antibodies for pCdc20 ( pT79 ) pApc1 ( pS314 / pS318 ) .

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E. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A-B56γ at 23˚C. Samples were taken at the indicated time points and analysed by SDS-PAGE and immunoblotting with antibodies, including phospho-site specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318).


of Ronin fl / del Ronin del / del ESC . Data represent mean ± SD , n = three independent experiments .

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Cell count of Ronin fl/del and Ronin del/del ESC. Data represent mean ± SD, n = three independent experiments.


Annexin V of Ronin fl / del Ronin del / del cultured for 6 days . Two - unpaired Student ' s t , data represent mean ± SD , n = three independent experiments .

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Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments.


Ronin fl / del Ronin del / del ESC stained for Ronin ( left panel ) or Oct4 ( right panel ) . nuclei are counterstained with DAPI .

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Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.


Haematoxylin / eosin of paraffin sections of Ronin fl / del ecto - Ronin teratomas .

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Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas.


In vitro differentiation of Ronin fl / del , ecto - Ronin ( grown in presence of Dox ) Ronin del / del ESC .

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In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.


Volcano plot of gene expression in Ronin del / del day 4 vs . Ronin fl / del ( left panel ) Ronin del / del day 8 vs . Ronin fl / del ESC ( right panel ) with annotated naïve primed pluripotency markers .

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Volcano plot of gene expression in Ronin del/del day 4 vs. Ronin fl/del (left panel) and Ronin del/del day 8 vs. Ronin fl/del ESC (right panel) with annotated naïve and primed pluripotency markers.


Volcano plot of gene expression in Ronin del / del day 8 vs . Ronin del / del day 4 ESC .

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Volcano plot of gene expression in Ronin del/del day 8 vs. Ronin del/del day 4 ESC.


of nascent synthesis using OP - puro . Ronin fl / del treated with synthesis inhibitor ( cycloheximide ) are used as negative control . nuclei are counterstained with DAPI . of OP - puro signal , normalised to DAPI . Ordinary one - way Anova data represent mean ± SD ; n = three independent experiments for each genotype .

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Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.   Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.


Transmission electron microscopy of mitochondrial structure in Ronin fl / del Ronin del / del ESC . Mitochondria analysed , n > 150 for each condition .

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Transmission electron microscopy analysis of mitochondrial structure in Ronin fl/del and Ronin del/del ESC. Mitochondria analysed, n > 150 for each condition.


of OCR . Kruskal - Wallis , data represent mean ± SD , n = 16 technical replicates per genotype in three independent experiments .

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Quantification of the OCR analysis. Kruskal-Wallis test, data represent mean ± SD, n = 16 technical replicates per genotype in three independent experiments.


Blastocyst ( E4 . 5 ) diapause stained for Ronin , Sox2 Sox17 . Egg cylinder stage ( E5 . 5 ) stained for Ronin Sox17 . Nuclei are counterstained with DAPI .

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Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI.


Transmission electron microscopy of mitochondrial in epiblast of E4 . 5 , diapause E5 . 5 . Mitochondria analysed , E4 . 5 ( n mitochondria = 55 ) , diapause ( n mitochondria = 62 ) , E5 . 5 ( n mitochondria = 65 ) .

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Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4.5, diapause and E5.5 embryo. Mitochondria analysed, E4.5 (n mitochondria = 55), diapause (n mitochondria = 62), E5.5 (n mitochondria = 65).


E4 . 5 chimeric stained for tdTomato DAPI . of donor ' contribution in E4 . 5 blastocysts , Mann - Whitney , data represent mean ± SD ; Ronin fl / del n = 26 , Ronin del / del n = 27 . Diapause stained for tdTomato DAPI . of donor ' contribution in diapause , unpaired Student ' s t , data represent mean ± SD , Ronin fl / del n = 4 , Ronin del / del n = 12 . E5 . 5 chimeric stained for tdTomato DAPI . of donor ' contribution in E5 . 5 , unpaired Student ' s t , data represent mean ± SD , Ronin fl / del n = 21 , Ronin del / del n = 15 . Reactivated E5 . 5 chimeric stained for tdTomato DAPI . of donor ' contribution in reactivated E5 . 5 , Mann - Whitney , data represent mean ± SD , Ronin fl / del n = 17 , Ronin del / del n = 12 .

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E4.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.   Diapause embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.   E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.   Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.


a ) Auto - ubiquitination assays in which E3 acts as both an E3 as proxy substrate were performed with GST - HHARIRBR , T7 - Triad1ΔAri , GST - ParkinRBR , Flag - BRCA1 / BARD1 either UbcH5WT or UbcH5L104Q as E2 . Products were visualized by western blotting against indicated tags on E3s . Times given are post - ATP addition .

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a) Auto-ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST-HHARIRBR, T7-Triad1ΔAri, GST-ParkinRBR, and Flag-BRCA1/BARD1 and either UbcH5WT or UbcH5L104Q as the E2. Products were visualized by western blotting against indicated tags on E3s. Times given are post-ATP addition.


b ) Overlay of ( 1H , 15N ) - HSQC - TROSY spectra of 15N - UbcH5 - O - 15N - Ub in absence ( black ) presence ( red ) of 0 . 5mol equiv . HHARI RING1 . A subset of UbcH5 peaks , but not Ub peaks , shift broaden upon HHARI RING1 binding .

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b) Overlay of (1H,15N)-HSQC-TROSY spectra of 15N-UbcH5-O-15N-Ub in the absence (black) and presence (red) of 0.5mol equiv. HHARI RING1. A subset of UbcH5 peaks, but not Ub peaks, shift and broaden upon HHARI RING1 binding.


a ) Histogram of CSPs between free 15N - UbcH7 15N - UbcH7 - O - Ub identifies UbcH7 residues affected by conjugation to Ub . Active site ( Ser86 ) is indicated with a star cross - over helix residues ( 101 - 113 ) are marked with a gray cylinder . INSET . CSPs greater than 1 stdv ( > 0 . 115 ppm ) are highlighted in yellow on a surface representation of UbcH7 ( PDB 1fbv ) . view on left shows surface that contains UbcH7 cross - over helix .

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a) Histogram of CSPs between free 15N-UbcH7 and 15N-UbcH7-O-Ub identifies UbcH7 residues affected by conjugation to Ub. Active site (Ser86) is indicated with a star and cross-over helix residues (101-113) are marked with a gray cylinder. INSET. CSPs greater than 1 stdv (> 0.115 ppm) are highlighted in yellow on a surface representation of UbcH7 (PDB 1fbv). The view on the left shows the surface that contains the UbcH7 cross-over helix.


b ) Region of 1H - 15N - HSQC TROSY spectra containing resonances of cross - over helix residues S107 Q106 are overlaid : unconjugated 15N - UbcH7 ( blue ) , 15N - UbcH7 - O - Ub ( black ) , 15N - UbcH7 - O - UbI44A ( red ) . Peaks representing S107 Q106 in context of UbcH7 ~ Ub - I44A ( red ) are closer to peaks observed in free UbcH7 ( blue ) than UbcH7 ~ Ub - WT ( black ) indicating that I44A of Ub disrupts interactions with cross - over helix of UbcH7 .

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

b) Region of 1H-15N-HSQC TROSY spectra containing resonances of the cross-over helix residues S107 and Q106 are overlaid: unconjugated 15N-UbcH7 (blue), 15N-UbcH7-O-Ub (black), and 15N-UbcH7-O-UbI44A (red). Peaks representing S107 and Q106 in the context of UbcH7~Ub-I44A (red) are closer to peaks observed in free UbcH7 (blue) than UbcH7~Ub-WT (black) indicating that I44A of Ub disrupts interactions with the cross-over helix of UbcH7.


a ) Region of 1H - 15N - HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid : free 15N - Ub ( blue ) , UbcH7 - O - 15N - Ub ( black ) , HHARI RING1 - bound UbcH7 - O - 15N - Ub ( red ) . Black arrow highlights perturbation upon conjugation to UbcH7 ( black arrow ) red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7 .

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

a) Region of 1H-15N-HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid: free 15N-Ub (blue), UbcH7-O-15N-Ub (black), and HHARI RING1-bound UbcH7-O-15N-Ub (red). Black arrow highlights perturbation upon conjugation to UbcH7 (black arrow) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7.