( A ) CP20 and OVCAR4 cells were transfected with either non - target miRNA control ( miR - CTL ) , miR - 15a , or miR - 195 . 72h following transfection , cells were lysed and immunoblotted for detection of MICU1 , BMI1 , BCL2 , and MFN2 . GAPDH and VDAC were used as loading control .

O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

(A) CP20 and OVCAR4 cells were transfected with either non-target miRNA control (miR-CTL), miR-15a, or miR-195. 72h following transfection, cells were lysed and immunoblotted for detection of MICU1, BMI1, BCL2, and MFN2. GAPDH and VDAC were used as loading control.


( B ) miR - 195 expression in FTE188 and ovarian cell lines as determined by RT - qPCR normalized with U6 . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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(B) miR-195 expression in FTE188 and ovarian cell lines as determined by RT-qPCR normalized with U6. Data are mean ± S.D., n=3 (Biological repeats). *P<0.05, Student's t-test.


( C ) Expression of MICU1 in FTE188 and various ovarian cell lines as determined by immunoblotting . Actin is used as the loading control .

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(C) Expression of MICU1 in FTE188 and various ovarian cell lines as determined by immunoblotting. Actin is used as the loading control.


( D ) Anti - miR - 195 was transfected in the FTE188 cell line , level of miR - 195 was measured using RT - qPCR ( left ) and MICU1 levels were measured using immunoblotting ( right ) . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Anti-miR-195 was transfected in the FTE188 cell line, level of miR-195 was measured using RT-qPCR (left) and MICU1 levels were measured using immunoblotting (right). Data are mean ± S.D., n=3 (Biological repeats). *P<0.05, Student's t-test.


( A , B ) CP20 , OVCAR4 , and OVSAHO cells were transfected with non - target micro RNA control ( miR - CTL ) or miR - 195 , 24 h post - transfection , cells were recounted and plated as single cells for anchorage - dependent ( A ) or anchorage - independent ( B ) clonal growth . After 10 ( CP20 ) or 14 ( OVCAR4 and OVSAHO ) days , colonies were quantified using an Optronix GelCount colony counter . The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control ( miR - CTL ) .

O O O O O B-CELL_LINE O B-CELL_LINE O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(A, B) CP20, OVCAR4, and OVSAHO cells were transfected with non-target micro RNA control (miR-CTL) or miR-195, 24 h post-transfection, cells were recounted and plated as single cells for anchorage-dependent (A) or anchorage-independent (B) clonal growth. After 10 (CP20) or 14 (OVCAR4 and OVSAHO) days, colonies were quantified using an Optronix GelCount colony counter. The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control (miR-CTL).


( C ) Migration of miR - CTL , miR - 195 , and miR - 195 + MICU1 ( pLYS1 - MICU1 - Flag ) transfected cells , towards serum gradient was examined and the number of cells per field was counted . The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control ( miR - CTL ) .

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(C) Migration of miR-CTL, miR-195, and miR-195+ MICU1 (pLYS1-MICU1-Flag) transfected cells, towards serum gradient was examined and the number of cells per field was counted. The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control (miR-CTL).


( D ) Invasion of miR - CTL , miR - 195 , and miR - 195 + MICU1 cells through fibronectin - coated filters was examined using Boyden chamber and number of cells per field was counted . Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control ( miR - CTL ) .

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(D) Invasion of miR-CTL, miR-195, and miR-195 + MICU1 cells through fibronectin-coated filters was examined using Boyden chamber and number of cells per field was counted. Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control (miR-CTL).


( B ) MICU1 3 ' UTR in LightSwitch ™ 3 ′ UTR Reporter Vector ( wild type or miR - 195 binding site deleted ) was co - transfected with either non - target miR control ( miR - CTL ) or different concentrations of miR - 195 . Post 24h transfection luciferase ( Renilla ) activity was measured . Relative light units ( RLU ) compared to miR - CTL were plotted . Data are mean ± S . D . n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O 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) MICU1 3'UTR in LightSwitch™ 3′UTR Reporter Vector (wild type or miR-195 binding site deleted) was co-transfected with either non- target miR control (miR-CTL) or different concentrations of miR-195. Post 24h transfection luciferase (Renilla) activity was measured. Relative light units (RLU) compared to miR-CTL were plotted. Data are mean ± S.D. n=3 (Biological repeats). *P<0.05, Student's t-test.


CP20 cells were transfected as indicated , 48 h post transfection 4x106 cells were permeabilized , and extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in miR - CTL and miR - 195 transfected cells . [ Ca2 + ] out pulses and FCCP were delivered as indicated .

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CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in miR-CTL and miR-195 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.


CP20 cells were transfected as indicated , 48 h post transfection 4x106 cells were permeabilized , and extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in control siRNA ( siCTL ) and siMICU1 transfected cells . [ Ca2 + ] out pulses and FCCP were delivered as indicated .

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CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in control siRNA (siCTL) and siMICU1 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.


( E ) Bar graph illustrating the number of Ca2 + pulses handled by control miR , miR - 195 , siCTL and siMICU1 transfected cells . Mean ± SEM ; n = 3 - 4 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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(E) Bar graph illustrating the number of Ca2+ pulses handled by control miR, miR-195, siCTL and siMICU1 transfected cells. Mean ±SEM; n=3-4 (Biological repeats). *P<0.05, Student's t-test.


( F ) Representative [ Ca2 + ] ( m ) traces after addition of FCCP ( 10 µM ) in permeabilized CP20 cells transfected with non - target miR - CTL or miR - 195 . ( G ) Quantification of resting matrix [ Ca2 + ] ( m ) after addition of FCCP . n = 5 ( Biological repeats ) * P < 0 . 05 , Student ' s t - test ,

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(F) Representative [Ca2+](m) traces after addition of FCCP (10 µM) in permeabilized CP20 cells transfected with non-target miR-CTL or miR-195. (G) Quantification of resting matrix [Ca2+](m) after addition of FCCP. n=5 (Biological repeats) *P<0.05, Student's t-test,


( H ) Representative traces of mitochondrial membrane potential ( ∆ Ψμ ) in CP20 cells transfected with miR - CTL or miR - 195 . ( I ) Quantification of relative ∆ Ψμ . ( n = 5 , Biological repeats ) .

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(H) Representative traces of mitochondrial membrane potential (∆Ψμ) in CP20 cells transfected with miR-CTL or miR-195. (I) Quantification of relative ∆Ψμ. (n=5, Biological repeats).


( J ) Intracellular lactate was measured in miR - CTL , miR - 195 , siCTL , and siMICU1 expressing OVCAR4 , CP20 , and OVSAHO cells . n = 3 , biological repeats * P < 0 . 05 , Student ' s t - test .

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(J) Intracellular lactate was measured in miR-CTL, miR-195, siCTL, and siMICU1 expressing OVCAR4, CP20, and OVSAHO cells. n=3, biological repeats *P<0.05, Student's t-test.


( K & L ) miR - CTL , miR - 195 , or miR - 195 + pLYS1 - MICU1 - Flag transfected cells were stained with MitoSOX Red , and mitochondrial ROS levels were determined by flow cytometry . The histogram shows representative staining and bar graph ( right ) shows results of three independent experiments . Mean ± SEM ; . n = 3 , biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - test .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

(K&L) miR-CTL, miR-195, or miR-195 + pLYS1-MICU1-Flag transfected cells were stained with MitoSOX Red, and mitochondrial ROS levels were determined by flow cytometry. The histogram shows representative staining and bar graph (right) shows results of three independent experiments. Mean ± SEM; . n=3, biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.


( A & B ) Stable cell lines for CP20 and OVCAR4 expressing GFP - miR - 195 or non - target miR - GFP were generated using lentiviral mediated transduction and were selected using puromycin ; representative images are shown . Scale bars , 50 µm .

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(A&B) Stable cell lines for CP20 and OVCAR4 expressing GFP-miR-195 or non-target miR-GFP were generated using lentiviral mediated transduction and were selected using puromycin; representative images are shown. Scale bars, 50 µm.


( C ) Relative miR - 195 expression was measured using RT - qPCR in stable cell lines , normalized with U6 . Mean ± SEM ; n = 3 , Biological repeats . * P < 0 . 05 , Student ' s t - test .

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(C) Relative miR-195 expression was measured using RT-qPCR in stable cell lines, normalized with U6. Mean ± SEM; n=3, Biological repeats. *P<0.05, Student's t-test.


( D ) CP20 and OVCAR4 stable cell lines were transfected as either empty vector ( EV ) or pLYS1 - MICU1 - Flag ( MICU1 ) and relative MICU1 levels were evaluated using immunoblotting .

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(D) CP20 and OVCAR4 stable cell lines were transfected as either empty vector (EV) or pLYS1-MICU1-Flag (MICU1) and relative MICU1 levels were evaluated using immunoblotting.


( E , F ) CP20 and OVCAR4 stable cell lines transfected as in ( D ) were counted and re - plated for anchorage - dependent ( E ) and independent clonal growth ( F ) . Quantification compared to the miR - CTL stable cells transfected with EV is shown . Mean ± SD ; n = 3 , Biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - test .

O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

(E, F) CP20 and OVCAR4 stable cell lines transfected as in (D) were counted and re-plated for anchorage-dependent (E) and independent clonal growth (F). Quantification compared to the miR-CTL stable cells transfected with EV is shown. Mean ± SD; n=3, Biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.


( A ) Female athymic mice were injected subcutaneously with either CP20 GFP - miR - CTL or CP20 - GFP - miR - 195 cells . Mice injected with the miR - 195 expressing cells ( miR - 195 ) had significantly smaller tumor volumes than control mice ( miR - CTL ) . Data are mean ± S . D , n = 10 . * P < 0 . 05 , Student ' s t - test .

O O O O O B-ORGANISM O O O O O B-CELL_LINE B-GENEPROD O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O 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) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 cells. Mice injected with the miR-195 expressing cells (miR-195) had significantly smaller tumor volumes than control mice (miR-CTL). Data are mean ± S.D, n=10. *P<0.05, Student's t-test.


( B ) Tumor doubling time was significantly longer in the mice injected with miR - 195 expressing cells than in control mice . Data are mean ± S . D , n = 10 . * P < 0 . 05 , Student ' s t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O O O 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) Tumor doubling time was significantly longer in the mice injected with miR-195 expressing cells than in control mice. Data are mean ± S.D, n=10. *P<0.05, Student's t-test.


( C ) Percent survival based on humane endpoint criteria , was calculated by the Kaplan - Meier method and P values determined by Log Rank Test .

O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(C) Percent survival based on humane endpoint criteria, was calculated by the Kaplan-Meier method and P values determined by Log Rank Test.


( D ) Four tumor samples from each group were analyzed for the expression of MICU1 , pPDH , PDH , PARP , and BCL2 using immunoblotting . α tubulin was used as the loading control .

O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O

(D) Four tumor samples from each group were analyzed for the expression of MICU1, pPDH, PDH, PARP, and BCL2 using immunoblotting. α tubulin was used as the loading control.


Representative histology images of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) cells with Ki67 expression ( Scale Bar = 50 μm ) ( E ) , CD31 positive vessels ( Scale Bar = 100 μm ) and TUNEL staining ( Scale Bar = 50 μm )

O B-EXP_ASSAY O O O O B-ORGANISM B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

Representative histology images of tumors from mice xenografts of CP20-GPF-miR-CTL (miR-CTL) or CP20-GPF-miR-195 (miR-195) cells with Ki67 expression (Scale Bar = 50 μm) (E), CD31 positive vessels (Scale Bar = 100 μm) and TUNEL staining (Scale Bar = 50 μm)


Representative histology images of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) cells with Ki67 expression ( Scale Bar = 50 μm ) ( F ) graph showing quantification of each marker in tumors from the experimental mice ; values are mean ± SD , n = 10 , * P < 0 . 05 , Student ' s t - test .

O B-EXP_ASSAY O O O O B-ORGANISM B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O 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 histology images of tumors from mice xenografts of CP20-GPF-miR-CTL (miR-CTL) or CP20-GPF-miR-195 (miR-195) cells with Ki67 expression (Scale Bar = 50 μm) (F) graph showing quantification of each marker in tumors from the experimental mice; values are mean ±SD, n=10, *P<0.05, Student's t-test.


( G ) Female athymic mice were injected subcutaneously with either CP20 GFP - miR - CTL or CP20 - GFP - miR - 195 or CP20 - GFP - miR - 195 + MICU1 cells and tumor volume was measured , values are mean ± SE , n = 10 . * P < 0 . 05 , Student ' s t - test .

O O O O O B-ORGANISM O O O O O B-CELL_LINE B-GENEPROD O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 or CP20- GFP-miR-195 + MICU1 cells and tumor volume was measured, values are mean ±SE, n=10. *P<0.05, Student's t-test.


Representative images of HeLa ( B ) or HCT - 8 ( C ) cells infected with Shigella WT pre - treated or not with arsenite , analyzed at the indicated times post - infection Cfu quantification of intracellular bacteria in HeLa and HCT - 8 cells pre - treated or not with arsenite and infected with

O O O B-CELL_LINE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O

Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella


Representative images ( E ) and cfu quantification ( F ) of intracellular bacteria in HeLa cells infected with Shigella WT after pre - treatment with TNF - α , H2O2 , anisomycin , hypoxia and corresponding controls , analyzed at 0 . 5

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Representative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia and corresponding controls, analyzed at 0.5 hpi


Representative images ( G ) and cfu quantification ( H ) of intracellular Shigella in HeLa cells pre - treated with arsenite , anisomycin , stressors plus NAC , and corresponding

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Representative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls


Representative images ( A ) and cfu quantification ( B ) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre - treated or not with arsenite . Ruffle formation induced by Shigella WT in panel ( A ) is indicated by white

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Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads


Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre - treated with anisomycin or DMSO ( control )

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Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control)


Representative images ( D ) and cfu quantification ( E ) of Shigella WT or ΔipaB mutant bound to HCT - 8 cells pre - treated or not with

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Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite


Representative image ) , cf and qRT - PCR quantification of intracellular Shigella in HeLa cells pre - treated with arsenite in the presence or not of amitriptyline , and corresponding

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Representative image ), cf and qRT-PCR quantification of intracellular Shigella in HeLa cells pre-treated with arsenite in the presence or not of amitriptyline, and corresponding controls


Representative image cf and qRT - PC quantification of intracellular Shigella in HeLa cells pre - treated wit anisomycin in the presence or not of amitriptyline , and corresponding

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Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells pre-treated wit anisomycin in the presence or not of amitriptyline, and corresponding controls


Representative image cf and qRT - PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre - treated or not with arsenite prior to

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Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection


3D reconstruction of representative images of ASM ( J , K ) or ceramide ( L , M ) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls . ASM , ceramide and Hoechst staining were surface converted by voxel

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3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide and Hoechst staining were surface converted by voxel distance


Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite , TNF - α , H2O2 or anisomycin ; β - actin was used as loading

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Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite, TNF-α, H2O2 or anisomycin; β-actin was used as loading control


Representative image of intracellular Shigella in HeLa cells pre - treated with arsenite in the absence or presence of the p38 inhibitor SB203580 , and corresponding

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Representative image of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


cfu quantificatio of intracellular Shigella in HeLa cells pre - treated with arsenite in the absence or presence of the p38 inhibitor SB203580 , and corresponding

cfu quantificatio of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre - treated or not with arsenite prior to

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Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not with arsenite prior to infection


cfu quantificatio of intracellular Shigella in HeLa cells pre - treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580 , and corresponding

cfu quantificatio of intracellular Shigella in HeLa cells pre-treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre - treated or not wit anisomycin prior to

Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not wit anisomycin prior to infection


3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite ) , in the presence or absence of SB203580 , and corresponding controls . Ceramide and Hoechst staining were surface converted by voxel

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3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance


3D reconstruction of representative images of ceramide staining in HeLa cells treated wit anisomyci ) , in the presence or absence of SB203580 , and corresponding controls . Ceramide and Hoechst staining were surface converted by voxel

3D reconstruction of representative images of ceramide staining in HeLa cells treated wit anisomyci ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance


Representative images ( A ) , cfu ( B and qRT - PCR quantification of intracellular Salmonella in HeLa cells pre - treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0 . 5

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Representative images (A), cfu (B and qRT-PCR quantification of intracellular Salmonella in HeLa cells pre-treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0.5 hpi


Representative images of bacteria bound to HeLa cells pre - treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10

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Representative images of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10 min


Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre - treated or not with arsenite followed by incubation with Salmonella WT ( E ) or Δ4 mutant strain ( F ) for 10

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Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT (E) or Δ4 mutant strain (F) for 10 min


Representative images ( G ) and cfu quantification ( H ) of bacteria bound to HeLa cells pre - treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC , ΔfliC / pFliC or ΔflhC for 10

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Representative images (G) and cfu quantification (H) of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC, ΔfliC/pFliC or ΔflhC for 10 min


Representative images ( I ) and cfu quantification ( J ) of Listeria bound to HeLa cells pre - treated or not with arsenite followed by incubation with the bacteria for 20

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Representative images (I) and cfu quantification (J) of Listeria bound to HeLa cells pre-treated or not with arsenite followed by incubation with the bacteria for 20 min


Representative images ( K ) and cfu quantification ( L ) of Yersinia bound / internalized by HeLa cells pre - treated or not with arsenite followed by 70 min incubation with the

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Representative images (K) and cfu quantification (L) of Yersinia bound/internalized by HeLa cells pre-treated or not with arsenite followed by 70 min incubation with the bacteria


Schematic representation of the experimental design for reinfection experiments . Cells were infected with Shigella - mCherry ( primary infection ) or mock - treated . At 3 hpi , cells were re - infected with Shigella expressing GFP ( secondary infection ) . Secondary infection was analyzed at 0 . 5 hpi ( i . e . 1 h after addition of bacteria to cells ) Representative image and cfu quantification of re - infection assays with Shigella W of HeLa cells primarily infected with Shigella WT or ΔipaB / Inv mutant strain , or mock -

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Schematic representation of the experimental design for reinfection experiments. Cells were infected with Shigella-mCherry (primary infection) or mock-treated. At 3 hpi, cells were re-infected with Shigella expressing GFP (secondary infection). Secondary infection was analyzed at 0.5 hpi (i.e. 1 h after addition of bacteria to cells) Representative image and cfu quantification of re-infection assays with Shigella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated


Representative image and cfu quantificatio of re - infection assays wit Salmonella W of HeLa cells primarily infected with Shigella WT or ΔipaB / Inv mutant strain , or mock -

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Representative image and cfu quantificatio of re-infection assays wit Salmonella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated


Representative images ( F ) and cfu quantification ( G ) of the re - infection assays with Salmonella ΔfliC , ΔfliC / pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock -

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Representative images (F) and cfu quantification (G) of the re-infection assays with Salmonella ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock-treated


Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment . I and J . Representative images ( I ) and cfu quantification ( J ) of the secondary infection with Shigella WT , in cells transfected with ASM siRNA or control

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Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment. I and J. Representative images (I) and cfu quantification (J) of the secondary infection with Shigella WT, in cells transfected with ASM siRNA or control siRNA


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Cfu quantification of the secondary infection with Shigella WT in HeLa cells following amitriptyline


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Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin transfectio with MAPK p38 siRN


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Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin SB203580 treatmen


3D reconstruction of representative images of ASM ( A ) or ceramide ( B ) staining in HeLa cells infected with Shigella WTor mock - treated , analyzed at 3 hpi . ASM , ceramide , Hoechst and Shigella staining were surface converted by voxel

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3D reconstruction of representative images of ASM (A) or ceramide (B) staining in HeLa cells infected with Shigella WTor mock-treated, analyzed at 3 hpi. ASM, ceramide, Hoechst and Shigella staining were surface converted by voxel distance


ASM enzymatic activity measurement in HeLa cells infected with Shigella - WT or mock - treated , analyzed at 3 hpi . The ASM enzymatic activity was determined in the membrane fraction corresponding to 3 . 0 × 105 cells per

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ASM enzymatic activity measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi. The ASM enzymatic activity was determined in the membrane fraction corresponding to 3.0×105 cells per condition


ceramide measurement in HeLa cells infected with Shigella - WT or mock - treated , analyzed at 3 hpi Ceramide is shown normalized to mock - treated

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ceramide measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi Ceramide is shown normalized to mock-treated cells


Representative spinning - disk confocal images of colon tissue sections of non - infected ( E and F ) and Shigella infected ( G and H ) guinea pigs , collected at 4 hpi . E and G are montages of multiple image fields ; dashed boxes in ( E and G ) are shown enlarged in ( F and H ) ,

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O 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 spinning-disk confocal images of colon tissue sections of non-infected (E and F) and Shigella infected (G and H) guinea pigs, collected at 4 hpi. E and G are montages of multiple image fields; dashed boxes in (E and G) are shown enlarged in (F and H), respectively


Mean fluorescence intensity of ceramide signal in surface epithelial cells ( outermost tissue layer ) of non - infected or Shigella - infected guinea pig colon tissue

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Mean fluorescence intensity of ceramide signal in surface epithelial cells (outermost tissue layer) of non-infected or Shigella-infected guinea pig colon tissue sections


( B ) Wild type mice received strain GFP - K79 via the tail vein and 1 hr later , animals were perfused and the meninges , cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria . The arrows represent bacteria co - localized with capillaries ( e . g . , within capillaries ) and arrowheads represent bacteria outside the capillaries ( e . g . , exited from the capillaries ) . A few bacteria were successfully passed through the meningeal and cortex capillaries , but no bacteria were demonstrated in the choroid plexus at this time .

O O O O O B-ORGANISM O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O

(B) Wild type mice received strain GFP-K79 via the tail vein and 1 hr later, animals were perfused and the meninges, cortex and choroid plexus were obtained for demonstration of intravenously injected bacteria. The arrows represent bacteria co-localized with capillaries (e.g., within capillaries) and arrowheads represent bacteria outside the capillaries (e.g., exited from the capillaries). A few bacteria were successfully passed through the meningeal and cortex capillaries, but no bacteria were demonstrated in the choroid plexus at this time.


( C ) Sulfo - NHS - biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood - brain barrier permeability , i . e . , extravasation is indicative of leakage from the intravascular lumen . Sulfo - NHS - Biotin was confined to the capillaries of the meninges and cortex ( as shown with arrows ) , and there was no evidence of extravasation . Scale bar = 100 μm .

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(C) Sulfo-NHS-biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood-brain barrier permeability, i.e., extravasation is indicative of leakage from the intravascular lumen. Sulfo-NHS-Biotin was confined to the capillaries of the meninges and cortex (as shown with arrows), and there was no evidence of extravasation. Scale bar = 100 μm.


( D ) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor ( gefitinib ) .

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(D) Relative invasion frequency of GBS strain K79 in HBMEC with or without EGFR inhibitor (gefitinib).


( E ) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control ( n = 5 ) or gefitinib ( 10 mg / kg ) ( n = 6 ) , infected with strain K79 for 1 h .

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(E) Bacterial counts recovered from the blood and brain in wild type mice receiving vehicle control (n = 5) or gefitinib (10 mg/kg) (n = 6), infected with strain K79 for 1 h.


( F ) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection .

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(F) Tyrosine phosphorylation of EGFR in HBMEC in response to GBS strain K79 infection at 15 min and 60 min post infection.


( G ) EGFR protein expression in EGFR knockout HBMEC using CRISPR / Cas9 ( left panel ) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC ( right panel ) . From left to right , p value of K79 is 0 . 0059 , p value of K160 is 0 . 00024 , p value of K161 is 0 . 00080 , p value of K181 is 0 . 00042 , p value of K226 is 0 . 00011 , p value of K237 is 0 . 00072 , p value of K238 is 0 . 0093 , p value of P539 is 0 . 00013 ( right panel ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-TISSUE O O B-ORGANISM O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EGFR protein expression in EGFR knockout HBMEC using CRISPR/Cas9 (left panel) and relative invasion frequency of 8 meningitis isolates of GBS strains in EGFR knockout and control HBMEC (right panel). From left to right, p value of K79 is 0.0059, p value of K160 is 0.00024, p value of K161 is 0.00080, p value of K181 is 0.00042, p value of K226 is 0.00011, p value of K237 is 0.00072, p value of K238 is 0.0093, p value of P539 is 0.00013 (right panel).


( H ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC .

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(H) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in EGFR knockout HBMEC compare to control HBMEC.


( I ) Bacterial counts recovered from the blood and brain of EGFR conditional knockout ( n = 5 ) and control mice ( n = 5 ) 1 h after intravenous inoculation with strain K79 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O B-TISSUE O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

(I) Bacterial counts recovered from the blood and brain of EGFR conditional knockout (n = 5) and control mice (n = 5) 1 h after intravenous inoculation with strain K79.


( A ) Tyrosine phosphorylation of EGFR in HBMEC pre - treated with 20 μM S1P2 antagonist ( JTE - 013 ) or vehicle control and infected with GBS strain K79 infection for 60 min . EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79 .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-CELL_LINE O O O O

(A) Tyrosine phosphorylation of EGFR in HBMEC pre-treated with 20 μM S1P2 antagonist (JTE-013) or vehicle control and infected with GBS strain K79 infection for 60 min. EGFR phosphorylation in control and S1P2 knockout HBMEC infected with K79.


( B ) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors . All the inhibitors were used at 10 μM . From left to right , p value of DJB - V - 39 is 0 . 0047 , p value of SKI is 0 . 0062 , p value of FTY720 is 0 . 0022 , and p value of HB56 is 0 . 0045 .

O O O O B-EXP_ASSAY O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O

(B) Relative invasion frequency of strain K79 in HBMEC with or without SphK1 and SphK2 inhibitors. All the inhibitors were used at 10 μM. From left to right, p value of DJB-V-39 is 0.0047, p value of SKI is 0.0062, p value of FTY720 is 0.0022, and p value of HB56 is 0.0045.


( C ) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR / Cas9 ( left panel ) . Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC . From left to right , p value of K79 is 0 . 0018 , p value of K160 is 0 . 0091 , p value of K161 is 0 . 0054 , p value of K181 is 0 . 00040 , p value of K226 is 0 . 00032 , p value of K237 is 0 . 040 , p value of K238 is 0 . 041 and p value of P539 is 0 . 0025 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-TISSUE O O B-ORGANISM O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) S1P2 protein expression in S1P2 knockout HBMEC using CRISPR/Cas9 (left panel). Relative invasion frequency of 8 meningitis isolates of GBS in S1P2 knockout HBMEC. From left to right, p value of K79 is 0.0018, p value of K160 is 0.0091, p value of K161 is 0.0054, p value of K181 is 0.00040, p value of K226 is 0.00032, p value of K237 is 0.040, p value of K238 is 0.041 and p value of P539 is 0.0025.


( D ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control .

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(D) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in S1P2 knockout HBMEC compare to control.


( E ) Bacterial counts recovered from the blood and brain of wild type ( n = 5 ) and S1P2 - / - mice ( n = 5 ) infected with strain K79 for 1h .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O B-TISSUE O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O

(E) Bacterial counts recovered from the blood and brain of wild type (n = 5) and S1P2-/- mice (n = 5) infected with strain K79 for 1h.


( A ) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM O O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-CELL_LINE O

(A) Serine phosphorylation of cPLA2α in response to GBS strain K79 in HBMEC pretreated with gefitinib or vehicle control and in EGFR knockout HBMEC.


( B ) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control , EGFR knockout and S1P2 knockout HBMEC .

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(B) Serine phosphorylation of cPLA2α in response to GBS strain K79 in control, EGFR knockout and S1P2 knockout HBMEC.


( C ) Bacterial counts recovered from the blood and brain in wild type mice ( n = 5 ) and CysLT2 - / - mice ( n = 5 ) infected with strain K79 for 1h .

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(C) Bacterial counts recovered from the blood and brain in wild type mice (n = 5) and CysLT2-/- mice (n = 5) infected with strain K79 for 1h.


( D ) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA ( left panel ) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC . t - test . From left to right , p value of K79 is 0 . 0039 , p value of K160 is 0 . 0082 , p value of K161 is 0 . 00091 , p value of K181 is 0 . 0087 , p value of K226 is 0 . 00020 , p value of K237 is 0 . 0039 , p value of K238 is 0 . 00066 , p value of P539 is 1 . 76E - 05 ( right panel ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CysLT1 protein expression in CysLT1 knockdown HBMEC using shRNA (left panel) and relative invasion frequency of 8 GBS isolates in CysLT1 knockdown and control HBMEC. t-test. From left to right, p value of K79 is 0.0039, p value of K160 is 0.0082, p value of K161 is 0.00091, p value of K181 is 0.0087, p value of K226 is 0.00020, p value of K237 is 0.0039, p value of K238 is 0.00066, p value of P539 is 1.76E-05 (right panel).


( E ) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control .

(E) GBS strain K79 traversal of HBMEC monolayer was significantly decreased in CysLT1 knockdown HBMEC compare to control.


( F ) Bacterial counts recovered from the blood and brain in wild type mice ( n = 7 ) and CysLT1 - / - mice ( n = 7 ) infected with strain K79 for 1h .

(F) Bacterial counts recovered from the blood and brain in wild type mice (n = 7) and CysLT1-/- mice (n = 7) infected with strain K79 for 1h.


( G ) Ezrin protein expression in ezrin knockdown HBMEC using shRNA ( left panel ) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC ( right panel ) .

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(G) Ezrin protein expression in ezrin knockdown HBMEC using shRNA (left panel) and relative invasion frequency of GBS strain K79 in ezrin knockdown and control HBMEC (right panel).


( H Ezrin phosphorylation in HBMEC pre - treated with CysLT1 antagonist ( montelukast ) and infected with GBS strain K79 for 60 min ( left panel )

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(H Ezrin phosphorylation in HBMEC pre-treated with CysLT1 antagonist (montelukast) and infected with GBS strain K79 for 60 min (left panel)


I ) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1 - / - and control mice infected with GBS strain K79 for 1h ( right panel ) .

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I) ezrin phosphorylation in the homogenates of brain capillaries of CysLT1-/- and control mice infected with GBS strain K79 for 1h (right panel).


( J ) Intracellular K79 co - localization with EGFR and ezrin in control and S1P2 knockout HBMEC ( as shown by arrows , cyan ) . Percentage of co - localization was calculated by counting numbers of all GBS and co - localized intracellular GBS from at least three representative fields , and expressing as numbers of co - localized GBS / all GBS x 100 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-ORGANISM O O B-ORGANISM O O O

(J) Intracellular K79 co-localization with EGFR and ezrin in control and S1P2 knockout HBMEC (as shown by arrows, cyan). Percentage of co-localization was calculated by counting numbers of all GBS and co-localized intracellular GBS from at least three representative fields, and expressing as numbers of co-localized GBS/all GBS x 100.


( A ) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79 . Data are presented as a Kaplan - Maier plot with a log rank test used to compare percentage of survival between the groups , CFX group n = 18 , and CFX + MONT group n = 10 * p = 0 . 015 . ( CFX : ceftriaxone ; MONT : montelukast ) .

O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

(A) Survival of wild type mice receiving drug administration for 7 days after infection with GBS strain K79. Data are presented as a Kaplan-Maier plot with a log rank test used to compare percentage of survival between the groups, CFX group n = 18, and CFX + MONT group n = 10*p = 0.015. (CFX: ceftriaxone; MONT: montelukast).


( B ) Nissl staining of the hippocampus CA1 , CA2 & CA3 regions ( low magnification ) and cortex region ( higher magnification ) ( left ) and histograms showing the number of Nissl - stained bodies ( right ) Scale bar , 50 μm . Data represent the means ± SEM from three independent experiments , with statistical analysis by One - way ANOVA , p value between control group and Ceftriaxone group is 0 . 00021 , p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0 . 013 .

O O O B-SUBCELLULAR B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O

(B) Nissl staining of the hippocampus CA1, CA2 & CA3 regions (low magnification) and cortex region (higher magnification) (left) and histograms showing the number of Nissl-stained bodies (right) Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value between control group and Ceftriaxone group is 0.00021, p value between Ceftriaxone group and Ceftriaxone + Montelukast group is 0.013.


( C - D ) Representative immunofluorescence images ( left ) and quantification ( right ) of TUNEL bodies , astrocyte ( GFAP ) , microglia ( Iba1 ) in cortex of wild type mice after 7 days of therapy . Scale bar , 50 μm . Data represent the means ± SEM from three independent experiments , with statistical analysis by One - way ANOVA , p value of TUNEL bodies between CFX group and MONT + CFX group is 0 . 0012 , p value of astrocyte between CFX group and MONT + CFX group is 0 . 0068 , and p value of microglia cells between CFX group and MONT + CFX group is 0 . 0029 . ( E ) Y - maze test for spatial learning and memory of different group of wild type mice . The total duration of time spent at novel arm ( left ) and the percentage of entries to novel arm of Y - maze ( right ) . Data represent the means ± SEM was statistically analyzed by One - way ANOVA , p value of total duration of time spent at novel arm between control group ( n = 6 ) and CFX group ( n = 5 ) is 0 . 00012 , p value between CFX group ( n = 5 ) and MONT + CFX group ( n = 6 ) is 0 . 00017 ; p value of total duration of % of entry to novel arm between control group ( n = 6 ) and CFX group ( n = 5 ) is 0 . 0102 , and p value between CFX group ( n = 5 ) and MONT + CFX group ( n = 6 ) is 0 . 0216 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O 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-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O

(C-D) Representative immunofluorescence images (left) and quantification (right) of TUNEL bodies, astrocyte (GFAP), microglia (Iba1) in cortex of wild type mice after 7 days of therapy. Scale bar, 50 μm. Data represent the means ± SEM from three independent experiments, with statistical analysis by One-way ANOVA, p value of TUNEL bodies between CFX group and MONT + CFX group is 0.0012, p value of astrocyte between CFX group and MONT + CFX group is 0.0068, and p value of microglia cells between CFX group and MONT + CFX group is 0.0029. (E) Y-maze test for spatial learning and memory of different group of wild type mice. The total duration of time spent at novel arm (left) and the percentage of entries to novel arm of Y-maze (right). Data represent the means ± SEM was statistically analyzed by One-way ANOVA, p value of total duration of time spent at novel arm between control group (n = 6) and CFX group (n = 5) is 0.00012, p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.00017; p value of total duration of % of entry to novel arm between control group (n = 6) and CFX group (n = 5) is 0.0102, and p value between CFX group (n = 5) and MONT + CFX group (n = 6) is 0.0216.


ZO - 1 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts .

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ZO-1 and GAPDH protein levels assessed in Epi cells by Western blot of whole protein extracts.


ZO - 1 quantification of IF images performed using the Fiji software and bar - plotted as normalized integrated densities per cell ( grey dots ) with bars corresponding to Mean ± Standard Error of the Mean ( SEM ) for at least 11 high - field units of at least 100 cells ; * P - value < 0 . 05 ( Student ' s t - test , N = 11 ) .

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ZO-1 quantification of IF images performed using the Fiji software and bar-plotted as normalized integrated densities per cell (grey dots) with bars corresponding to Mean ± Standard Error of the Mean (SEM) for at least 11 high-field units of at least 100 cells; * P-value < 0.05 (Student's t-test, N=11).


LMS ( red ) and HMS ( black ) gene expression quantification in Epi cells by random - primed RT - qPCR relative to RPL11 and represented as log2 fold change to CTR . Each dot corresponds to an independent replicate , bars represent Mean ± Standard Deviation values .

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LMS (red) and HMS (black) gene expression quantification in Epi cells by random-primed RT-qPCR relative to RPL11 and represented as log2 fold change to CTR. Each dot corresponds to an independent replicate, bars represent Mean ± Standard Deviation values.


Metagene profiling within the 10 kb window around TSS of H3K27ac , LSD1 and H3K4me2 over IgG in Epi cells expressing none ( CTR ) or different HOTAIR variants ( HOT , HOTΔP and HOTΔL ) . bp stands for base pairs .

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Metagene profiling within the 10 kb window around TSS of H3K27ac, LSD1 and H3K4me2 over IgG in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.


Heatmap of unsupervised hierarchical clustering based on H3K27ac mean values at gene promoters showing separation of two phenotypic groups .

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Heatmap of unsupervised hierarchical clustering based on H3K27ac mean values at gene promoters showing separation of two phenotypic groups.


Metagene profiling of H3K27ac , LSD1 and H3K4me2 over IgG within the 5 kb window of enhancers in Epi cells expressing none ( CTR ) or different HOTAIR variants ( HOT , HOTΔP and HOTΔL ) . bp stands for base pairs .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

Metagene profiling of H3K27ac, LSD1 and H3K4me2 over IgG within the 5 kb window of enhancers in Epi cells expressing none (CTR) or different HOTAIR variants (HOT, HOTΔP and HOTΔL). bp stands for base pairs.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 103 PFU of CHIKV . ( E ) Viral tissue burdens were analyzed at 1 day post inoculation ( dpi ) by FFA . Mean ± SEM .

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WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of CHIKV. (E) Viral tissue burdens were analyzed at 1 day post inoculation (dpi) by FFA. Mean ± SEM.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 103 PFU of ONNV ( F ) Tissues and serum were collected at 1 dpi and analyzed by plaque assay .

O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of ONNV (F) Tissues and serum were collected at 1 dpi and analyzed by plaque assay.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 103 PFU of RRV ( G ) . Tissues and serum were collected at 1 dpi and analyzed by plaque assay .

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WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of RRV (G). Tissues and serum were collected at 1 dpi and analyzed by plaque assay.


( A ) WT and Clec4F - DTR + C57BL / 6 mice were treated with diphtheria toxin ( DT ) either intraperitoneally ( i . p . ) 24 h prior to inoculation ( n = 10 , data pooled from two experiments , black dots ) or intravenously ( i . v . ) 48 and 24 h prior to inoculation ( n = 4 , one experiment , grey dots ) to deplete KCs and inoculated i . v . with 108 CHIKV particles . Viral genomes in the inoculum and serum at 45 minutes ( min ) - post inoculation were determined by RT - qPCR . Mean ± SD . Data are pooled from three experiments , n = 14 . Mann - Whitney test ; * * * * P < 0 . 0001 .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O B-ORGANISM O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) WT and Clec4F-DTR+ C57BL/6 mice were treated with diphtheria toxin (DT) either intraperitoneally (i.p.) 24 h prior to inoculation (n=10, data pooled from two experiments, black dots) or intravenously (i.v.) 48 and 24 h prior to inoculation (n=4, one experiment, grey dots) to deplete KCs and inoculated i.v. with 108 CHIKV particles. Viral genomes in the inoculum and serum at 45 minutes (min)-post inoculation were determined by RT-qPCR. Mean ± SD. Data are pooled from three experiments, n= 14. Mann-Whitney test; ****P < 0.0001.


( C ) WT or CD169 - DTR + mice were treated i . p . with DT prior to i . v . inoculation of 108 CHIKV particles . Viral genomes were quantified as in ( A ) . Mean ± SD . Data are pooled from two experiments , n = 7 - 8 . Mann - Whitney test ; * * * P < 0 . 001 .

O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O 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) WT or CD169-DTR+ mice were treated i.p. with DT prior to i.v. inoculation of 108 CHIKV particles. Viral genomes were quantified as in (A). Mean ± SD. Data are pooled from two experiments, n=7-8. Mann-Whitney test; ***P < 0.001.


( D ) Livers from WT , Clec4F - DTR + or CD169 - DTR + mice treated i . p . with DT , or WT mice treated i . v . with PLL or CLL were analyzed by IHC to visualize F4 / 80 + macrophages ( brown ) . Two representative sections for each group are shown .

O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O

(D) Livers from WT, Clec4F-DTR+ or CD169-DTR+ mice treated i.p. with DT, or WT mice treated i.v. with PLL or CLL were analyzed by IHC to visualize F4/80+ macrophages (brown). Two representative sections for each group are shown.


( A ) WT or Clec4F - DTR + C57BL / 6 mice were treated i . v . with DT prior to s . c . inoculation in the left rear footpad with 103 PFU of CHIKV . Infectious virus was quantified at 24 hpi by FFA . Mean ± SEM . Data are pooled from two experiments , n = 8 . Mann - Whitney test ; P > 0 . 05 .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) WT or Clec4F-DTR+ C57BL/6 mice were treated i.v. with DT prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus was quantified at 24 hpi by FFA. Mean ± SEM. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.


( B ) WT C57BL / 6 mice were treated i . v . with PLL or CLL 42 h prior to s . c . inoculation in the left rear footpad with 103 PFU of CHIKV . Infectious virus at 24 hpi was quantified by FFA . Mean ± SD . Data are pooled from two experiments , n = 8 . Mann - Whitney test ; * * * P < 0 . 001 .

O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) WT C57BL/6 mice were treated i.v. with PLL or CLL 42 h prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; ***P < 0.001.


( B ) WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R . Viral genomes in the dLN and serum at 2 hpi were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 10 . Two - way ANOVA with Bonferroni ' s multiple comparisons test ; * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O O B-ORGANISM O B-ORGANISM B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=10. Two-way ANOVA with Bonferroni's multiple comparisons test; ***P < 0.001, ****P < 0.0001.


( C ) WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 108 particles of RRV . Viral genomes in the dLN and serum were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 8 - 9 . Mann - Whitney test ; * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of RRV. Viral genomes in the dLN and serum were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8-9. Mann-Whitney test; ***P < 0.001, ****P < 0.0001.


( A ) CD169 - DTR + mice were treated i . p . with PBS or DT prior to collection of the popliteal LN . Frozen LN sections were stained for CD169 + macrophages ( green ) , B220 + B cells ( blue ) or ERTR - 7 + stromal cells ( red ) . Image shown is representative of three biological replicates . Scale bars : 200 μm .

O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(A) CD169-DTR+ mice were treated i.p. with PBS or DT prior to collection of the popliteal LN. Frozen LN sections were stained for CD169+ macrophages (green), B220+ B cells (blue) or ERTR-7+ stromal cells (red). Image shown is representative of three biological replicates. Scale bars: 200 μm.


( B ) WT or CD169 - DTR + C57BL / 6 mice were treated i . p . with DT prior to s . c . inoculation in the left rear footpad with 108 particles of WT CHIKV . Viral genomes in the dLN and serum at 2 hpi were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 8 . Mann - Whitney test ; P > 0 . 05 .

O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR O O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) WT or CD169-DTR+ C57BL/6 mice were treated i.p. with DT prior to s.c. inoculation in the left rear footpad with 108 particles of WT CHIKV. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the rear feet with 5 * 104 PFU of CHIKV - E2 - mCherry and the popliteal dLNs were collected at 2 hpi . ( A ) Frozen dLN sections were stained for Lyve - 1 + LECs ( white ) , MARCO ( green ) , B220 ( blue ) and mCherry + CHIKV particles ( red ) . Scale bars : 200 μm .

O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the rear feet with 5*104 PFU of CHIKV-E2-mCherry and the popliteal dLNs were collected at 2 hpi. (A) Frozen dLN sections were stained for Lyve-1+ LECs (white), MARCO (green), B220 (blue) and mCherry+ CHIKV particles (red). Scale bars: 200 μm.


( H , I ) MARCO ( H ) and Mxra8 ( I ) expression is shown for MARCO + LECs for mock - infected cells and CHIKV - infected cells classified as either CHIKV - low or CHIKV - high . P values were calculated using a two - sided Wilcoxon rank - sum test with Bonferroni correction . In the boxplot , the central lines , the box limits , and the whiskers represent medians , the interquartile range ( IQR ) , and min / max values that are not outliers , respectively . Outliers are shown as points and include any values that are more than 1 . 5x IQR away from the box .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MARCO (H) and Mxra8 (I) expression is shown for MARCO+ LECs for mock-infected cells and CHIKV-infected cells classified as either CHIKV-low or CHIKV-high. P values were calculated using a two-sided Wilcoxon rank-sum test with Bonferroni correction. In the boxplot, the central lines, the box limits, and the whiskers represent medians, the interquartile range (IQR), and min/max values that are not outliers, respectively. Outliers are shown as points and include any values that are more than 1.5x IQR away from the box.


( B ) HCT116 cells in which eIF3k was homozygously modified with the mAID domain to trigger auxin - dependent degradation were exposed to 500 μM indole - 3 - acetic acid ( IAA ) for the periods shown , and cell lysate was subjected to immunoblotting with the indicated

O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O

(B) HCT116 cells in which eIF3k was homozygously modified with the mAID domain to trigger auxin-dependent degradation were exposed to 500 μM indole-3-acetic acid (IAA) for the periods shown, and cell lysate was subjected to immunoblotting with the indicated antibodies


( A ) Parental HCT116 and the five different conditional eIF3 - mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated . Metabolic activity ( i . e . NAD ( P ) H ) as a proxy of cell proliferation was determined by MTT assay . Graphs represent means ± SD , n = 5 - 6 . Asterisks denote : * p < 0 . 005 , * * p < 0 . 00005 , * * * p < 0 .

O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Parental HCT116 and the five different conditional eIF3-mAID cell lines were treated with the vehicle DMSO or with IAA for the periods indicated. Metabolic activity (i.e. NAD(P)H) as a proxy of cell proliferation was determined by MTT assay. Graphs represent means ± SD, n = 5 - 6. Asterisks denote: * p < 0.005 , ** p < 0.00005, *** p < 0.000005


( D ) 1 x 106 eIF3 - mAID cells were injected into nude mice . When tumors reached a diameter of ~ 4 mm , mice were treated with vehicle or 500 mg / kg IAA , and tumor growth was measured for 2 weeks . Graphs represent means ± SD , n = 5 to 7 . Numbers indicate p

O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

(D) 1 x 106 eIF3-mAID cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5 to 7. Numbers indicate p values


( E ) Expression of the indicated eIF3 proteins in 97 colon adenocarcinomas versus 100 normal colonic mucosae and 110 clear cell renal cell carcinomas versus 84 normal kidney samples . Log2 spectral count data from CPTAC via UALCAN

O O O B-EXP_ASSAY O O O O O O O B-DISEASE I-DISEASE O O O B-TISSUE I-TISSUE O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O B-TISSUE O O O O O O O O O O

(E) Expression of the indicated eIF3 proteins in 97 colon adenocarcinomas versus 100 normal colonic mucosae and 110 clear cell renal cell carcinomas versus 84 normal kidney samples. Log2 spectral count data from CPTAC via UALCAN portal


( A ) The indicated conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits . Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits . Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO . Bars represent means ± SD , n = 3 . Numbers indicate p values ( unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O 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 conditional eIF3 cell lines were exposed to 500 μM IAA for 12 h to induce the degradation of the respective eIF3 subunits. Cell lysates were subjected to immunoblotting to quantify the expression of the indicated eIF3 subunits. Signals were normalized to the reference signal of actin and plotted relative to the vehicle DMSO. Bars represent means ± SD, n = 3. Numbers indicate p values (unpaired Student's t-test).


( B ) The indicated cell lines were exposed to IAA for 12 h , and cell lysates were separated by sucrose density gradient centrifugation . The monosomal ( M ) and polysomal ( P ) peaks were quantified , and P / M ratios relative to the vehicle DMSO were plotted . Bars represent means ± SD , n = 3 . Numbers indicate p values ( unpaired Student ' s t - test ) .

O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O 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) The indicated cell lines were exposed to IAA for 12 h, and cell lysates were separated by sucrose density gradient centrifugation. The monosomal (M) and polysomal (P) peaks were quantified, and P/M ratios relative to the vehicle DMSO were plotted. Bars represent means ± SD, n = 3. Numbers indicate p values (unpaired Student's t-test).


( C ) Scatter plots of changes in polysomal mRNA versus total mRNA upon depletion of the indicated eIF3 subunits .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

(C) Scatter plots of changes in polysomal mRNA versus total mRNA upon depletion of the indicated eIF3 subunits.


( A ) eIF3k - mAID cells were maintained in media containing different concentrations of FBS and DMSO or IAA as indicated , and cell numbers were determined at various time points . Data represent means ± SD , n = 3 biological replicates . Numbers indicate p

O O O B-GENEPROD O O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(A) eIF3k-mAID cells were maintained in media containing different concentrations of FBS and DMSO or IAA as indicated, and cell numbers were determined at various time points. Data represent means ± SD, n = 3 biological replicates. Numbers indicate p values


( B ) eIF3k - mAID cells were serum starved by maintaining in media containing 0 % FBS for 24 h . During the last 12 h of starvation , DMSO or IAA was added as indicated . Cells were re - stimulated with media containing 10 % FBS , and the expression of the indicated proteins was followed of a period of 120 minutes . The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species . Bars represent means ± SD , n = 3 Numbers indicate p values ( unpaired Student ' s t - test ) .

O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) eIF3k-mAID cells were serum starved by maintaining in media containing 0% FBS for 24 h. During the last 12 h of starvation, DMSO or IAA was added as indicated. Cells were re-stimulated with media containing 10% FBS, and the expression of the indicated proteins was followed of a period of 120 minutes. The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).


( C ) eIF3k - mAID cells were exposed to IAA or DMSO for 12 hours , followed by the addition of increasing concentrations of rapamycin for 1 h . The expression of the indicated proteins was determined by immunoblotting . The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species . Bars represent means ± SD , n = 3 Numbers indicate p values ( unpaired Student ' s t - test ) .

O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) eIF3k-mAID cells were exposed to IAA or DMSO for 12 hours, followed by the addition of increasing concentrations of rapamycin for 1 h. The expression of the indicated proteins was determined by immunoblotting. The data from triplicate experiments were quantified and plotted as relative ratios of phosphorylated to unphosphorylated species. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).


( C ) Total ribosome occupancy of the indicated mRNAs in eIF3k - mAID cells exposed to DMSO or IAA for 12 hours was determined by RT - qPCR of RNA across a sucrose density gradient Bars represent means ± SD , n = 3 ; numbers indicate p values ( unpaired Student ' s t - test ) . Triplicate RT - qPCR data across the sucrose gradient are shown below the bar graphs .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(C) Total ribosome occupancy of the indicated mRNAs in eIF3k-mAID cells exposed to DMSO or IAA for 12 hours was determined by RT-qPCR of RNA across a sucrose density gradient Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test). Triplicate RT-qPCR data across the sucrose gradient are shown below the bar graphs.


( D ) Equal numbers of eIF3k - mAID cells stably expressing ectopic RPS15A ( pCDH - RPS15A ) or empty vector ( pCDH ) were plated and counted over a period of 6 days . Graphs represent means ± SD , n = 3 . Numbers indicate p

O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O 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) Equal numbers of eIF3k-mAID cells stably expressing ectopic RPS15A (pCDH-RPS15A) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate p values


( G ) Equal numbers of eIF3k - mAID cells stably expressing ectopic RPS4X ( pCDH - RPS4X ) or empty vector ( pCDH ) were plated and counted over a period of 6 days . Graphs represent means ± SD , n = 3 . Numbers indicate p values Total ribosome content of these cells was determined as described in ( A ) .

O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O

(G) Equal numbers of eIF3k-mAID cells stably expressing ectopic RPS4X (pCDH-RPS4X) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate p values Total ribosome content of these cells was determined as described in (A).


( B ) Parental eIF3k - mAID cells or S15A - eIF3KO cells ( clones # 45 and # 361 ) were maintained in standard media , and cell numbers were determined at various time points . Data represent means ± SD , n = 3 . Asterisks denote : * p < 0 . 005 , * * p < 0 . 00005 , * * * p < 0 .

O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Parental eIF3k-mAID cells or S15A-eIF3KO cells (clones #45 and # 361) were maintained in standard media, and cell numbers were determined at various time points. Data represent means ± SD, n = 3. Asterisks denote: * p < 0.005, ** p < 0.00005, *** p < 0.000005


( D ) Relative translational efficiencies ( TEs ) of mRNAs encoding RPS15A , RPS4X , and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines . RT - qPCR was performed on total RNA and on RNA contained within polysomal fractions > 2 ribosomes , and TE was calculated according to the formula TE = polysomal mRNA / total mRNA . Bars represent means ± SD , n = 3 ; numbers indicate p values ( unpaired Student ' s t - test ) .

O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O 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) Relative translational efficiencies (TEs) of mRNAs encoding RPS15A, RPS4X, and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines. RT-qPCR was performed on total RNA and on RNA contained within polysomal fractions > 2 ribosomes, and TE was calculated according to the formula TE = polysomal mRNA / total mRNA. Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test).


( A ) Parental eIF3k - mAID cells or S15A - eIF3KO cells ( clones # 45 and # 361 ) were maintained in media with DMSO or IAA , and cell numbers were determined at various time points . Data represent means ± SD ( too small to be visible ) , n = 3 . All p values were > 0 . 45 except at the single time point where indicated otherwise .

O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Parental eIF3k-mAID cells or S15A-eIF3KO cells (clones #45 and # 361) were maintained in media with DMSO or IAA, and cell numbers were determined at various time points. Data represent means ± SD (too small to be visible), n = 3. All p values were > 0.45 except at the single time point where indicated otherwise.


( D ) Total ribosome occupancy of the indicated mRNAs in S15A - eIF3KO ( clone # 361 ) cells exposed to DMSO or IAA for 12 hours was determined by RT - qPCR of RNA across a sucrose density gradient Bars represent means ± SD , n = 3 ; numbers indicate p values ( unpaired Student ' s t - test ) . Triplicate RT - qPCR data across the sucrose gradient are shown below the bar graphs .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(D) Total ribosome occupancy of the indicated mRNAs in S15A-eIF3KO (clone #361) cells exposed to DMSO or IAA for 12 hours was determined by RT-qPCR of RNA across a sucrose density gradient Bars represent means ± SD, n = 3; numbers indicate p values (unpaired Student's t-test). Triplicate RT-qPCR data across the sucrose gradient are shown below the bar graphs.


( E ) RNA immunoprecipitation . eIF3k - mAID and S15A - eIF3KO ( clone # 361 ) cells were exposed to DMSO or IAA for 12 hours . Cell lysates were employed in immunoprecipitation with eIF3c antibodies and co - precipitated mRNAs were quantified by qPCR . Bars represent means ± SD , n = 4 ; numbers indicate p values ( unpaired Student ' s t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

(E) RNA immunoprecipitation. eIF3k-mAID and S15A-eIF3KO (clone #361) cells were exposed to DMSO or IAA for 12 hours. Cell lysates were employed in immunoprecipitation with eIF3c antibodies and co-precipitated mRNAs were quantified by qPCR. Bars represent means ± SD, n = 4; numbers indicate p values (unpaired Student's t-test).


( A ) The indicated eIF3 - mAID cell lines were treated with DMSO or IAA for 12 hours , followed by exposure to 2 μg / ml tunicamycin for up to 72 hours . Metabolic activity ( i . e . NAD ( P ) H ) as a proxy of cell viability was determined by CCK8 assay . Graphs represent the percentage of change in cell viability upon downregulating eIF3 subunits with IAA . Date are means ± SD , n = 3 . ( B ) Same experiment as in ( A ) but cells were exposed to increasing concentrations of the oxidative stress inducer tert - butyl hydroperoxide ( TBHP ) for 2 hours .

O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O

(A) The indicated eIF3-mAID cell lines were treated with DMSO or IAA for 12 hours, followed by exposure to 2 μg/ml tunicamycin for up to 72 hours. Metabolic activity (i.e. NAD(P)H) as a proxy of cell viability was determined by CCK8 assay. Graphs represent the percentage of change in cell viability upon downregulating eIF3 subunits with IAA. Date are means ± SD, n = 3. (B) Same experiment as in (A) but cells were exposed to increasing concentrations of the oxidative stress inducer tert-butyl hydroperoxide (TBHP) for 2 hours.


( C ) The indicated eIF3 - mAID cell lines were treated with DMSO or IAA for 12 hours , followed by exposure to 2 μg / ml tunicamycin for 24 hours . The expression of individual eIF3 subunits , ribosomal proteins , and the ER stress marker BIP were assessed by immunoblotting . Tubulin is show for reference . The data from triplicate experiments were quantified and to avoid overcrowding of the graph data obtained from IAA - treated cells were not plotted . Bars represent means ± SD , n = 3 Numbers indicate p values ( unpaired Student ' s t - test ) .

O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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) The indicated eIF3-mAID cell lines were treated with DMSO or IAA for 12 hours, followed by exposure to 2 μg/ml tunicamycin for 24 hours. The expression of individual eIF3 subunits, ribosomal proteins, and the ER stress marker BIP were assessed by immunoblotting. Tubulin is show for reference. The data from triplicate experiments were quantified and to avoid overcrowding of the graph data obtained from IAA-treated cells were not plotted. Bars represent means ± SD, n = 3 Numbers indicate p values (unpaired Student's t-test).


Comparison of constitutively active rhodopsin mutants M257Y6 . 40 ( left [ 21 ] ) , T94I2 . 61 ( middle ) and G90D2 . 57 ( right [ 16 ] ) . All - trans ( yellow ) and cis retinal isomers ( orange ) , the side of retinal attachment K2967 . 43 ( blue slate ) , the retinal counterion E1133 . 28 ( blue slate ) , GαCT peptides ( cyan ) and the constitutively activating mutations M257Y6 . 40 , T94I2 . 61 , and G90D2 . 57 ( green ) are shown as spheres . Palmitoylation at C323H8 and glycosylation at N15NT are shown as sticks . The lower panels compare the M257Y6 . 40 , T94I2 . 61 and G90D2 . 57 retinal - binding pockets . All constructs also contain an additional thermostabilizing disulfide bridge , depicted as ( c - c ) , to enhance the stability [ 18 ] and crystallizability [ 19 ] without changing the rhodopsin activation pathway [ 20 ] .

O O O O B-GENEPROD O O O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O 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-GENEPROD O O O O O O

Comparison of constitutively active rhodopsin mutants M257Y6.40 (left [21]), T94I2.61 (middle) and G90D2.57 (right [16]). All-trans (yellow) and cis retinal isomers (orange), the side of retinal attachment K2967.43 (blue slate), the retinal counterion E1133.28 (blue slate), GαCT peptides (cyan) and the constitutively activating mutations M257Y6.40, T94I2.61, and G90D2.57 (green) are shown as spheres. Palmitoylation at C323H8 and glycosylation at N15NT are shown as sticks. The lower panels compare the M257Y6.40, T94I2.61 and G90D2.57 retinal-binding pockets. All constructs also contain an additional thermostabilizing disulfide bridge, depicted as (c-c), to enhance the stability [18] and crystallizability [19] without changing the rhodopsin activation pathway [20].


Kinetic characterization of the rhodopsin dark state . Thermal decay of dark state rhodopsin : A ) WT , B ) T94I and C ) G90D rhodopsin . The spectra are normalized to the OD280 and OD500 / OD480 at t = 0 . D ) Absorption maximum plotted as a function of time and fitted to a single exponential decay .

O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetic characterization of the rhodopsin dark state. Thermal decay of dark state rhodopsin: A) WT, B) T94I and C) G90D rhodopsin. The spectra are normalized to the OD280 and OD500/OD480 at t=0. D) Absorption maximum plotted as a function of time and fitted to a single exponential decay.


Retinal isomerization in the rhodopsin dark state : E ) WT , F ) T94I and G ) G90D rhodopsin , H ) The fraction of 11 - cis retinal is plotted as a function of time and fitted to a single exponential decay .

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Retinal isomerization in the rhodopsin dark state: E) WT, F) T94I and G) G90D rhodopsin, H) The fraction of 11-cis retinal is plotted as a function of time and fitted to a single exponential decay.


SB stability in dark state rhodopsin : I ) WT , J ) T94I and K ) G90D rhodopsin . L ) OD440 is plotted as a function of time and fitted to a single exponential decay . All decay rates were measured at 55 C .

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SB stability in dark state rhodopsin: I) WT, J) T94I and K) G90D rhodopsin. L) OD440 is plotted as a function of time and fitted to a single exponential decay. All decay rates were measured at 55 C.