Overall survival stratified by tumor mutational burden ( TMB ) across treatment groups in 3 , 217 patients . Samples were classified as TMB - high if the amount of non - synonymous mutations per expressed MB ( rnaMB ) was ≥ the median number of non - synonymous mutations per rnaMB across the whole SCAN - B cohort ( 0 . 082 mutations per rnaMB ) and TMB - low otherwise . In each Kaplan - Meier plot , TMB - low cases are plotted in blue , TMB - high cases are plotted in red , the log - rank P value is given , and the hazard ratio ( HR ) for TMB - high is given with a 95 % CI and after uni - variable and multi - variable ( MV ) Cox regression adjustment . Covariables included in the MV analysis were age at diagnosis , lymph node status , tumor size , and the variables denoted by the following symbols : ¶ , ER , PgR , HER2 , and NHG ; ¤ , ER , PgR , and NHG ; $ , HER2 and NHG ; # , NHG . Abbreviations : TMB , tumor mutational burden ; ER , estrogen receptor ; PgR , progesterone receptor ; HER2 , human epidermal growth factor receptor 2 ; NHG , Nottingham histological grade ; HoR , hormone receptor ; TNBC , triple - negative breast cancer .

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Overall survival stratified by tumor mutational burden (TMB) across treatment groups in 3,217 patients. Samples were classified as TMB-high if the amount of non-synonymous mutations per expressed MB (rnaMB) was ≥ the median number of non-synonymous mutations per rnaMB across the whole SCAN-B cohort (0.082 mutations per rnaMB) and TMB-low otherwise. In each Kaplan-Meier plot, TMB-low cases are plotted in blue, TMB-high cases are plotted in red, the log-rank P value is given, and the hazard ratio (HR) for TMB-high is given with a 95% CI and after uni-variable and multi-variable (MV) Cox regression adjustment. Covariables included in the MV analysis were age at diagnosis, lymph node status, tumor size, and the variables denoted by the following symbols: ¶, ER, PgR, HER2, and NHG; ¤, ER, PgR, and NHG; $, HER2 and NHG; #, NHG. Abbreviations: TMB, tumor mutational burden; ER, estrogen receptor; PgR, progesterone receptor; HER2, human epidermal growth factor receptor 2; NHG, Nottingham histological grade; HoR, hormone receptor; TNBC, triple-negative breast cancer.


E , Immunoblot analysis of cells described in ( D ) , harvested at the indicated time points after G1 / S release . Exemplary blot from three independent experiments .

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E, Immunoblot analysis of cells described in (D), harvested at the indicated time points after G1/S release. Exemplary blot from three independent experiments.


F , DUB activity assay for OTUD6B in asynchronous , G1 / S - or mitotically synchronized and released A549 cells using HA - ubiquitin vinyl - sulfone to isolate active forms of DUBs . Analysis was performed by immunoblotting using the indicated antibodies .

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F, DUB activity assay for OTUD6B in asynchronous, G1/S- or mitotically synchronized and released A549 cells using HA-ubiquitin vinyl-sulfone to isolate active forms of DUBs. Analysis was performed by immunoblotting using the indicated antibodies.


B , In - vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA . Cells were transfected with the indicated siRNAs and overexpression constructs , then treated with MG132 for 3 hrs . Lysis and IP was done under denaturing conditions followed by WB analysis . Data information : For B , exemplary blot of two independent experiments .

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B, In-vivo ubiquitylation analyses of LIN28B in HEK293T cells in which OTUD6B was silenced by siRNA. Cells were transfected with the indicated siRNAs and overexpression constructs, then treated with MG132 for 3 hrs. Lysis and IP was done under denaturing conditions followed by WB analysis. Data information: For B, exemplary blot of two independent experiments.


C , In - vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B ( OTUD6B - C158A ) overexpressing cells . HEK293T cells were transfected with indicated combinations of FLAG - LIN28B , HA - Ubiquitin , OTUD6B , OTUD6B - C158A and EV control and treated with MG132 for 3 hrs 24 hrs later . Denatured WCE were subjected to FLAG - IP . WCE and IP were analyzed by immunoblotting including a K48 - specific ubiquitin antibody . Exemplary blots from three independent experiments are shown .

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C, In-vivo ubiquitylation assay of LIN28B in OTUD6B and a catalytically inactive variant of OTUD6B (OTUD6B-C158A) overexpressing cells. HEK293T cells were transfected with indicated combinations of FLAG-LIN28B, HA-Ubiquitin, OTUD6B, OTUD6B-C158A and EV control and treated with MG132 for 3 hrs 24 hrs later. Denatured WCE were subjected to FLAG-IP. WCE and IP were analyzed by immunoblotting including a K48-specific ubiquitin antibody. Exemplary blots from three independent experiments are shown.


D , Pull - down of endogenous LIN28B from HEK293T WCE using bacterially purified GST - OTUD6B . Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies . Data information : For D , exemplary blot of two independent experiments .

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D, Pull-down of endogenous LIN28B from HEK293T WCE using bacterially purified GST-OTUD6B. Pulldowns and WCE were analyzed by immunoblot using the indicated antibodies. Data information: For D, exemplary blot of two independent experiments.


E , Immunoblot analysis of an in - vitro deubiquitylation assay using GST - purified OTUD6B from bacteria and FLAG - purified ubiquitylated LIN28B from HEK293T . FLAG - IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG - LIN28B and HA - Ubiquitin . Purified FLAG - LIN28B was eluted from the beads and incubated with GST or GST - OTUD6B proteins followed by immunoblot analysis . Data information : For E : exemplary blot of two independent experiments .

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E, Immunoblot analysis of an in-vitro deubiquitylation assay using GST-purified OTUD6B from bacteria and FLAG-purified ubiquitylated LIN28B from HEK293T. FLAG-IP was performed under denaturing conditions from HEK293T WCEs expressing FLAG-LIN28B and HA-Ubiquitin. Purified FLAG-LIN28B was eluted from the beads and incubated with GST or GST-OTUD6B proteins followed by immunoblot analysis. Data information: For E: exemplary blot of two independent experiments.


A , Co - IP of FLAG - OTUD6B with endogenous LIN28B from MM1 . S cells synchronized in G1 / S and 12 hrs post release . MM1 . S cells stably expressing FLAG - OTUD6B or FLAG - EV were synchronized at G1 / S and harvested at the indicated time points after release . IPs were preformed and analyzed together with WCE by immunoblotting . Data information : All blots are representations of three independent experiments .

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A, Co-IP of FLAG-OTUD6B with endogenous LIN28B from MM1.S cells synchronized in G1/S and 12 hrs post release. MM1.S cells stably expressing FLAG-OTUD6B or FLAG-EV were synchronized at G1/S and harvested at the indicated time points after release. IPs were preformed and analyzed together with WCE by immunoblotting. Data information: All blots are representations of three independent experiments.


B , Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle . RPMI ( B ) cells were synchronized at the G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized cells were released , harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies . Data information : All blots are representations of three independent experiments .

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B, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. RPMI (B) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.


C , Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle . A549 ( C ) cells were synchronized at the G1 / S transition by a double thymidine block or in mitosis by a sequential thymidine / nocodazole block . Synchronized cells were released , harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies . Data information : All blots are representations of three independent experiments .

C, Immunoblot analysis of LIN28B and OTUD6B protein levels throughout the cell cycle. A549 (C) cells were synchronized at the G1/S transition by a double thymidine block or in mitosis by a sequential thymidine/nocodazole block. Synchronized cells were released, harvested at the indicated time points and analyzed by immunoblot using the indicated antibodies. Data information: All blots are representations of three independent experiments.


D , Immunoblot analysis of LIN28B protein half - life in G1 / S - synchronized ( upper panel ) and asynchronous ( lower panel ) A549 cells upon OTUD6B depletion . Cells transfected with the respective siRNAs were synchronized in G1 / S - phase or not and treated with cycloheximide ( CHX ) as indicated . Data information : All blots are representations of three independent experiments .

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D, Immunoblot analysis of LIN28B protein half-life in G1/S-synchronized (upper panel) and asynchronous (lower panel) A549 cells upon OTUD6B depletion. Cells transfected with the respective siRNAs were synchronized in G1/S-phase or not and treated with cycloheximide (CHX) as indicated. Data information: All blots are representations of three independent experiments.


D , Immunoblot analysis of WCE obtained from a representative experiment depicted in ( C ) .

D, Immunoblot analysis of WCE obtained from a representative experiment depicted in (C).


C , Immunoblot analysis of RPMI8226 , KMS12BM and MM1 . S cells expressing the indicated shRNAs . Depicted are examples of three independent experiments per cell line . Specification of MYC rearrangements : RPMI8226 ( der ( 17 ) t ( ? 8 ; 17 ) ( q21 . 2 ; q25 ) ) , KMS12BM ( der ( 1 ; 8 ) ( q10 ; q10 ) x2 ) , MM1 . S ( der3t ( 3 ; 8 ) ) .

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C, Immunoblot analysis of RPMI8226, KMS12BM and MM1.S cells expressing the indicated shRNAs. Depicted are examples of three independent experiments per cell line. Specification of MYC rearrangements: RPMI8226 (der(17)t(?8;17)(q21.2;q25)), KMS12BM (der(1;8)(q10;q10)x2), MM1.S (der3t(3;8)).


A , Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis . mRNA expression in primary CD138 + MM cells was quantified by real - time qPCR ( n = 89 patients ) . Data are fit by linear regression ( black line ) ; Pearson r , Pearson correlation coefficient ; P < 0 . 0001 ; by linear regression and Pearson correlation .

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A, Correlation between MYC and OTUD6B expression in MM patient samples at diagnosis. mRNA expression in primary CD138+ MM cells was quantified by real-time qPCR (n = 89 patients). Data are fit by linear regression (black line); Pearson r, Pearson correlation coefficient; P < 0.0001; by linear regression and Pearson correlation.


Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing , indicated by increased % callus mineralization , at day 28 after operation in a dose - dependent manner . Data are presented as mean ± SEM . Treatment with 1 ng TNF versus PBS control , * * * * P < 0 . 0001 by one - way ANOVA with Dunnett ' s multiple comparisons test .

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Addition of rhTNF at the fracture site on days 0 and 1 led to augmented healing, indicated by increased % callus mineralization, at day 28 after operation in a dose-dependent manner. Data are presented as mean ± SEM. Treatment with 1 ng TNF versus PBS control, ****P < 0.0001 by one-way ANOVA with Dunnett's multiple comparisons test.


Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing , indicated by % callus mineralization . Data are presented as mean ± SEM . 1 ng TNF versus PBS control treatment on days 0 and 1 , * * * P = 0 . 0009 by one - way ANOVA with Dunnett ' s multiple comparisons test .

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Addition of rhTNF at the fracture site must be given within the first 24 h to augment healing, indicated by % callus mineralization. Data are presented as mean ± SEM. 1 ng TNF versus PBS control treatment on days 0 and 1, ***P = 0.0009 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with systemic anti - TNF or local rmIL - 10 led to impaired fracture repair at day 28 , indicated by % callus mineralization . Data are presented as mean ± SEM . Treatment with PBS control versus anti - TNF , * P = 0 . 037 , PBS control versus rmIL - 10 , * P = 0 . 037 , by unpaired two - sided t - tests .

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Treatment with systemic anti-TNF or local rmIL-10 led to impaired fracture repair at day 28, indicated by % callus mineralization. Data are presented as mean ± SEM. Treatment with PBS control versus anti-TNF, *P = 0.037, PBS control versus rmIL-10, *P = 0.037, by unpaired two-sided t-tests.


Representative micro - CT images at the fracture site showing ( from left to right ) : lateral view of tibia , cross - section , and cross and longitudinal sections with color overlay and 3D reconstruction . In the color overlays , the shade of blue corresponds to percentage mineralization : light blue denotes soft immature callus , whereas dark blue denotes hard mineralized callus . Scale bar , 2 mm .

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Representative micro-CT images at the fracture site showing (from left to right): lateral view of tibia, cross-section, and cross and longitudinal sections with color overlay and 3D reconstruction. In the color overlays, the shade of blue corresponds to percentage mineralization: light blue denotes soft immature callus, whereas dark blue denotes hard mineralized callus. Scale bar, 2 mm.


Representative ISH image ( light field ) showing TNF expression at the murine fracture site at 24 h after fracture . Scale bar , 250 μm . Red box indicates region of interest .

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Representative ISH image (light field) showing TNF expression at the murine fracture site at 24 h after fracture. Scale bar, 250 μm. Red box indicates region of interest.


High - power micrographs of region of interest from ( E ) : at 24 h after fracture , mTNF expression ( white signal on dark field , above ) co - localized with polymorphonuclear cells found on the adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm . Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti - neutrophil elastase .

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High-power micrographs of region of interest from (E): at 24 h after fracture, mTNF expression (white signal on dark field, above) co-localized with polymorphonuclear cells found on the adjacent H&amp;E section (below). Scale bar, 25 μm. Neutrophils were identified by their polymorphonuclear morphology as well as positive dark brown staining with anti-neutrophil elastase.


High - power micrographs : at 7 days , TNF expression ( white signal on dark field , above ) co - localized with F4 / 80 - positive cells ( stained dark brown ) extravasating from a blood vessel on the adjacent H & amp ; amp ; E section ( below ) . Scale bar , 25 μm .

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High-power micrographs: at 7 days, TNF expression (white signal on dark field, above) co-localized with F4/80-positive cells (stained dark brown) extravasating from a blood vessel on the adjacent H&amp;E section (below). Scale bar, 25 μm.


Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model . Compared to control and number of blood neutrophils at 3 h * * * P = 0 . 0006 and at 6 h * P = 0 . 042 , by one - way ANOVA with Dunnett correction .

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Neutrophils were mobilized into the murine systemic circulation within 30 min of injury in our murine fracture model. Compared to control and number of blood neutrophils at 3 h ***P = 0.0006 and at 6 h *P = 0.042, by one-way ANOVA with Dunnett correction.


Depletion of neutrophils using anti - Ly6G . Intravenous anti - Ly6G treatment led to depletion of neutrophils in the systemic circulation ( left ) as well the local peri - fracture soft tissues ( right ) . Left : counts of neutrophils in the blood harvested by cardiac puncture at 3 h post - injury . Data are presented as mean ± SEM ( * * P = 0 . 0074 using unpaired one - tailed t - test ) . Right : counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti - Ly6G antibody versus IgG control at day 1 and day 7 post - fracture . At day 1 , number of neutrophils in control group as detected by Ly6G and NE in control versus treatment : * * * * P = 0 . 0001 for both Ly6G and NE , by unpaired two - tailed t - test . At day 7 , number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups : * * * * P = 0 . 0001 for Ly6G and * P = 0 . 020 for NE , by unpaired two - tailed t - test . Number of neutrophils at day 1 versus day 7 in control groups , * P = 0 . 037 for Ly6G and * * * P = 0 . 0006 for NE , by unpaired two - tailed t - test .

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Depletion of neutrophils using anti-Ly6G. Intravenous anti-Ly6G treatment led to depletion of neutrophils in the systemic circulation (left) as well the local peri-fracture soft tissues (right). Left: counts of neutrophils in the blood harvested by cardiac puncture at 3 h post-injury. Data are presented as mean ± SEM (**P = 0.0074 using unpaired one-tailed t-test). Right: counts of positively stained infiltrative neutrophils in the adjacent muscle to the fracture site comparing neutrophil depletion with anti-Ly6G antibody versus IgG control at day 1 and day 7 post-fracture. At day 1, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment: ****P = 0.0001 for both Ly6G and NE, by unpaired two-tailed t-test. At day 7, number of neutrophils in control group as detected by Ly6G and NE in control versus treatment groups: ****P = 0.0001 for Ly6G and *P = 0.020 for NE, by unpaired two-tailed t-test. Number of neutrophils at day 1 versus day 7 in control groups, *P = 0.037 for Ly6G and ***P = 0.0006 for NE, by unpaired two-tailed t-test.


Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti - Ly6G and anti - neutrophil elastase primary antibodies at day 1 post - fracture following treatment with anti - Ly6G antibody or IgG control . Scale bar , 100 μm .

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Representative sections showing local infiltration of neutrophils in the adjacent muscle stained using anti-Ly6G and anti-neutrophil elastase primary antibodies at day 1 post-fracture following treatment with anti-Ly6G antibody or IgG control. Scale bar, 100 μm.


Depletion of neutrophils using anti - Ly6G led to impaired fracture healing . Representative section stained with Masson ' s trichrome at day 14 comparing fracture healing in mouse treated with isotype control ( top ) versus anti - Ly6G ( bottom ) . Black and white images in the right column are identical to the color images in the left column ; they have been labeled to clearly demonstrate the anatomical structures . Control section shows advanced mineralized callus , while treatment section shows a large immature unmineralized callus . Muscle fibers : red ; collagen , bone , and mineralized callus : green . Scale bar , 1 mm .

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Depletion of neutrophils using anti-Ly6G led to impaired fracture healing. Representative section stained with Masson's trichrome at day 14 comparing fracture healing in mouse treated with isotype control (top) versus anti-Ly6G (bottom). Black and white images in the right column are identical to the color images in the left column; they have been labeled to clearly demonstrate the anatomical structures. Control section shows advanced mineralized callus, while treatment section shows a large immature unmineralized callus. Muscle fibers: red; collagen, bone, and mineralized callus: green. Scale bar, 1 mm.


Anti - Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro - CT images . Anti - Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control . Data are presented as mean ± SEM . * * * P = 0 . 0006 by unpaired two - sided t - test . Scale Bar , 2 mm .

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Anti-Ly6G treatment led to impaired fracture healing as shown by the reduced % callus mineralization at day 28 after surgery and representative micro-CT images. Anti-Ly6G treatment led to delayed mineralization and remodeling of the fracture callus compared to control. Data are presented as mean ± SEM. ***P = 0.0006 by unpaired two-sided t-test. Scale Bar, 2 mm.


Anti - Ly6G treatment depletes local monocytes / macrophages at the fracture site . Counts of positively stained infiltrative F4 / 80 + cells in the adjacent muscle to the fracture site following treatment with control or anti - Ly6G at day 1 and day 7 . Data are presented as mean ± SEM . * * P = 0 . 005 at day 1 and * * P = 0 . 002 at day 7 , and * P = 0 . 013 for control at day 1 versus control at day 7 , by unpaired two - sided t - test .

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Anti-Ly6G treatment depletes local monocytes/macrophages at the fracture site. Counts of positively stained infiltrative F4/80+ cells in the adjacent muscle to the fracture site following treatment with control or anti-Ly6G at day 1 and day 7. Data are presented as mean ± SEM. **P = 0.005 at day 1 and **P = 0.002 at day 7, and *P = 0.013 for control at day 1 versus control at day 7, by unpaired two-sided t-test.


Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils ( Ly6G + , CD11b + cells ) and monocytes / macrophages ( Ly6G − , CD11b + , CD115 + cells ) in the cellular infiltrate . These effects were abrogated by the addition of anti - CCL2 . Neutrophils : FS versus FS + TNF * P = 0 . 015 , FS + TNF versus FS + TNF + anti - CCL2 * P = 0 . 028 , and FS + TNF + anti - CCL2 versus FS + TNF + IgG * P = 0 . 042 by one - way ANOVA followed by Bonferroni ' s multiple comparison test . Monocytes / macrophages : FS versus FS + TNF * * P = 0 . 0063 , FS + TNF versus FS + TNF + anti - CCL2 * * * * P < 0 . 0001 , and FS + TNF + anti - CCL2 v FS + TNF + IgG * P = 0 . 015 by one - way ANOVA followed by Bonferroni ' s multiple comparisons test .

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

Addition of 1 ng rhTNF to fracture supernatant in the air pouch model led to increased numbers of neutrophils (Ly6G+, CD11b+ cells) and monocytes/macrophages (Ly6G−, CD11b+, CD115+ cells) in the cellular infiltrate. These effects were abrogated by the addition of anti-CCL2. Neutrophils: FS versus FS + TNF *P = 0.015, FS + TNF versus FS + TNF + anti-CCL2 *P = 0.028, and FS + TNF + anti-CCL2 versus FS + TNF + IgG *P = 0.042 by one-way ANOVA followed by Bonferroni's multiple comparison test. Monocytes/macrophages: FS versus FS + TNF **P = 0.0063, FS + TNF versus FS + TNF + anti-CCL2 ****P < 0.0001, and FS + TNF + anti-CCL2 v FS + TNF + IgG *P = 0.015 by one-way ANOVA followed by Bonferroni's multiple comparisons test.


Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch . " Neat " indicates level of CCL2 in fracture supernatant before injection into air pouch . Data are presented as mean ± SEM . FS versus FS + TNF * * P = 0 . 0020 by one - way ANOVA with Bonferroni ' s multiple comparisons test .

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 B-MEASURED_VAR 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

Addition of 1 ng rhTNF to fracture supernatant increased CCL2 protein levels in the air pouch. "Neat" indicates level of CCL2 in fracture supernatant before injection into air pouch. Data are presented as mean ± SEM. FS versus FS + TNF **P = 0.0020 by one-way ANOVA with Bonferroni's multiple comparisons test.


Addition of rhTNF to enriched bone marrow - derived murine neutrophils pre - exposed to fracture supernatant in vitro led to up - regulation of CCL2 production at 1 h . Data are presented as mean ± SEM . No TNF versus TNF 0 . 01 ng / ml * P = 0 . 012 , TNF 0 . 1 ng / ml * * * P < 0 . 0001 , TNF 1 . 0 ng * * * P = 0 . 0001 , TNF 10 ng / ml * * * P < 0 . 0001 , and TNF 100 ng / ml * P = 0 . 023 by one - way ANOVA with Dunnett ' s multiple comparisons test .

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

Addition of rhTNF to enriched bone marrow-derived murine neutrophils pre-exposed to fracture supernatant in vitro led to up-regulation of CCL2 production at 1 h. Data are presented as mean ± SEM. No TNF versus TNF 0.01 ng/ml *P = 0.012, TNF 0.1 ng/ml ***P < 0.0001, TNF 1.0 ng ***P = 0.0001, TNF 10 ng/ml ***P < 0.0001, and TNF 100 ng/ml *P = 0.023 by one-way ANOVA with Dunnett's multiple comparisons test.


Treatment with CCR2 antagonist , INCB3344 , led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation . Data are presented as mean ± SEM . * P = 0 . 011 unpaired two - sided t - test . Representative micro - CT images are shown . Scale bar , 2 mm .

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

Treatment with CCR2 antagonist, INCB3344, led to impaired fracture healing compared to vehicle control as shown by the reduced % callus mineralization at day 28 after operation. Data are presented as mean ± SEM. *P = 0.011 unpaired two-sided t-test. Representative micro-CT images are shown. Scale bar, 2 mm.


rhTNF treatment augmented early phase fracture healing in osteoporotic mice . Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28 . Data are presented as mean ± SEM . At day 14 , PBS control versus TNF , * * P = 0 . 0099 ; at day 28 , PBS control versus TNF , nsP = 0 . 37 , by unpaired two - sided t - test . Representative micro - CT images at day 14 are shown . rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS - treated control group . Scale bar , 2 mm .

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

rhTNF treatment augmented early phase fracture healing in osteoporotic mice. Treatment with 1 ng rhTNF at the fracture site on days 0 and 1 led to increased % callus mineralization at day 14 but no difference at day 28 indicating accelerated early healing but the same final result at day 28. Data are presented as mean ± SEM. At day 14, PBS control versus TNF, **P = 0.0099; at day 28, PBS control versus TNF, nsP = 0.37, by unpaired two-sided t-test. Representative micro-CT images at day 14 are shown. rhTNF treatment led to mature callus bridging across the fracture which was absent in the PBS-treated control group. Scale bar, 2 mm.


( A - D ) A . castellanii was infected ( MOI 5 ) with L . pneumophila JR32 producing ( A ) GFP under the control of PflaA ( Ptac - mCherry - PflaA - gfp ) and mCherry constitutively , ( B ) GFP under the control of PralF ( PralF - gfp ) and stained with DAPI , ( C ) GFP under the control of PsidC ( PsidC - gfp ) and stained with DAPI , or ( D ) Timer constitutively ( pNP107 ) . The infected amoebae were fixed at different time points post - infection , analyzed by confocal microscopy and 3D reconstructed ( scale bars : 5 µm ) . Motile and virulent bacteria ( GFP - positive ) emerge at the colony cluster periphery only after bacterial replication has ceased ( red fluorescent Timer ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer).


( G ) Transmission electron micrographs of A . castellanii infected with L . pneumophila . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila wild - type strain JR32 and subjected to high - pressure freezing and electron microscopy . Many LCVs were still intact at 42 h post infection ( left panel ; arrow : intact LCV membrane , arrowhead plasma membrane ) , and occasionally , the bacteria were flagellated ( middle panel , arrow ) . Some LCVs were already ruptured , and the bacteria were released into the host cell cytosol ( right panel ; arrows : ruptured LCV membrane ) . Scale bars : 2 µm , 0 . 5 µm ( insets ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transmission electron micrographs of A. castellanii infected with L. pneumophila. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila wild-type strain JR32 and subjected to high-pressure freezing and electron microscopy. Many LCVs were still intact at 42 h post infection (left panel; arrow: intact LCV membrane, arrowhead plasma membrane), and occasionally, the bacteria were flagellated (middle panel, arrow). Some LCVs were already ruptured, and the bacteria were released into the host cell cytosol (right panel; arrows: ruptured LCV membrane). Scale bars: 2 µm, 0.5 µm (insets).


( A ) L . pneumophila subpopulation proteomics . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) . After cell lysis , released intracellular bacteria were sorted by flow cytometry in mCherry - positive / GFP - negative and mCherry - positive / GFP - positive L . pneumophila subpopulations , and their proteome was determined and compared . Protein abundance in each subset is depicted as a volcano plot . Proteins enriched in the GFP - producing subpopulation have a positive log2 ratio ( q - value ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) L. pneumophila subpopulation proteomics. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry-positive/GFP-negative and mCherry-positive/GFP-positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP-producing subpopulation have a positive log2 ratio (q-value).


( D , E ) Expression of select L . pneumophila genes in amoebae . A . castellanii was infected ( MOI 5 ; 6 h and 48 h ) with L . pneumophila JR32 harboring reporter constructs for ( D ) PfleQ - mCherry - PflaA - gfp or ( E ) PlegC8 - mCherry - PflaA - gfp , respectively . The infected amoebae were fixed and imaged by confocal microscopy ( scale bars : 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 O O O O O O O O O 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, E) Expression of select L. pneumophila genes in amoebae. A. castellanii was infected (MOI 5; 6 h and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) PfleQ-mCherry-PflaA-gfp or (E) PlegC8-mCherry-PflaA-gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).


( A - E ) Representative live cell microscopy images of D . discoideum infected with PflaA - GFP - producing L . pneumophila . D . discoideum Ax3 producing cytosolic mCherry ( pDM1042 ) was infected ( MOI 5 ) with ( A , B ) wild - type L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) , ( C , D ) JR32 ( Ptac - mCerulean - PflaA - gfp ) or ( E ) ∆ flaA ( Ptac - mCerulean - PflaA - gfp ) and embedded in agarose for 48 h . PflaA - GFP - positive L . pneumophila ( A ) escapes the LCV ( 0 sec ) , followed by other bacteria , which are contained in the cytosol and localize to the plasma membrane , where a PflaA - GFP - positive L . pneumophila first leaves the ruptured host cell ( 185 sec ) , or ( B ) triggers host cell death ( loss of cytosolic mCherry ) upon exit of the amoeba . PflaA - GFP - positive L . pneumophila ( C ) escape the LCV after 75 sec and spread within the cytosol for several minutes , and ( D ) are the first to exit the host cell , leading to the loss of cytosolic mCherry and host cell death . ( E ) L . pneumophila ∆ flaA lyses the LCV and host cell less vigorously . Scale bars : 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 O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(A-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm.


( B ) L . pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae . A . castellanii amoebae were infected ( MOI 5 , 52 h ) with L . pneumophila JR32 , ∆ flaA or ∆ lqsA harboring Ptac - mCherry - PflaA - gfp and embedded in 0 . 5 % agarose / PYG medium . The fluorescence images are representative for the L . pneumophila strains indicated : JR32 ( n = 25 ) , ∆ flaA ( n = 13 ) or ∆ lqsA ( n = 19 ) . Scale bars : 20 µm .

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

(B) L. pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. A. castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆flaA or ∆lqsA harboring Ptac-mCherry-PflaA-gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 (n = 25), ∆flaA (n = 13) or ∆lqsA (n = 19). Scale bars: 20 µm.


( A , B ) Role of the Lqs system for PflaA expression in intracellular L . pneumophila . A . castellanii was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , and ( A ) fixed and analyzed by confocal microscopy ( representative 3D reconstructions , scale bars : 10 µm ) , or ( B ) lysed , fixed and analyzed by flow cytometry .

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

(A, B) Role of the Lqs system for PflaA expression in intracellular L. pneumophila. A. castellanii was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, and (A) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 10 µm), or (B) lysed, fixed and analyzed by flow cytometry.


( C - F ) Role of the Lqs system for LCV escape and cytosolic localization of L . pneumophila . ( C , D ) A . castellanii or ( E , F ) D . discoideum was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , fixed and treated with an anti ‐ ubiquitin antibody . Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy . Representative images of ( C ) A . castellanii ( scale bars : 10 µm ) or ( E ) D . discoideum ( scale bars : 5 μm ) infected with L . pneumophila JR32 escaping or not from the LCV are shown . The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells : ( D ) JR32 : n = 482 ; ΔlqsA : n = 834 ; ΔlqsR : n = 572 ; ΔlqsS : n = 729 ; ΔlqsT : n = 1469 ; ΔlqsS - ΔlqsT : n = 693 ; ( F ) : JR32 : n = 136 ; ΔlqsA : n = 201 ; ΔlqsR : n = 303 ; ΔlqsS : n = 183 ; ΔlqsT : n = 534 ; ΔlqsS - ΔlqsT : n = 180 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila. (C, D) A. castellanii or (E, F) D. discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, fixed and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; ΔlqsA: n = 834; ΔlqsR: n = 572; ΔlqsS: n = 729; ΔlqsT: n = 1469; ΔlqsS-ΔlqsT: n = 693; (F): JR32: n = 136; ΔlqsA: n = 201; ΔlqsR: n = 303; ΔlqsS: n = 183; ΔlqsT: n = 534; ΔlqsS-ΔlqsT: n = 180.


( G , H ) D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - GFP and cytosolic - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , ∆ lqsS - ∆ lqsT , or ∆ flaA harboring Ptac - mCerulean . Representative images of ( G ) D . discoideum infected with L . - pneumophila JR32 in intact cells with intact LCV ( 18 . 5 % ) , lysed cells and lysed LCVs ( 60 . 2 % ) , intact cells with lysed LCVs ( 15 . 5 % ) and lysed cells with intact LCVS ( 5 . 8 % ) are shown ( scale bars : 5 µm ) . ( H ) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane : JR32 : n = 426 ; ΔlqsA : n = 468 ; ΔlqsR : n = 344 ; ΔlqsS : n = 325 ; ΔlqsT : n = 287 ; ΔlqsS - ΔlqsT : n = 289 ; ΔflaA : n = 248 .

O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) D. discoideum producing the LCV/PtdIns(4)P probe P4C-GFP and cytosolic-mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, ∆lqsS-∆lqsT, or ∆flaA harboring Ptac-mCerulean. Representative images of (G) D. discoideum infected with L.-pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%) and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; ΔlqsA: n = 468; ΔlqsR: n = 344; ΔlqsS: n = 325; ΔlqsT: n = 287; ΔlqsS-ΔlqsT: n = 289; ΔflaA: n = 248.


D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila wild - type strains ( JR32 , Corby ) , or phospholipase triple mutants ( ∆ plcABC , ∆ plaACD ) harboring the Ptac - mCerulean - PflaA - gfp reporter construct , and fixed and treated with an anti ‐ ubiquitin antibody , LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact , P4C - mCherry - positive LCVs or ubiquitin - decorated , cytosolic bacteria . ( A ) Representative images of amoebae harboring L . pneumophila confined within LCVs or released from the LCV to the cytosol are shown ( scale bars : 5 μm ) .

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

D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm).


( A ) Pancreatic cancer cells ( PANC - 1 ) and normal human fibroblasts ( NHFs ) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points , cells were incubated with WST - 1 cell viability dye for 4 hours . Absorbance was read at 450 nm and results were expressed as a percent of the control . Values are expressed as mean ± SD from quadruplicates ; ( i ) PANC - 1 treated with NSC 51046 ( Interaction between each concentration : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( ii ) NHF treated with NSC 51046 ( Interaction between each concentration : * * * * p < 0 . 0001 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iii ) PANC - 1 treated with Green 1 ( Interaction between each concentration : p = 0 . 9955 ; Control versus column factor : * * * * p < 0 . 0001 ) ; ( iv ) NHF treated with Green 1 ( Interaction between each concentration : p = 0 . 3642 ; Control versus column factor : * p = 0 . 0370 )

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(A) Pancreatic cancer cells (PANC-1) and normal human fibroblasts (NHFs) were treated with increasing concentrations of Green 1 and NSC 51046 and following treatment at the indicated time points, cells were incubated with WST-1 cell viability dye for 4 hours. Absorbance was read at 450 nm and results were expressed as a percent of the control. Values are expressed as mean ± SD from quadruplicates; (i) PANC-1 treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (ii) NHF treated with NSC 51046 (Interaction between each concentration: ****p<0.0001; Control versus column factor: ****p<0.0001); (iii) PANC-1 treated with Green 1 (Interaction between each concentration: p = 0.9955; Control versus column factor: ****p<0.0001); (iv) NHF treated with Green 1 (Interaction between each concentration: p = 0.3642; Control versus column factor: *p = 0.0370)


( B ) Following treatment of human acute T cell leukemia cells ( Jurkat ) with Green 1 , a trypan blue excluding assay was carried out . Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer . Results are expressed as percent of trypan blue positive cells , indicating dead cells with non - intact cell membranes . Values are expressed as mean ± SD from two independent experiments .

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(B) Following treatment of human acute T cell leukemia cells (Jurkat) with Green 1, a trypan blue excluding assay was carried out. Cells were stained with trypan blue cell membrane impermeable dye and the numbers of trypan blue positive cells were obtained using a hemocytometer. Results are expressed as percent of trypan blue positive cells, indicating dead cells with non-intact cell membranes. Values are expressed as mean ± SD from two independent experiments.


Following treatment with either Green 1 ( A ) Pancreatic cancer cells ( BxPC - 3 and PANC - 1 ) and normal human and fetal fibroblasts ( NHFs and NFFs ) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis . Brightly stained , condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis .

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Following treatment with either Green 1(A) Pancreatic cancer cells (BxPC-3 and PANC-1) and normal human and fetal fibroblasts (NHFs and NFFs) were incubated with Hoechst 33342 dye to characterize nuclear morphology and detect the induction of apoptosis. Brightly stained, condensed nuclei accompanied by apoptotic bodies are indicative of apoptosis.


( B ) PANC - 1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046 . Cells were counterstained with Hoechst 33342 dye and propidium iodide ( PI ) to visualize nuclear morphology and cell death . Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine .

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(B) PANC-1 cells were incubated with Annexin V dye to verify apoptotic induction caused by NSC 51046. Cells were counterstained with Hoechst 33342 dye and propidium iodide (PI) to visualize nuclear morphology and cell death. Images shown are representative of two independent experiments at 48 hours following treatment with NSC 51046 and colchicine.


( A ) Pancreatic cancer cells , human leukemia cells ( Jurkat ) and NHFs were incubated with Monodansylcadaverine ( MDC ) to stain autophagic vacuoles and propidium iodide ( PI ) for cell death to determine if autophagic induction led to cell death . Bright , punctate staining is indicative of autophagy , as seen in our positive control ( Tamoxifen treated cells ) . Images were taken at 400X magnification on a fluorescent microscope .

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(A) Pancreatic cancer cells, human leukemia cells (Jurkat) and NHFs were incubated with Monodansylcadaverine (MDC) to stain autophagic vacuoles and propidium iodide (PI) for cell death to determine if autophagic induction led to cell death. Bright, punctate staining is indicative of autophagy, as seen in our positive control (Tamoxifen treated cells). Images were taken at 400X magnification on a fluorescent microscope.


( B ) Western blot analyses of Beclin - 1 expression and LC3 conversion were conducted on PANC - 1 cells treated with 5 . 0 µM Green 1 at various time points . β - actin was measured as an internal control . Samples treated for 3 and 6 hours were compared to the early control , while the sample treated for 48 hours was compared to the 48 - hour control . Results are expressed as mean ± SD from two independent experiments for both Western blots .

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(B) Western blot analyses of Beclin-1 expression and LC3 conversion were conducted on PANC-1 cells treated with 5.0 µM Green 1 at various time points. β-actin was measured as an internal control. Samples treated for 3 and 6 hours were compared to the early control, while the sample treated for 48 hours was compared to the 48-hour control. Results are expressed as mean ± SD from two independent experiments for both Western blots.


( A ) Colchicine derivatives ( Green 1 and NSC 51046 ) were incubated with porcine tubulin for an hour in a 96 - well plate . The absorbance ( OD 340 nm ) was obtained every minute during the one hour incubation . Colchicine was used for comparison to the derivatives .

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(A) Colchicine derivatives (Green 1 and NSC 51046) were incubated with porcine tubulin for an hour in a 96-well plate. The absorbance (OD 340 nm) was obtained every minute during the one hour incubation. Colchicine was used for comparison to the derivatives.


( B ) Isolated mitochondria from PANC - 1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase ( HRP ) . Results were compared to control untreated mitochondria , colchicine treated mitochondria and positive control , paraquat ( PQ ) . Fluorescence readings were taken in 5 min intervals for 4 hours at Ex . 560 nm and Em . 590 nm and expressed as relative fluorescence units ( RFU ) . Results are a representative of three independent experiments .

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(B) Isolated mitochondria from PANC-1 cells were treated with Green 1 and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase (HRP). Results were compared to control untreated mitochondria, colchicine treated mitochondria and positive control, paraquat (PQ). Fluorescence readings were taken in 5 min intervals for 4 hours at Ex. 560 nm and Em.590 nm and expressed as relative fluorescence units (RFU). Results are a representative of three independent experiments.


Following acclimatization , CD - 1 nu / nu mice were separated into two groups , one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS ( 10 µL in 200 µL PBS ) , while the second group received subcutaneous injections of Green 1 ( 10 mg / kg / day for a total volume of 10 µL Green 1 / 200 µL PBS ) three times a week for a period of one month . ( A ) Mice weights were recorded twice a week for the duration of the study .

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Following acclimatization, CD-1 nu/nu mice were separated into two groups, one group was injected subcutaneously in the right and left hind flanks with Me2SO in PBS (10 µL in 200 µL PBS), while the second group received subcutaneous injections of Green 1 (10 mg/kg/day for a total volume of 10 µL Green 1/200 µL PBS) three times a week for a period of one month. (A) Mice weights were recorded twice a week for the duration of the study.


( B ) Following the study , mice were sacrificed and their organs were obtained for histopathological assessment . Hematoxylin and Eosin staining was carried out on the tissue samples .

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(B) Following the study, mice were sacrificed and their organs were obtained for histopathological assessment. Hematoxylin and Eosin staining was carried out on the tissue samples.


, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates . Colour represents the scaled reduced cost value . A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate .

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, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.


, Protein fold changes of the CcpA mutant over wild type . Log2 fold changes were calculated from LFQ values p values were calculated based on 2 - sided two sample t - tests with a FDR threshold of 0 . 05 and S 0 equal to 1 ( Tusher et al , 2001 ) . Proteins outside the significance lines ( FDR = 0 . 05 , S 0 = 1 ) are significantly changed in expression . Example sets of proteins are highlighted and are labeled with their gene names .

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, Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names.


( C ) Change in the expression levels of 36 genes defining the host - targeted anti - viral signature . log2 fold change at 24 hours post SARS - CoV - 2 infection from A549 cells are shown .

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(C) Change in the expression levels of 36 genes defining the host-targeted anti-viral signature. log2 fold change at 24 hours post SARS-CoV-2 infection from A549 cells are shown.


( D ) Change in the expression levels of 17 genes defining anti - cytokine signature ; log2 fold change at 24 hours post high SARS - CoV - 2 infection from A549 - ACE2 cells are shown .

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(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.


( A ) Representative fluorescence images of Mock , SARS - CoV - 2 infected ( Ctrl ) , and Salmeterol - treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress . Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments : Segmentation images show how cells were segmented and identified as spike positive . Purple , nuclei ; cyan , spike . Scale bar , 100 μm .

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(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.


( B ) Violin plots of Vero - E6 cells labeled for Spike protein . The Multi - wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell . The plots show the population distribution of the integrated signal for all of the treatments . The Boxes in the plot show the interquartile range ( IQR ) with the top and bottom edges marking the 75th and 25th percentiles , respectively . The horizontal line in the box is the median value , and the whiskers are defined to be 1 . 5 IQR . The ordinate is a log scale . The effect of the treatment is assessed quantitatively by changes in the median signal level , and qualitatively by observing changes in the modes . The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut - off to quantify the number of cells that are positive or negative for the Spike signal . The statistics table below the plots shows the number of cells counted in each treatment group , and the median of the population .

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(B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.


HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates , pretreated with 7 - point gradients of test compounds for 1 - 2 h , and co - cultured for 4 hours with HEK293 donor cells expressing SARS - CoV - 2 spike and GFP , or donor cells expressing GFP only ( no spike ) . Images of GFP - positive objects were acquired on a confocal high - content imager and analyzed for syncytia formation and integrated GFP area ( total GFP ) as a measure of cytotoxicity , using a CNT algorithm as described in the Materials and Methods . Data are the aggregate of 8 independent biological repeats ; where errors are shown they represent SD from matching concentrations in at least three experiments .

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HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.


V . cholerae wild - type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase ( OD600 of 1 . 5 ) and induced with L - arabinose ( 0 . 2 % final conc . ) . Expression of MicV and VrrA was monitored on Northern blots . 5S rRNA served as loading control .

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V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.


V . cholerae wild - type , ΔvchM and ΔvchM ΔrpoE strains harboring PmicV : : mKate2 plasmids were grown in M9 medium . Samples were collected during various growth phases and analyzed for fluorescence .

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V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.


V . cholerae wild - type and ΔrpoE strains carrying PmicV : : mKate2 plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type and ΔrpoE strains carrying PvrrA : : mKate2 ( E ) plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type , ΔmicV , ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


V . cholerae ΔvrrA ΔmicV strains carrying pBAD - micV , pBAD - vrrA or an empty vector control ( pCtr ) were cultivated to early stationary phase ( OD600 of 1 . 5 ) in LB . Cells were treated with L - arabinose ( 0 . 2 % final conc . ) and RNA samples were collected at the indicated time points after induction . Northern blot analysis was performed to determine VrrA , MicV and ompT levels . 5S rRNA served as loading control . For comparison , RNA samples of a wild - type strain carrying pCtr were collected during various growth phases , which indicated ~ 18 - fold and ~ 7 - fold higher levels of VrrA and MicV expressed from the pBAD plasmids , respectively ( see Source data for quantifications ) .

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V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).


V . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to ( B ) : regulated by both

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V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAs


V . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to regulated only by

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V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by MicV


V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by VrrA


V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : gfp or ompT M1 * : : gfp fusions ( D ) , ushA : : gfp or ushA M1 * : : gfp fusions ( E ) , or lpp : : gfp or lpp M2 * : : gfp fusions ( F ) and an empty vector control ( pCtr ) , the micV expression plasmids ( pMicV , pMicV M1 ) or the vrrA expression plasmids ( pVrrA , pVrrA M2 ) were grown in M9 and GFP fluorescence was measured . M1 and M2 denote the mutations indicated in ( A , B , C ) . Fluorescence of the control strains was set to 1 .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1) or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.


V . cholerae ΔrpoE , ΔrpoE ΔvrrA , ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD - rpoE or an empty vector control ( pCtr ) were grown to OD600 of 1 . 5 and L - arabinose ( 0 . 2 % final conc . ) was added . RNA samples were collected at the indicated time points and monitored for ompT ( G ) , ushA ( H ) , or lpp ( I ) levels using qRT - PCR .

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

V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.


V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : 3XFLAG gene and pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and analyzed for MicV , VrrA and RybB expression on Northern blots . OmpT : : 3xFLAG production was tested on Western blots . RNAPα and 5S rRNA served as loading controls for Western and Northern blots , respectively .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.


E . coli wild - type strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and investigated on Northern blots and SDS - PAGE , respectively . For comparison , we included the E . coli insertional mutant strains ompA : : kanR and ompC : : kanR for specific assignment of OmpA and OmpC bands .

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

E. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.


V . cholerae wild - type and ΔrpoE strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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

V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library after consecutive selection experiments ( Sel1 , Sel2 , Sel3 ) were grown in LB to OD600 of 0 . 2 . Cells were treated with ethanol ( 3 . 5 % final conc . ) for 6 hours . Serial dilutions were prepared and spotted onto agar plates . R1 and R2 indicate two independent 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

V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.


V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library before ( input ) or after consecutive ethanol selection experiments ( Sel1 , Sel2 , Sel3 ) were cultivated in LB to OD600 of 2 . 0 . Membrane fractions were analyzed by SDS - PAGE . The indicated bands were analyzed by mass spectrometry .

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

V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.


V . cholerae ΔvrrA ΔmicV cells expressing the ompA : : 3xFLAG gene and carrying an empty vector control ( pCtr ) , or plasmids producing the 15 most highly enriched sRNA variants ( sRNA variants 1 - 15 ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and tested for sRNA and OmpA : : 3xFLAG expression on Northern and Western blots , respectively ( with RNAPα and 5S rRNA as loading controls ) .

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 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

V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).


V . cholerae ΔvrrA ΔmicV strains carrying the ompA : : gfp fusion and an empty vector control ( pCtr ) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.


V . cholerae wild - type , ΔrpoE , ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


( A ) Immunoblot showing the activation of PI3K / AKT pathway ( by assessing the levels of p - AKT Ser473 ) of ECs 24h after induction of Pik3caH1047R expression .

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

(A) Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.


( B ) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression . Differentially expressed genes ( DEGs ) ( p adj < 0 . 05 ) in Pik3caH1047R over Pik3caWT ECs at 24h ( grey , unchanged ; orange , downregulated ; purple , upregulated ) . n = 4 biological replicates per genotype .

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 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

(B) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (p adj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24h (grey, unchanged; orange, downregulated; purple, upregulated). n=4 biological replicates per genotype.


( D ) Immunoblot showing the activation of PI3K / AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation ( - ) and 30 min stimulation with 20 % fetal bovine serum ( FBS ) ( + ) .

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(D) Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation (-) and 30 min stimulation with 20% fetal bovine serum (FBS) (+).


( E , F ) ( E ) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum ( - ) or 20 % serum ( + ) for 24h . ( F ) Percentage of proliferative ECs ( in S and G2M phases ) from Fig 1E . Error bars are s . e . m . n ≥ 3 biological replicates per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 and * * * * p < 0 . 0001 were considered statistically significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum (-) or 20% serum (+) for 24h. (F) Percentage of proliferative ECs (in S and G2M phases) from Fig 1E. Error bars are s.e.m. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 and ****p < 0.0001 were considered statistically significant.


( B ) Representative images of control and EC - Pik3caH1047R / WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels . Scale bars : 150 μm .

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(B) Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 μm.


( D ) Quantification of IB4 - positive areas per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype .

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

(D) Quantification of IB4-positive areas per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype.


( E ) Representative images of EC - mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP . Scale bars : 150 μm . ( F ) Quantification of GFP / IB4 ratio per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

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 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

(E) Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 μm. (F) Quantification of GFP/IB4 ratio per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( A , B ) Representative images of EC - Pik3caH1047R / WT ( A ) and EC - mTmG ( B ) P6 retinas from mice treated with decreasing doses of 4 - OHT on P1 . Retinas were stained for blood vessels ( IB4 ) and GFP as indicated . Scale bars : 150 μm .

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

(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 μm.


( C ) Quantification of GFP / IB4 ratio of EC - mTmG retinas . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

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

(C) Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( D ) Quantification of IB4 - positive area per retina in Pik3caH1047R / WT retinas . Data presented as a percentage of the control for each 4 - OHT dose . Error bars are s . e . m . n = 4 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

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

(D) Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are s.e.m. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( A ) Representative images of EC - Pik3caH1047R / WT P6 retinas isolated from mice treated with 0 . 125 mg / kg of 4 - OHT on P1 . Retinas were immunostained for blood vessels ( IB4 ) . Red asterisks show arteries and arterioles and yellow arrowheads veins . Scale bars : 150 μm ( left panel ) and 30 μm ( right , high magnification panels ) . ( B ) Representative images of P6 retinas from control and EC - Pik3caH1047R / WT mice treated with 0 . 125 mg / kg 4 - OHT on P1 , following immunostaining for p - S6 ( S235 / 236 ) and blood vessels ( IB4 ) . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( C ) Representative control and EC - Pik3caH1047R / WT P6 retinas immunostained for blood vessels ( IB4 ) , EC nuclei ( Erg ) and EdU . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( D - G ) Quantification of ( D ) p - S6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) , ( E ) EC proliferation by EdU staining , ( F ) EC number by Erg positive cells and ( G ) retinal vascularity by IB4 - positive area in control and EC - Pik3caH1047R / WT retinas . Error bars are s . e . m . n > 5 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 was considered statistically significant .

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

(A) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.


( B , C ) Representative images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panel ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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 B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.