Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 and the time from NEBD to exit was measured in each condition . Red circles represent cells still arrested in mitosis when the filming stopped .

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

Tethering of Mad1 (Mad1 485-718) to Ndc80 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Tethering of Mad1 ( Mad1 485 - 718 ) to Ndc80 , KNL1 ( Zhang et al , 2017 ) or Bub1 1 - 553ΔCD1 and the time from NEBD to exit was measured in each condition . Red circles represent cells still arrested in mitosis when the filming stopped .

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

Tethering of Mad1 (Mad1 485-718) to Ndc80, KNL1 (Zhang et al, 2017) or Bub1 1-553ΔCD1 and the time from NEBD to exit was measured in each condition. Red circles represent cells still arrested in mitosis when the filming stopped.


Localization of Venus - tagged Mad1 in parental HeLa cells , Bub1 CR cells and Rod CR cells in the presence of nocodazole . Scale bar , 5 μm .

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

Localization of Venus-tagged Mad1 in parental HeLa cells, Bub1 CR cells and Rod CR cells in the presence of nocodazole. Scale bar, 5 μm.


Still images from FRAP experiments using Venus - tagged Mad1 in the indicated conditions . Image before bleach is shown and then time following bleach . Arrowheads indicate kinetochore pairs . Scale bar , 5 μm for whole cell image and 1 μm for the zoom in on kinetochores .

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

Still images from FRAP experiments using Venus-tagged Mad1 in the indicated conditions. Image before bleach is shown and then time following bleach. Arrowheads indicate kinetochore pairs. Scale bar, 5 μm for whole cell image and 1 μm for the zoom in on kinetochores.


Analysis of Rod levels in cells used for FRAP experiment .

Analysis of Rod levels in cells used for FRAP experiment.


Venus - tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells ( left ) or in cells arrested with nocodazole ( right ) . Data combined from 3 independent experiments and standard deviation 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Venus-tagged Mad1 kinetochore intensity versus time from bleaching shortly after NEBD in unperturbed cells (left) or in cells arrested with nocodazole (right). Data combined from 3 independent experiments and standard deviation indicated.


A HeLa cell line stably expressing Mad1 - BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole . Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies . Representative of 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 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

A HeLa cell line stably expressing Mad1-BirA was depleted of Rod and synchronized in mitosis using thymidine and nocodazole. Biotin was added to media where indicated and biotinylated proteins were purified and analyzed by western blot with the indicated antibodies. Representative of 2 independent experiments.


The duration from NEBD to exit in each indicated conditions was recorded by time - lapse microscopy .

The duration from NEBD to exit in each indicated conditions was recorded by time-lapse microscopy.


Mitotic duration in the indicated conditions expressing Mad1 - Bub1 ( Mad1 485 - 718 fused to Bub1 1 - 553ΔCD1 ) fusion protein . The time from NEBD to exit is indicated . Red circles represent cells still arrested in mitosis when the filming stopped .

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

Mitotic duration in the indicated conditions expressing Mad1-Bub1 (Mad1 485-718 fused to Bub1 1-553ΔCD1) fusion protein. The time from NEBD to exit is indicated. Red circles represent cells still arrested in mitosis when the filming stopped.


Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs . ( D ) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition . Scale bar , 5 μm .

Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. (D) Representative immunofluorescence images of cells stained for YFP and Mad1 in each condition. Scale bar, 5 μm.


Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs . Mad1 kinetochore levels were measured and normalized to Bub1 ( YFP ) level . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown .

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

Cells depleted of Rod using RNAi and transfected with the indicated Bub1 constructs. Mad1 kinetochore levels were measured and normalized to Bub1 (YFP) level. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time - lapse microscopy . Mean ( red line ) and standard error of mean ( black bar ) 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

Bub1 CR cells complemented with the indicated Bub1 constructs and time from NEBD to exit in nocodazole measured by time-lapse microscopy. Mean (red line) and standard error of mean (black bar) indicated.


Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs . Mad1 levels are normalized to YFP signal . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown .

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

Kinetochore levels of Mad1 in Bub1 CR cells complemented with the indicated YFP tagged Bub1 constructs. Mad1 levels are normalized to YFP signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Still images of unperturbed mitosis of HeLa cells transfected with Venus - Bub1 4xCD1 . CFP - Histone 3 was used as the chromosome marker ( the middle panel ) and Venus - Bub1 4xCD1 was shown in the bottom panel . Scale bar , 5 μm .

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

Still images of unperturbed mitosis of HeLa cells transfected with Venus-Bub1 4xCD1. CFP-Histone 3 was used as the chromosome marker (the middle panel) and Venus-Bub1 4xCD1 was shown in the bottom panel. Scale bar, 5 μm.


HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis . The time from NEBD to exit was analyzed in each condition . Mean ( red line ) and standard error of mean ( black bar ) indicated . ( Mann Whitney test , ns : non - significant ) . A representative result from at least 3 independent experiments is shown .

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

HeLa cells transfected with the indicated Bub1 constructs and time from NEBD to exit determined during an unperturbed mitosis. The time from NEBD to exit was analyzed in each condition. Mean (red line) and standard error of mean (black bar) indicated. (Mann Whitney test, ns: non-significant). A representative result from at least 3 independent experiments is shown.


Localization of Venus - Rod during unperturbed mitosis in control depleted or Bub1 depleted cells .

Localization of Venus-Rod during unperturbed mitosis in control depleted or Bub1 depleted cells.


HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus - Bub1 constructs . Immunofluorescence images were shown for Bub1 and ZW10 localization . Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions .

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

HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus-Bub1 constructs. Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions.


HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization . Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions . with the indicated Bub1 fusion

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

HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions. with the indicated Bub1 fusion proteins


( B ) Representative images of β - Gal staining ( left panel ) and quantification of β - Gal positive cells ( right panel ) of BMSCs isolated from 2 ‑ month - old and 24 - month - old mice . Scale bar : 100 μm . β - Gal , beta ‑ galactosidase .

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

(B) Representative images of β-Gal staining (left panel) and quantification of β-Gal positive cells (right panel) of BMSCs isolated from 2‑month-old and 24-month-old mice. Scale bar: 100 μm. β-Gal, beta‑galactosidase.


( C ) Representative images of Alizarin Red staining at 21 days of osteogenic induction ( left panel ) and quantification of calcification ( right panel ) of BMSCs isolated from 2 - month - old and 24 - month - old mice by detecting the amount of Alizarin Red extracted from the matrix .

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

(C) Representative images of Alizarin Red staining at 21 days of osteogenic induction (left panel) and quantification of calcification (right panel) of BMSCs isolated from 2-month-old and 24-month-old mice by detecting the amount of Alizarin Red extracted from the matrix.


( A - B ) Representative images ( A ) of immunofluorescence staining and quantification ( B ) of YBX1 ( green ) in primary BMSCs .

O O O 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-B) Representative images (A) of immunofluorescence staining and quantification (B) of YBX1 (green) in primary BMSCs.


( C - D ) Representative immunohistochemical staining images of YBX1 ( red ) and LEPR ( green ) ( C ) and quantification of YBX1 + LEPR + cell numbers ( D ) in femoral bone marrow . MP , metaphysis ; T Ar , Tissue area . Arrows point to YBX1 + LEPR + cells .

(C-D) Representative immunohistochemical staining images of YBX1 (red) and LEPR (green) (C) and quantification of YBX1+ LEPR + cell numbers (D) in femoral bone marrow. MP, metaphysis; T Ar, Tissue area. Arrows point to YBX1+ LEPR + cells.


( E ) Age ‑ associated changes in YBX1 levels in human BMSCs from 30 males ( left panel ) and 30 females ( right panel ) .

O O O O 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) Age‑associated changes in YBX1 levels in human BMSCs from 30 males (left panel) and 30 females (right panel).


( F and G ) Representative images of Alizarin Red staining ( F ) and quantification of calcification ( G ) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) Representative images of Alizarin Red staining (F) and quantification of calcification (G) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction.


( H and I ) Representative images of Oil Red O staining ( H ) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix ( I ) in BMSCs after 10 days of adipogenic induction .

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

(H and I) Representative images of Oil Red O staining (H) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix (I) in BMSCs after 10 days of adipogenic induction.


( J and K ) Representative images of β - Gal staining ( J ) and quantification ( K ) of β - Gal positive cells among BMSCs .

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

(J and K) Representative images of β-Gal staining (J) and quantification (K) of β-Gal positive cells among BMSCs.


( L - M ) Relative mRNA levels of osteogenic differentiation related genes ( L ) , adipogenic differentiation and senescence related genes ( M ) between BMSCs transfected with adenovirus driven control and Ybx1 shRNA .

O O O O O O O O O O O O O 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-M) Relative mRNA levels of osteogenic differentiation related genes (L), adipogenic differentiation and senescence related genes (M) between BMSCs transfected with adenovirus driven control and Ybx1 shRNA.


( B - F ) Representative μCT images , Scale bar : 1mm . ( B ) and quantitative μCT analysis of trabecular bone microarchitecture ( C - F ) in distal femora from 3 - and 12 - month - old male Ybx1Prx1 - CKO mice and Ybx1flox / flox mice ( BV / TV , bone volume per tissue volume ; Tb . N , trabecular number ; Tb . Th , trabecular thickness ; Tb . Sp , trabecular separation ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative μCT analysis of trabecular bone microarchitecture (C-F) in distal femora from 3- and 12-month-old male Ybx1Prx1-CKO mice and Ybx1flox/flox mice (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).


( G ) Representative images of H & E staining ( Hematoxylin - Eosin Staining ) in distal femora . Scale bar : 100μm . ( H ) Quantification of the number of adipocytes related to the tissue area ( N . adipocytes / T . Ar ) . ( I ) Representative images of osteocalcin ( OCN ) immunohistochemical staining in distal femora . Arrows point to osteocalcin positive cells . Scale bar : 50 μ ( J ) Quantification of osteocalcin positive cells on the bone surface . Number of OCN + cells per bone perimeter ( N . Ocn + / B . Pm ) . ( K ) Representative images of TRAP staining in distal femora . Scale bar : 100 μm . ( L ) Quantification of TRAP positive cells on the bone surface . Number of TRAP + cells per bone perimeter ( N . Trap + / B . Pm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of H&E staining (Hematoxylin-Eosin Staining) in distal femora. Scale bar: 100μm. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin (OCN) immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. Scale bar: 50 μ (J) Quantification of osteocalcin positive cells on the bone surface. Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. Scale bar: 100 μm. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm).


( M ) Representative images of calcein double labeling of trabecular bone . Scale bar : 50 μm . ( N ) Quantification of the bone formation rate ( BFR ) based on calcein double labeling . ( O ) Quantification of the mineral apposition rate ( MAR ) based on calcein double labeling .

(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50 μm. (N) Quantification of the bone formation rate (BFR) based on calcein double labeling. (O) Quantification of the mineral apposition rate (MAR) based on calcein double labeling.


( E - F ) Representative μCT images , Scale bar : 1mm . ( E ) and quantitative μCT analysis of trabecular bone microarchitecture ( F ) in distal femora from 24 - month - old mice with rAAV8 - GFP - Ybx1 or rAAV8 - GFP transfection ( BV / TV , bone volume per tissue volume ; Tb . N , trabecular number ; Tb . Th , trabecular thickness ; Tb . Sp , trabecular separation ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative μCT images, Scale bar: 1mm. (E) and quantitative μCT analysis of trabecular bone microarchitecture (F) in distal femora from 24-month-old mice with rAAV8-GFP-Ybx1 or rAAV8-GFP transfection (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).


( G ) Representative images of H & E staining in distal femora . ( H ) Quantification of the number of adipocytes related to the tissue area ( N . adipocytes / T . Ar ) . ( I ) Representative images of osteocalcin immunohistochemical staining in distal femora . Arrows point to osteocalcin positive cells . ( J ) Quantification of osteocalcin positive cells in bone surface . Number of OCN + cells per bone perimeter ( N . Ocn + / B . Pm ) . ( K ) Representative images of TRAP staining in distal femora . ( L ) Quantification of TRAP positive cells on the bone surface . Number of TRAP + cells per bone perimeter ( N . Trap + / B . Pm ) . ( M ) Representative images of calcein double labeling of trabecular bone . ( N ) Quantification of the bone formation rate ( BFR ) and mineral apposition rate ( MAR ) based on calcein double labeling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of H&E staining in distal femora. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. (J) Quantification of osteocalcin positive cells in bone surface . Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). (M) Representative images of calcein double labeling of trabecular bone. (N) Quantification of the bone formation rate (BFR) and mineral apposition rate (MAR) based on calcein double labeling.


( C ) Semi - quantitative PCR showing the different isoforms of Fn1 , Nrp2 , Sp7 , and Sirt2 from BMSCs of Ybx1Prx1 - CKO mice and Ybx1flox / flox mice .

O O O O O O O O O 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) Semi-quantitative PCR showing the different isoforms of Fn1, Nrp2, Sp7, and Sirt2 from BMSCs of Ybx1Prx1-CKO mice and Ybx1flox/flox mice.


( D - G ) Representative images of Alizarin Red staining ( D and F ) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix ( E and G ) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Representative images of Alizarin Red staining (D and F) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (E and G) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1.


( H - I ) Representative images ( H ) and quantification of Oil Red O staining ( I ) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction .

O O O O O O O O O O O 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-I) Representative images (H) and quantification of Oil Red O staining (I) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction.


( J - K ) Representative images of β - Gal staining ( J ) and quantification ( K ) of β - Gal positive cells in BMSCs transfected with different isoforms of Sirt2 .

(J-K) Representative images of β-Gal staining (J) and quantification (K) of β-Gal positive cells in BMSCs transfected with different isoforms of Sirt2.


( L - T ) BMSCs were isolated from Ybx1Prx1 - CKO mice and Ybx1flox / flox mice , among them , the BMSCs from Ybx1flox / flox mice were transfected with the blank control , BMSCs from Ybx1Prx1 - CKO mice were transfected with the blank control or different isoforms of the target genes . ( L - P ) Representative images of Alizarin Red staining ( L and O ) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix ( M and P ) . ( Q - R ) Representative images ( Q ) and quantification of Oil Red O staining ( R ) in BMSCs after 10 days of adipogenic induction . ( S - T ) Representative images of β - Gal staining ( S ) and quantification ( T ) of β - Gal positive cells among BMSCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-T) BMSCs were isolated from Ybx1Prx1-CKO mice and Ybx1flox/flox mice, among them, the BMSCs from Ybx1flox/flox mice were transfected with the blank control, BMSCs from Ybx1Prx1-CKO mice were transfected with the blank control or different isoforms of the target genes. (L-P) Representative images of Alizarin Red staining (L and O) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (M and P). (Q-R) Representative images (Q) and quantification of Oil Red O staining (R) in BMSCs after 10 days of adipogenic induction. (S-T) Representative images of β-Gal staining (S) and quantification (T) of β-Gal positive cells among BMSCs.


( D ) Representative images of Alizarin Red staining ( up panel ) , Oil Red O staining ( middle panel , scale bar : 100 μm ) and β - Gal staining ( bottom panel , scale bar : 100 μm ) in BMSCs treated with different compounds . ( E - G ) Quantification of calcium mineralization based on Alizarin Red staining ( E ) , quantification of Oil Red O based on Oil Red O staining ( F ) , and quantification of β - Gal positive cells based on β ‑ Gal staining in BMSCs treated with different compounds ( G ) .

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

(D) Representative images of Alizarin Red staining (up panel), Oil Red O staining (middle panel, scale bar: 100 μm) and β-Gal staining (bottom panel, scale bar: 100 μm) in BMSCs treated with different compounds. (E-G) Quantification of calcium mineralization based on Alizarin Red staining (E), quantification of Oil Red O based on Oil Red O staining (F), and quantification of β-Gal positive cells based on β‑Gal staining in BMSCs treated with different compounds (G).


( K ) Co - IP of His - FBXO33 with HA - YBX1 and a series of mutant HA - YBX1 proteins following transfection into BMSCs .

(K) Co-IP of His-FBXO33 with HA-YBX1 and a series of mutant HA-YBX1 proteins following transfection into BMSCs.


( M ) Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin .

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

(M) Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin.


( N ) Semi - quantitative PCR showed the isoforms of Fn1 , Nrp2 and Sirt2 in cultured BMSCs isolated from 2 - month - old or 24 - month - old mice then treated with or without sciadopitysin .

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

(N) Semi-quantitative PCR showed the isoforms of Fn1, Nrp2 and Sirt2 in cultured BMSCs isolated from 2-month-old or 24-month-old mice then treated with or without sciadopitysin.


( B - F ) Representative μCT images , Scale bar : 1mm . ( B ) and quantitative analysis of trabecular bone microarchitecture ( C - F ) in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle ( BV / TV , bone volume per tissue volume ; Tb . N , trabecular number ; Tb . Th , trabecular thickness ; Tb . Sp , trabecular separation ) .

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

(B-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative analysis of trabecular bone microarchitecture (C-F) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).


( G , H ) Representative images ( G ) and quantification ( H ) of osteocalcin ( OCN ) positive cells in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle . Number of Ocn + cells per bone perimeter ( N . Ocn + / B . Pm ) . Arrows point to osteocalcin positive cells . Scale bar : 50

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

(G, H) Representative images (G) and quantification (H) of osteocalcin (OCN) positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of Ocn+ cells per bone perimeter (N. Ocn+/B.Pm). Arrows point to osteocalcin positive cells. Scale bar: 50 μ


( I , J ) Representative images ( I ) of H & E staining in distal femora and quantification ( J ) of the number of adipocytes related to the tissue area ( right panel , N . adipocytes / T . Ar ) in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle . Scale bar : 100 μm .

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

(I, J) Representative images (I) of H&E staining in distal femora and quantification (J) of the number of adipocytes related to the tissue area (right panel, N. adipocytes/T.Ar) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Scale bar: 100 μm.


( K , L ) Representative images ( K ) and quantification ( L ) of TRAP positive cells in distal femora of 25 - month - old mice administered with sciadopitysin or vehicle . Number of TRAP + cells per bone perimeter ( N . Trap + / B . Pm ) . Scale bar : 50 μm .

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

(K, L) Representative images (K) and quantification (L) of TRAP positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). Scale bar: 50 μm.


( M ) Representative images of calcein double labeling of trabecular bone . Scale bar : 50μm . ( N , O ) Quantification of the bone formation rate ( BFR , N ) and mineral apposition rate ( MAR , O ) based on calcein double labeling .

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

(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50μm. (N, O) Quantification of the bone formation rate (BFR, N) and mineral apposition rate (MAR, O) based on calcein double labeling.


Representative density plots of Ca2 + flux from conjugated OptoCAR ‑ T cells over time at 37ºC . Conjugated cells were stimulated with dimerizer addition for 0 ( F ) , 5 ( G ) , 10 ( H ) , 15 ( I ) or 30 ( J ) minutes prior to disrupting signaling by illuminating cells .

O O O O B-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O

Representative density plots of Ca2+ flux from conjugated OptoCAR‑T cells over time at 37ºC. Conjugated cells were stimulated with dimerizer addition for 0 (F), 5 (G), 10 (H), 15 (I) or 30 (J) minutes prior to disrupting signaling by illuminating cells.


OptoCAR ‑ T cells were conjugated with ligand - presenting cells and activated as in the upper regime shown in ( A ) . The median of ppERK intensity distribution was plotted with time , after subtraction of background intensity . Bars show mean ± SEM ( n = 3 ) . An equivalent experiment was performed as in ( B ) but following the middle regime shown in ( A ) , with sample illumination after 5 minutes for the remainder of the dataset . Indicated half - life was calculated from the time taken for the median ppERK to decrease to 50 % of maximum . Bars show mean ± SEM ( n = 4 ) . An equivalent experiment was performed as in ( B ) but following the lower regime shown in ( A ) , with sample illumination after 5 minutes for 3 minutes . Bars show mean ± SEM ( n = 3 ) .

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

OptoCAR‑T cells were conjugated with ligand-presenting cells and activated as in the upper regime shown in (A). The median of ppERK intensity distribution was plotted with time, after subtraction of background intensity. Bars show mean ± SEM (n=3). An equivalent experiment was performed as in (B) but following the middle regime shown in (A), with sample illumination after 5 minutes for the remainder of the dataset. Indicated half-life was calculated from the time taken for the median ppERK to decrease to 50% of maximum. Bars show mean ± SEM (n=4). An equivalent experiment was performed as in (B) but following the lower regime shown in (A), with sample illumination after 5 minutes for 3 minutes. Bars show mean ± SEM (n=3).


Representative fluorescent Western blot showing the dynamics of FOS phosphorylation ( p - FOSS32 ) on light - modulated control . Conjugated OptoCAR ‑ T cells were stimulated for a defined period after dimerizer addition ( denoted above blots ) before either being left in the dark ( dark line ) or illuminated ( blue line ) . Full blot image with total protein normalization control shown in Figure EV3A . Representative fluorescent Western blot showing the dynamics of FOS expression ( FOSTotal ) on light - modulated control , from the same dataset as in ( A ) . Full blot image with total protein normalization control shown in Figure EV3B .

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

Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A. Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.


RT - qPCR analysis of relative mRNA levels for four representative genes following OptoCAR - mediated T cell activation . OptoCAR ‑ T cell conjugates were activated in the dark ( signaling competent ) state for a period of 3 hours before light - mediated disruption of signaling ( open circles ) or maintained for a further 60 minutes in the dark ( filled circles ) . The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3 - 4 hours . Individual datasets are presented as gray lines , with bars showing mean ± SEM ( n = 3 ) . Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1 .

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

RT-qPCR analysis of relative mRNA levels for four representative genes following OptoCAR-mediated T cell activation. OptoCAR‑T cell conjugates were activated in the dark (signaling competent) state for a period of 3 hours before light-mediated disruption of signaling (open circles) or maintained for a further 60 minutes in the dark (filled circles). The relative mRNA values were calculated by subtracting baseline value before scaling to mean output between 3-4 hours. Individual datasets are presented as gray lines, with bars showing mean ± SEM (n=3). Raw cRQ plots for all datasets are provided in Appendix Fig S2 and RQ values can be found in Dataset EV1.


Single pulses of varying duration were applied to OptoCAR ‑ T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling , with illumination started once the signal pulse had ended . All values have been corrected for background GFP fluorescence from non - activated OptoCAR ‑ T cells and shown relative to the output at 24 hours . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 ) Equivalent datasets as in ( B ) for the geometric mean of CD69 upregulation . A representative dataset is provided in Figure EV4F . Bars show mean ± SEM ( n = 3 )

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

Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)


The cRQ values from RT - qPCR of indicated cells . Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells . Conjugated OptoCARCD19 ‑ T cells were also assayed either before or after 24 hours stimulation in the dark state . Individual values from three biological replicates are shown , with mean depicted by bar plot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot.


A . In vivo genetic complementation of the E . coli MC4100 wild - type ( WT ) and secB or / and tig knock - out derivatives by either an empty vector or one carrying the secB or tig ( encoding TF ) genes or derivatives ( see " Plasmid " table in Appendix ) , as indicated . Serial dilutions of a culture ( OD600 = 0 . 5 ) were spotted ( 12μl ) on LB - Ampicillin plates containing or not anhydrotetracycline ( AHT ; 5 ng / ml ) and grown at 16 ° C or 30 ° C ( as indicated ) . n = 3 - 7 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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. In vivo genetic complementation of the E. coli MC4100 wild-type (WT) and secB or/and tig knock-out derivatives by either an empty vector or one carrying the secB or tig (encoding TF) genes or derivatives (see "Plasmid" table in Appendix), as indicated. Serial dilutions of a culture (OD600= 0.5) were spotted (12μl) on LB-Ampicillin plates containing or not anhydrotetracycline (AHT; 5 ng/ml) and grown at 16°C or 30°C (as indicated). n= 3-7 biological replicates.


C . SecA ATPase activity determination in solution ( basal ; B ) , or plus SecYEG IMVs ; ( membrane ; M ) or plus proOmpA ( pre - treated with 10 mM DTT ; 5 mM EDTA ; 20 min ; 4 ° C ; translocation ; T ) . n = 21 biological replicates . Mean values ( ± SEM ) shown .

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

C. SecA ATPase activity determination in solution (basal; B), or plus SecYEG IMVs; (membrane; M) or plus proOmpA (pre-treated with 10 mM DTT; 5 mM EDTA; 20 min; 4°C; translocation; T). n= 21 biological replicates. Mean values (± SEM) shown.


D . proOmpA translocation ATPase activity stimulation of SecA ( as in C ) in the absence or presence of TF2 or / and SecB4 , added at the indicated molar excess over proOmpA1 . The T / M ratios were calculated ; the one in the absence of chaperones was considered as 100 % , all other values were expressed as % of this . n = 2 - 4 biological replicates . Mean values ( ± SEM ) shown . Unpaired parametric t - test , 95 % confidence interval : ns : not significant ( p = 0 . 1032 ) ; * * * * : p < 0 . 0001 ; * * : p = 0 . 0037 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. proOmpA translocation ATPase activity stimulation of SecA (as in C) in the absence or presence of TF2 or/and SecB4, added at the indicated molar excess over proOmpA1. The T/M ratios were calculated; the one in the absence of chaperones was considered as 100%, all other values were expressed as % of this. n= 2-4 biological replicates. Mean values (± SEM) shown. Unpaired parametric t-test, 95% confidence interval: ns: not significant (p = 0.1032); ****: p < 0.0001; **: p = 0.0037.


E . Titrated TF - mediated inhibition ( 0 - 6 µM range ) of proOmpA - stimulated translocation ATPase activity ( as in C ) . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) and plotted ( n = 1 - 4 biological replicates ; mean values ± SEM ) versus the log10 [ TF2 ] . The Ki was determined using a variable slope fit ( log [ inhibitor ] versus normalized response ; GraphPad Prism ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Titrated TF-mediated inhibition (0-6 µM range) of proOmpA-stimulated translocation ATPase activity (as in C). Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 1-4 biological replicates; mean values ± SEM) versus the log10[TF2]. The Ki was determined using a variable slope fit (log[inhibitor] versus normalized response; GraphPad Prism).


F . SecB - mediated relief of TF - mediated inhibition of proOmpA translocation ATPase activity ( as in C ) . TF2 ( 1μM ) and SecB4 ( 0 - 6 µM ) were used . The T / M ratios were calculated , normalized ( as in D ) and plotted ( n = 2 - 3 biological replicates ; mean values ± SEM ) versus the log10 [ SecB4 ] . An apparent relief constant ( Krel ) was determined ( as in E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB-mediated relief of TF-mediated inhibition of proOmpA translocation ATPase activity(as in C). TF2 (1μM) and SecB4 (0-6 µM) were used. The T/M ratios were calculated, normalized (as in D) and plotted (n= 2-3 biological replicates; mean values ± SEM) versus the log10[SecB4]. An apparent relief constant (Krel) was determined (as in E).


A . TF - proOmpA physical interaction in solution . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) with the indicated molar excess of TF2 . Soluble proteins were analyzed on 10 % native - PAGE ( 4 mA ; 16h ; 4 ° C ) and Coomassie - blue stained . A representative experiment is shown ; n = 4 biological replicates

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TF-proOmpA physical interaction in solution. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) with the indicated molar excess of TF2. Soluble proteins were analyzed on 10% native-PAGE (4 mA; 16h; 4°C) and Coomassie-blue stained. A representative experiment is shown; n= 4 biological replicates


C . TF solubilizes proOmpA * . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 37 ° C ; 60 min ) alone or in the presence of TF . Proteins in the soluble fraction were analyzed on 15 % SDS - PAGE and Coomassie - blue stained . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 5 biological replicates

C. TF solubilizes proOmpA*. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 37°C; 60 min) alone or in the presence of TF. Proteins in the soluble fraction were analyzed on 15% SDS-PAGE and Coomassie-blue stained. Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 5 biological replicates


D . proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity . proOmpA ( 0 . 5 μM ) was either diluted directly ( lane 1 ) or after preincubation alone ( 10 min ; buffer D ; < 0 . 2 M urea ; either at 4 ° C or 37 ° C ) into reactions containing SecYEG - SecA alone or together with TF2 or SecB4 ( 2 and 4 x molar excess over proOmpA , respectively ) . T / M ratios ( as in Fig . 1D ) are shown ( n = 8 biological replicates ; mean values ± SEM ; Unpaired parametric t - test , 95 % confidence interval : * * : p = 0 . 0017 ; * * * * : p < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. proOmpA preincubated at 37oC fails to stimulate SecA ATPase activity. proOmpA (0.5 μM) was either diluted directly (lane 1) or after preincubation alone (10 min; buffer D; < 0.2 M urea; either at 4°C or 37°C) into reactions containing SecYEG-SecA alone or together with TF2 or SecB4 (2 and 4 x molar excess over proOmpA, respectively). T/M ratios (as in Fig. 1D) are shown (n= 8 biological replicates; mean values ± SEM; Unpaired parametric t-test, 95% confidence interval: **: p = 0.0017; ****: p < 0.0001)


E . proOmpA pre - incubated at 37oC does not interact with TF . proOmpA1 ( 5 μM ) was preincubated ( Buffer D ; < 0 . 2 M urea ; 37 ° C ; 10 min ) or not ( lane 3 ) , prior to addition of the indicated molar excess of TF2 ( analyzed as in A ) . Representative experiment shown ; n = 4 biological replicates

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. proOmpA pre-incubated at 37oC does not interact with TF. proOmpA1 (5 μM) was preincubated (Buffer D; < 0.2 M urea; 37°C; 10 min) or not (lane 3), prior to addition of the indicated molar excess of TF2 (analyzed as in A). Representative experiment shown; n= 4 biological replicates


B . SecB protects proOmpA * from aggregation . proOmpA * 1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; 37 ° C ; 60 min ) alone or in the presence of the indicated molar excess of SecB4 , and proOmpA * in the soluble fraction ( after centrifugation ) was analyzed by SDS - PAGE ( as in Fig . 2C ) . Lane 2 : 2 . 84 μg proOmpA * . Representative experiment shown ; n = 4 biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB protects proOmpA* from aggregation. proOmpA*1 (5 μM) was incubated (50 μl; Buffer C; 37°C; 60 min) alone or in the presence of the indicated molar excess of SecB4, and proOmpA* in the soluble fraction (after centrifugation) was analyzed by SDS-PAGE (as in Fig. 2C). Lane 2: 2.84 μg proOmpA*. Representative experiment shown; n= 4 biological replicates


C . SecB physically interacts with proOmpA . proOmpA1 ( 5 μM ) was incubated ( 50 μl ; Buffer C ; < 0 . 2 M urea ; 4 ° C ; 60 min ) , with the indicated molar excess of SecB4 ( filled arrow ) , analyzed by native - PAGE as in Fig . 2A , and stained as indicated . brace : higher order proOmpA species . Representative experiment shown ; n = 5 biological replicates

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB physically interacts with proOmpA. proOmpA1 (5 μM) was incubated (50 μl; Buffer C; < 0.2 M urea; 4°C; 60 min), with the indicated molar excess of SecB4 (filled arrow), analyzed by native-PAGE as in Fig. 2A, and stained as indicated. brace: higher order proOmpA species. Representative experiment shown; n= 5 biological replicates


B . Blow up of the ESI - mass spectrum of the TF : proOmpA : SecB4 super - complex from A ( complete dataset in Fig . EV3 ) . Left : charge state distributions , at the indicated m / z range . Right : masses deconvoluted from the mass spectra ( as in Fig . 2B ) . Inset cartoon depicts stoichiometry . Representative spectrum shown ; n = 2 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blow up of the ESI-mass spectrum of the TF:proOmpA:SecB4 super-complex from A (complete dataset in Fig. EV3). Left: charge state distributions, at the indicated m/z range. Right: masses deconvoluted from the mass spectra (as in Fig. 2B). Inset cartoon depicts stoichiometry. Representative spectrum shown; n=2 biological replicates.


C . TF : proOmpA : SecB supercomplex analysis using GPC - MALS in Buffer A . TF1 : proOmpA1 ( 10 µM each ; green ) , SecB4 ( 40 µM ; blue ) and TF1 : proOmpA1 : SecB4 ( 10 , 10 and 40 µM , respectively ; black ) . n = 5 biological replicates ; mean mass : 137 kDa ( ± 6 kDa ) representative experiment shown ; red circles : determined mass ( kDa ) ; AU : arbitrary units .

C. TF:proOmpA:SecB supercomplex analysis using GPC-MALS in Buffer A. TF1:proOmpA1 (10 µM each; green), SecB4 (40 µM; blue) and TF1:proOmpA1:SecB4 (10, 10 and 40 µM, respectively; black). n= 5 biological replicates; mean mass: 137 kDa (± 6 kDa) representative experiment shown; red circles: determined mass (kDa); AU: arbitrary units.


A . SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA ( noC - tail ) in the presence of TF . proOmpA translocation ATPase activity stimulation ( as in Fig . 1C ; SecB molar excess over proOmpA indicated ) . T / M ratio of SecA ( noC - tail ) in the absence of TF or SecB = 100 % ; lane 1 ; all other values were expressed as a % of this value . n = 4 biological replicates ; mean values ± SEM . Unpaired parametric t - test , 95 % confidence interval : * * : p = 0 . 0048 ; * * * * : p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SecB fails to restore proOmpA translocation ATPase activity stimulation of SecA(noC-tail) in the presence of TF. proOmpA translocation ATPase activity stimulation (as in Fig. 1C; SecB molar excess over proOmpA indicated). T/M ratio of SecA(noC-tail) in the absence of TF or SecB= 100%; lane 1; all other values were expressed as a % of this value. n= 4 biological replicates; mean values ± SEM. Unpaired parametric t-test, 95% confidence interval: **: p= 0.0048; ****: p < 0.0001.


B . Excess of SecB inhibits the proOmpA - stimulated translocation ATPase activity of SecA ( noCtail ) . ATPase measured as in Fig . 1C ; SecB concentration range : 0 - 6 µM . Values were normalized ( effectmax = 100 % ; effectmin = 0 % ) and plotted ( n = 3 biological replicates ; mean values ± SEM ) versus the log10 [ SecB4 ] . The Ki was determined as in Fig . 1E .

B. Excess of SecB inhibits the proOmpA-stimulated translocation ATPase activity of SecA(noCtail). ATPase measured as in Fig. 1C; SecB concentration range: 0-6 µM. Values were normalized (effectmax=100%; effectmin=0%) and plotted (n= 3 biological replicates; mean values ± SEM) versus the log10[SecB4]. The Ki was determined as in Fig. 1E.


C . TF and SecB - regulated , SecA - dependent in vitro translocation of proOmpA into SecYEG / A - IMVs , analyzed after proteolytic treatment . Translocated , protease - resistant proOmpA was visualized using immunostaining ( α - His ) in the absence or presence of TF2 or / and SecB4 ( as indicated ) . Translocation in the absence of TF or SecB was considered 100 % ( lane 3 ) ; all other values were expressed as a percentage of this . A summary of all repeats is presented ( mean ± SEM ; n = 6 biological replicates ; ns : not significant ( p = 0 . 1568 for lane 3 vs 4 ; p = 0 . 4835 for lane 3 vs 6 ) ; Unpaired parametric t - test , 95 % confidence interval : * * * * : p < 0 . 0001 ) with a representative western blot ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TF and SecB-regulated, SecA-dependent in vitro translocation of proOmpA into SecYEG/A-IMVs, analyzed after proteolytic treatment. Translocated, protease-resistant proOmpA was visualized using immunostaining (α-His) in the absence or presence of TF2 or/and SecB4 (as indicated). Translocation in the absence of TF or SecB was considered 100% (lane 3); all other values were expressed as a percentage of this. A summary of all repeats is presented (mean ± SEM; n= 6 biological replicates; ns: not significant (p = 0.1568 for lane 3 vs 4; p = 0.4835 for lane 3 vs 6); Unpaired parametric t-test, 95% confidence interval: ****: p < 0.0001) with a representative western blot (bottom).


Immunofluorescence staining of apoE44 ( n = 3 ) and apoE23 ( n = 2 ) genotyped iNPH patient right frontal cortex biopsy samples Two representative microscopy images from six separate images show : ( A ) Colocalization of ( red ) apoE44 and ( green ) FH in the presence of ( white ) Aβ plaques . Scale bar = 50 μm . ( B ) Colocalization of ( red ) apoE23 and ( green ) FH around brain capillaries . The blue nuclei were detected using DAPI staining . ( C ) Colocalization of ( red ) apoE44 and ( green ) FH in the presence of ( white ) Aβ plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer ' s clinical syndrome ( ACS ) . Colocalization of apoE on Aβ is shown with an arrow . The negative staining control is shown . ( D ) ApoE - FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 ( n = 3 ) and apoE23 ( n = 2 ) genotyped iNPH patient biopsy samples . Six microscope images from Alzheimer ' s clinical syndrome ( ACS ) biopsy sample are included in the apoE44 dataset ( red dots ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O 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 staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples Two representative microscopy images from six separate images show: (A) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques. Scale bar = 50 μm. (B) Colocalization of (red) apoE23 and (green) FH around brain capillaries. The blue nuclei were detected using DAPI staining. (C) Colocalization of (red) apoE44 and (green) FH in the presence of (white) Aβ plaques from a biopsy sample obtained from an iNPH patient diagnosed with Alzheimer's clinical syndrome (ACS). Colocalization of apoE on Aβ is shown with an arrow. The negative staining control is shown. (D) ApoE-FH colocalization analysis of all detected colocalized foci in six microscope images from the apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient biopsy samples. Six microscope images from Alzheimer's clinical syndrome (ACS) biopsy sample are included in the apoE44 dataset (red dots).


Immunofluorescence staining of apoE44 ( n = 3 ) and apoE23 ( n = 2 ) genotyped iNPH patient right frontal cortex biopsy samples ( I ) MST showing binding of NT647 - labeled full - length FH to different apoE isoforms , ( J ) NT647 - labeled apoE2 ( 1 - 165 ) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1 - 4 and 19 - 20 and ( K ) NT647 - labeled recombinant N - terminal apoE2 ( 1 - 165 ) to increasing concentrations of FH5 - 7 402Y and FH5 - 7 402H fragments .

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Immunofluorescence staining of apoE44 (n=3) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples (I) MST showing binding of NT647-labeled full-length FH to different apoE isoforms, (J) NT647-labeled apoE2(1-165) fragment to increasing concentrations of FH402Y and FH402H variants and FH domains 1-4 and 19-20 and (K) NT647-labeled recombinant N-terminal apoE2(1-165) to increasing concentrations of FH5-7 402Y and FH5-7 402H fragments.


( B ) Different mixtures of apoE isoforms , FH , FH19 - 20 and Aβ1 - 42 were incubated for 72 h , and the presence of stable complexes was analyzed by running the sample in Tris - glycine PAGE for ( left and middle ) 90 or ( right ) 60 min at 100 V in the presence of 1 Bolt ™ LDS Sample Buffer ( Thermo Fisher ) . Gels were subjected to silver staining ( left ) or WB using anti - Aβ antibody ( middle and right ) . ApoE isoform and incubation time are shown on the top . The presence of each protein is indicated with arrows . Aβ1 - 42 aggregate and the stable complexes formed between apoE , Aβ1 - 42 and FH are shown near the top of the gel , while apoE2 / Aβ1 - 42 formed in the absence of FH are shown in several bands below apoE / Aβ1 - 42 / FH complexes .

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(B) Different mixtures of apoE isoforms, FH, FH19-20 and Aβ1-42 were incubated for 72 h, and the presence of stable complexes was analyzed by running the sample in Tris-glycine PAGE for (left and middle) 90 or (right) 60 min at 100 V in the presence of 1 Bolt™ LDS Sample Buffer (Thermo Fisher). Gels were subjected to silver staining (left) or WB using anti-Aβ antibody (middle and right). ApoE isoform and incubation time are shown on the top. The presence of each protein is indicated with arrows. Aβ1-42 aggregate and the stable complexes formed between apoE, Aβ1-42 and FH are shown near the top of the gel, while apoE2/Aβ1-42 formed in the absence of FH are shown in several bands below apoE/Aβ1-42/FH complexes.


( C ) Single - molecule TIRF micrograph of Hylite 488 labeled Aβ1 - 42 and Alexa Fluor 568 C5 Maleimide labeled apoE2 . Scale bar = 5 μm . ( D ) Jitter plot of proportion of colocalized Aβ1 - 42 / apoE2 and Aβ1 - 42 / apoE3 over multiple fields of view in the presence and absence of FH . Statistics was calculated using Student ' s t - test from multiple images ( n = 12 ) in each sample . Error bars indicate SD values . (

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(C) Single-molecule TIRF micrograph of Hylite 488 labeled Aβ1-42 and Alexa Fluor 568 C5 Maleimide labeled apoE2. Scale bar = 5 μm. (D) Jitter plot of proportion of colocalized Aβ1-42/apoE2 and Aβ1-42/apoE3 over multiple fields of view in the presence and absence of FH. Statistics was calculated using Student's t-test from multiple images (n=12) in each sample. Error bars indicate SD values. (


( E ) Jitter plot of Aβ1 - 42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH . Statistics of multiple images was calculated using two - sided Student ' s t - test . Statistics was calculated using Student ' s t - test from all detected colocalized foci in multiple images ( n = 12 ) in each sample . Error bars indicate SD values .

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(E) Jitter plot of Aβ1-42 foci stoichiometry in number of fluorophores colocalized or not with apoE2 and apoE3 and in the presence and absence of FH. Statistics of multiple images was calculated using two-sided Student's t-test. Statistics was calculated using Student's t-test from all detected colocalized foci in multiple images (n=12) in each sample. Error bars indicate SD values.


( A and B ) Immunofluorescence staining of apoE44 ( n = 2 ) and apoE23 ( n = 2 ) genotyped iNPH patient right frontal cortex biopsy samples showing localization of FH ( green ) with Iba - 1 stained cells ( red ) and clustering around cells and brain capillaries . Presence of ( white ) Aβ plaques in apoE44 samples and ( arrow ) FH in both samples are shown . . C - E ( C ) Iba - 1 , ( D ) Aβ and ( E ) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 ( n = 2 ) and apoE23 ( n = 2 ) genotyped iNPH patient biopsy samples .

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(A and B) Immunofluorescence staining of apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient right frontal cortex biopsy samples showing localization of FH (green) with Iba-1 stained cells (red) and clustering around cells and brain capillaries. Presence of (white) Aβ plaques in apoE44 samples and (arrow) FH in both samples are shown.. C-E (C) Iba-1, (D) Aβ and (E) FH intensity analysis was calculated from all detected foci in six microscope images from the apoE44 (n=2) and apoE23 (n=2) genotyped iNPH patient biopsy samples.


( F ) Flow cytometric experiments showing binding of ( left ) 405 - labeled apoE4 and ( right ) 405 - labeled apoE2 to activated or inactivated CR3 on U937 empty and U937 - CR3 cells in the presence of FH , FH fragments 19 - 20 and 5 - 7 , BSA and Aβ1 - 42 .

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(F) Flow cytometric experiments showing binding of (left) 405-labeled apoE4 and (right) 405-labeled apoE2 to activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH, FH fragments 19-20 and 5-7, BSA and Aβ1-42.


( I ) Binding of 488 - labeled Aβ1 - 42 to ( left ) activated or inactivated CR3 on U937 empty and U937 - CR3 cells in the presence of FH19 - 20 , BSA , with combinations of apoE4 and FH , apoE2 and FH or apoE3 and FH . The fluorescence intensities of 488 - Aβ1 - 42 in each experiment was normalized against activated U937 empty cells sample .

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(I) Binding of 488-labeled Aβ1-42 to (left) activated or inactivated CR3 on U937 empty and U937-CR3 cells in the presence of FH19-20, BSA, with combinations of apoE4 and FH, apoE2 and FH or apoE3 and FH. The fluorescence intensities of 488-Aβ1-42 in each experiment was normalized against activated U937 empty cells sample.


( A ) Immunofluorescence staining of iNPH patient biopsy samples ( apoE33 ) showing ( arrow ) colocalization of complement activator , ( red ) C1q , and complement regulator ( green ) FH . ( B ) ( arrow ) Colocalization of ( green ) apoE and ( red ) C1q on ( white ) Aβ plaques . Colocalization analysis is calculated from colocalized foci in two separate microscope images .

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(A) Immunofluorescence staining of iNPH patient biopsy samples (apoE33) showing (arrow) colocalization of complement activator, (red) C1q, and complement regulator (green) FH. (B) (arrow) Colocalization of (green) apoE and (red) C1q on (white) Aβ plaques. Colocalization analysis is calculated from colocalized foci in two separate microscope images.


( D ) C3a formation in supernatant of C3 , factor B ( fB ) , factor D ( fD ) and factor I ( fI ) incubated beads coupled with apoE2 / 488 - Aβ1 - 42 , apoE4 / 488 - Aβ1 - 42 , apoE2 / 488 - Aβ1 - 42 / FH , apoE4 / 488 - Aβ1 - 42 / FH , 488 - Aβ1 - 42 / FH complexes . Beads with488 - Aβ1 - 42 / BSA complexes shows the level of C3a formation without inhibitor . Release of C3a is calculated as relative C3a increase % compared to the absorbance measured from 488 - Aβ1 - 42 / FH sample .

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(D) C3a formation in supernatant of C3, factor B (fB), factor D (fD) and factor I (fI) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH complexes. Beads with488-Aβ1-42/BSA complexes shows the level of C3a formation without inhibitor. Release of C3a is calculated as relative C3a increase % compared to the absorbance measured from 488-Aβ1-42/FH sample.


( G ) ( left ) Phagocytosis of complement C3 , fB , fD and fI ( C ) or only buffer ( no C ) incubated beads coupled with apoE2 / 488 - Aβ1 - 42 , apoE4 / 488 - Aβ1 - 42 , apoE2 / 488 - Aβ1 - 42 / FH , apoE4 / 488 - Aβ1 - 42 / FH , 488 - Aβ1 - 42 / FH and 488 - Aβ1 - 42 / BSA ( oligomerized Aβ = OAβ ) or with non - oligomerized 488 - Aβ1 - 42 ( monomeric Aβ = MAβ ) . Also , samples without apoE or FH were included in the assay ( buffer ) . ( right ) Cell permeability of C3 , fB , fD and fI incubated beads coupled with apoE2 / 488 - Aβ1 - 42 , apoE4 / 488 - Aβ1 - 42 , apoE2 / 488 - Aβ1 - 42 / FH , apoE4 / 488 - Aβ1 - 42 / FH , 488 - Aβ1 - 42 / FH and 488 - Aβ1 - 42 / BSA complexes ( OAβ ) or with MAβ . Also , cells incubated with only buffer were included in the assay ( buffer ) . Cell permeability is calculated by flow cytometry as % of DAPI stained cells in the cell population . The effect of CR3 expression in the phagocytosis was measured using PMA activated U937 cells with ( U937 CR3 cells ) or without ( U937 empty cells ) CR3 expression .

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(G) (left) Phagocytosis of complement C3, fB, fD and fI (C) or only buffer (no C) incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA (oligomerized Aβ = OAβ) or with non-oligomerized 488-Aβ1-42 (monomeric Aβ = MAβ). Also, samples without apoE or FH were included in the assay (buffer). (right) Cell permeability of C3, fB, fD and fI incubated beads coupled with apoE2/488-Aβ1-42, apoE4/488-Aβ1-42, apoE2/488-Aβ1-42/FH, apoE4/488-Aβ1-42/FH, 488-Aβ1-42/FH and 488-Aβ1-42/BSA complexes (OAβ) or with MAβ. Also, cells incubated with only buffer were included in the assay (buffer). Cell permeability is calculated by flow cytometry as % of DAPI stained cells in the cell population. The effect of CR3 expression in the phagocytosis was measured using PMA activated U937 cells with (U937 CR3 cells) or without (U937 empty cells) CR3 expression.


( A ) 488 - Aβ1 - 42 phagocytosis in the presence of FH and apoE isoform . Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample .

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(A) 488-Aβ1-42 phagocytosis in the presence of FH and apoE isoform. Flow cytometry data are presented as mean fluorescence intensity of the cell population of the sample.


( B ) Comparison of mRNA transcripts ( two individual samples obtained from three times repeated experiments ) and MS1 intensities ( from 8 apoE44 / apoE43 and 9 apoE33 / 23 carriers ) between genes or proteins that showed differential expression in vitro ( Fig . 5B ) and in vivo .

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(B) Comparison of mRNA transcripts (two individual samples obtained from three times repeated experiments) and MS1 intensities (from 8 apoE44/apoE43 and 9 apoE33/23 carriers) between genes or proteins that showed differential expression in vitro (Fig. 5B) and in vivo.


( D ) ( left ) Combined analysis of complement activation markers C3 , C4 , CFH and Clusterin obtained from the detected MS1 spectra between samples from apoE44 ( n = 3 ) , apoE43 ( n = 5 ) , apoE33 ( n = 7 ) and apoE23 ( n = 2 ) carriers ( four markers / patient ) . ( center ) Combined analysis of complement activation markers C3 , C4 , CFH and Clusterin obtained from the detected MS1 spectra between samples that were positive ( n = 10 ) or negative ( n = 7 ) for Aβ pathology ( four markers / patient ) . ( right ) Expression levels of Phosphodiesterase A control , showing similar expression levels in frontal cortex , between samples from apoE44 ( n = 3 ) , apoE43 ( n = 5 ) , apoE33 ( n = 7 ) and apoE23 ( n = 2 ) carriers The data were normalized against neutral apoE33 samples with no apoE association .

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(D) (left) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples from apoE44 (n=3), apoE43 (n=5), apoE33 (n=7) and apoE23 (n=2) carriers (four markers/patient). (center) Combined analysis of complement activation markers C3, C4, CFH and Clusterin obtained from the detected MS1 spectra between samples that were positive (n=10) or negative (n=7) for Aβ pathology (four markers/patient). (right) Expression levels of Phosphodiesterase A control, showing similar expression levels in frontal cortex, between samples from apoE44 (n=3), apoE43 (n=5), apoE33 (n=7) and apoE23 (n=2) carriers The data were normalized against neutral apoE33 samples with no apoE association.


( 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 .

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(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 .

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(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 .

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(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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 the 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 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 .

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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 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 .

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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 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 .

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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 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.


A , B . Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos . Bead tracks every 500 ms over 2 s are shown ( A ) . Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants , which can be fully rescued by co - injecting with Xenopus ccdc108 mRNA ( B ) . 150 beads from 15 embryos for each condition . Arrowheads indicate beads that showed no movement on the embryo surface through the recording . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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A, B. Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos. Bead tracks every 500 ms over 2 s are shown (A). Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants, which can be fully rescued by co-injecting with Xenopus ccdc108 mRNA (B). 150 beads from 15 embryos for each condition. Arrowheads indicate beads that showed no movement on the embryo surface through the recording. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


C , D . Defective cilia formation in ccdc108 morphants . Embryos at stage 27 were fixed and stained with the acetylated tubulin ( Ac - tub ) antibody and / or Cep164 antibody . mRNA of a membrane - bound form of GFP ( mGFP ) was co - injected with each morpholino to indicate targeted cells . Representative confocal images ( C ) and 3D - SIM images ( D ) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos . ( C ) 20 images of 20 embryos and ( D ) > 80 MCCs from 6 embryos for each condition . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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C, D. Defective cilia formation in ccdc108 morphants. Embryos at stage 27 were fixed and stained with the acetylated tubulin (Ac-tub) antibody and/or Cep164 antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino to indicate targeted cells. Representative confocal images (C) and 3D-SIM images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


F . Defective cilia formation occurs in ccdc108 CRISPR mutants . Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 ( 108sg ) and fixed at stage 27 . Representative 3D - SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants . > 70 MCCs from 6 embryos for each condition . Cell membranes ( mGFP , purple ) , cilia ( Ac - tub , green ) and basal bodies ( Cep164 , yellow ) were labeled with indicated antibodies . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

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F. Defective cilia formation occurs in ccdc108 CRISPR mutants. Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 (108sg) and fixed at stage 27. Representative 3D-SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants. > 70 MCCs from 6 embryos for each condition. Cell membranes (mGFP, purple), cilia (Ac-tub, green) and basal bodies (Cep164, yellow) were labeled with indicated antibodies. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


G . In situ hybridization chain reaction ( HCR ) reveals that ccdc108 mRNA is specially detected in MCCs . Embryos at stage 35 were fixed and subjected to in situ HCR ( red ) . Embryos were then incubated with the Ac - tub antibody ( cyan ) and DAPI ( gray ) .

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G. In situ hybridization chain reaction (HCR) reveals that ccdc108 mRNA is specially detected in MCCs. Embryos at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the Ac-tub antibody (cyan) and DAPI (gray).


C , D . Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts ( C ) and olfactory placodes ( D ) . Zebrafish embryos were fixed and stained with the Ac - tub antibody ( green ) , phalloidin ( purple ) and Hoechst ( blue ) . Data information : Quantitative data were from three independent repeats . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . Mean ± s . e . m values are presented .

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C, D. Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts (C) and olfactory placodes (D). Zebrafish embryos were fixed and stained with the Ac-tub antibody (green), phalloidin (purple) and Hoechst (blue). Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). Mean ± s.e.m values are presented.


E . Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes .

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E. Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes.


H . Representative 3D - SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs . mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining . GFP - CETN1 ( blue ) marked mEPCs infected with lentivirus . Basal bodies ( CEP164 , purple ) and cilia ( Ac - tub , green ) were labeled with indicated antibodies . > 60 MCCs from three independent repeats for each condition were counted . Data information : Quantitative data were from three independent repeats . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . mean ± s . d . values are presented .

H. Representative 3D-SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs. mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining. GFP-CETN1 (blue) marked mEPCs infected with lentivirus. Basal bodies (CEP164, purple) and cilia (Ac-tub, green) were labeled with indicated antibodies. > 60 MCCs from three independent repeats for each condition were counted. Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). mean ± s.d. values are presented.


A . Depletion of Ccdc108 causes disorganized basal body distribution in MCCs . Representative 3D - SIM images ( x - y ) and 3D - reconstructions ( x - z ) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells . Cell membranes ( mGFP , blue ) , basal bodies ( Cetn1 , green ) and distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations . Orange arrowheads and numbers mark individual centriole . > 70 MCCs from 6 embryos for each condition were counted . Data information : Quantitative data were from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

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A. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


B . Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs . Orange arrowheads mark centrioles . Percentage of centrioles docked to plasma membrane in each field were scored . At least 7 cells for each sample were counted .

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B. Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs. Orange arrowheads mark centrioles. Percentage of centrioles docked to plasma membrane in each field were scored. At least 7 cells for each sample were counted.