f8 ( ai ) Western blot analysis of tubulin , LC3 - I and LC3 - II in zebrafish larvae where ATG7 was downregulated , as indicated . ( ii ) Western blot analysis of CALM - HA , actin and LC3 - II in zebrafishlarvae where CALM - HA was expressed , as indicated . The larvae were treated with ammonium chloride ( NH4Cl ) , as indicated . ( SE , short exposure ; LE , longer exposure ) . Quantification of LC3 - II / actin ratio is shown . Data are representative of three independent experiments and shown as mean ± s . d . ( * P0 . 05 ; two - tailed t - test ) . ( b ) Modulation of autophagy alters Dendra - tau clearance dynamics . The fluorescence intensity of each individual cell was quantified at each timepoint ( n ≥ 31 cells , ≥ 9 larvae per treatment group ) and mean cell intensity values for each drug treatment at each timepoint were calculated . Images were taken immediately after photoconversion and at 24 , 30 and 48 h intervals thereafter . Rapamycin treatment significantly increased the rate of Dendra - tau clearance . Ammonium chloride ( NH4Cl ) treatment significantly decreases the rate of Dendra - tau clearance ( * * P0 . 01 , * * * P0 . 001 , one - way analysis of variance ( ANOVA ) ) . Error bars are ± s . e . m . ( c ) Dendra - tau clearance in the presence of CALM : representative images of CALM with mosaic expression of Dendra - tau and full - length Dendra - tau taken immediately after photoconversion and at 6 , 24 , 30 , 48 and 54 h after conversion . The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co - expressing Dendra - tau and either full - length CALM or Δ - ANTH CALM ( larvae - ANTH - HA ) constructs ( n ≥ 100 cells , ≥ 9 larvae per treatment group ) at each time point was calculated . ( i ) Expression of full - length HA significantly delayed the clearance of CALM at all time points compared with Δ - ANTH CALM ( * * * P0 . 001 , one - way ANOVA ) . One graph , representative of three independent experiments , is presented . ( CALM - ANTH - HA : 132 cells , 20 fishes ; CALM - CALM : 18 cells , 8 fishes ) . Another two experiments are shown in Supplementary Fig . S5C . Error bars are mean ± s . d . ( ii ) Treatment with CALM alters the dynamics of CALM clearance . Expression of full - length CALM significantly delayed the clearance of CALM at all time points compared with Δ - ANTH CALM , as observed in i ( P0 . 001 , one - way ANOVA ) . However , treatment of Δ - ANTH CALM injected CALM with CALM slows the CALM clearance by 70 % to a level comparable to that observed in larvae injected with full - length Dendra - tau . Ammonium chloride treatment of Dendra - tau injected with full - length null results in a modest ( 22 % ) decrease in Dendra - tau clearance , suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect ( n ≥ 25 cells , n ≥ 9 Dendra - tau per treatment group ) . Error bars are mean ± s . d . Note that i and ii are distinct experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f8(ai) Western blot analysis of tubulin, LC3-I and LC3-II in zebrafish larvae where ATG7 was downregulated, as indicated. (ii) Western blot analysis of CALM-HA, actin and LC3-II in zebrafishlarvae where CALM-HA was expressed, as indicated. The larvae were treated with ammonium chloride (NH4Cl), as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is shown. Data are representative of three independent experiments and shown as mean ±s.d. (*P0.05; two-tailed t-test).(b) Modulation of autophagy alters Dendra-tau clearance dynamics. The fluorescence intensity of each individual cell was quantified at each timepoint (n≥ 31 cells, ≥9 larvae per treatment group) and mean cell intensity values for each drug treatment at each timepoint were calculated. Images were taken immediately after photoconversion and at 24, 30 and 48 h intervals thereafter. Rapamycin treatment significantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatment significantly decreases the rate of Dendra-tau clearance (**P0.01, ***P0.001, one-way analysis of variance (ANOVA)). Error bars are ±s.e.m.(c) Dendra-tau clearance in the presence of CALM: representative images of CALM with mosaic expression of Dendra-tau and full-length Dendra-tau taken immediately after photoconversion and at 6, 24, 30, 48 and 54 h after conversion. The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co-expressing Dendra-tau and either full-length CALM or Δ-ANTH CALM (larvae-ANTH-HA) constructs (n≥ 100 cells, ≥9 larvae per treatment group) at each time point was calculated. (i) Expression of full-length HA significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM (***P0.001, one-way ANOVA). One graph, representative of three independent experiments, is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-CALM: 18 cells, 8 fishes). Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii) Treatment with CALM alters the dynamics of CALM clearance. Expression of full-length CALM significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM, as observed in i (P0.001, one-way ANOVA). However, treatment of Δ-ANTH CALM injected CALM with CALM slows the CALM clearance by 70% to a level comparable to that observed in larvae injected with full-length Dendra-tau. Ammonium chloride treatment of Dendra-tau injected with full-length null results in a modest (22%) decrease in Dendra-tau clearance, suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect (n≥25 cells, n≥9 Dendra-tau per treatment group). Error bars are mean ±s.d. Note that i and ii are distinct experiments.


f9 ( a ) rho : : GFP - tau fishes were incubated from 3 to 9 d . p . f . in either dimethyl sulphoxide ( DMSO ) or rapamycin ( i ) or from 3 to 7 d . p . f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin ( ii ) . Images through the central retina at 9 d . p . f . ( top panel ; ( i ) ) reveal rod degeneration ( arrow ) throughout the retina in control ( DMSO - treated ) larvae , whereas rod photoreceptors are present throughout the retina , particularly in the central region following treatment with rapamycin ( arrow ) . Images taken through the central retina at 7 d . p . f . ( bottom panel ; ( ii ) ) show normal photoreceptors in the marginal zones ( arrows ) and only limited numbers in the central region . NH4Cl exacerbates degeneration - photoreceptors are absent from the central retina and reduced / absent from marginal zones ( arrows ) . Sections were stained with anti - rhodopsin ( 1D1 ) antibody . GFP labels whole rod photoreceptors , whereas rhodopsin is present in the rod outer segment . GFP co - localizes with the red rhodopsin label in all experimental conditions . Scale bars , 50 μm . Quantification of rodphotoreceptor degeneration ( n = 10 larvae per group ; * * * P0 . 001 ; # # # P0 . 001 , two - tailed unpaired t - test ) . Error bars are mean ± s . d . ( b ) Full - length CALM electroporation into rho : : GFP larvae did not cause degeneration . , while full - length CALM electroporation into rho : : GFP - tau larvae exacerbated photoreceptor degeneration . Central retina sections in the region of the optic nerve head are presented . Scale bars , 50 μm . Data represent the ratio ( in % ) of the intensity of the GFP signal between CALM - HA electroporated eye versus control eye for five individuals per transgenic line . * * * P0 . 05 ; two - tailed t - test . Error bars are mean ± s . d . ( c ) NH4Cl treatment of rho : : GFP - tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control ( non - electroporated ) side but did not alter the degeneration caused by CALM electroporation . Rapamycin treatment of Rapamycin : : GFP - tau immediately after unilateral CALM electroporation rescued photoreceptors on the control ( non - electroporated ) side but not on the CALM - electroporated side . To demonstrate that loss of GFP corresponds to loss of photoreceptors , sections were stained with anti - rhodopsin ( 1D1 ) antibody . Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish ; Mann - Whitney test was used to compare drug treatment in the right eye of different fishes . * P0 . 05 . Scale bars , 50 μm . Error bars are mean ± s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f9(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH4Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 μm. Quantification of rodphotoreceptor degeneration (n=10 larvae per group; ***P0.001; ###P0.001, two-tailed unpaired t-test). Error bars are mean ±s.d.(b) Full-length CALM electroporation into rho::GFP larvae did not cause degeneration., while full-length CALM electroporation into rho::GFP-tau larvae exacerbated photoreceptor degeneration. Central retina sections in the region of the optic nerve head are presented. Scale bars, 50 μm. Data represent the ratio (in %) of the intensity of the GFP signal between CALM-HA electroporated eye versus control eye for five individuals per transgenic line. ***P0.05; two-tailed t-test. Error bars are mean ±s.d.(c) NH4Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of Rapamycin::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann-Whitney test was used to compare drug treatment in the right eye of different fishes. *P0.05. Scale bars, 50 μm. Error bars are mean ±s.e.m.


f10 ( a ) Histological section to demonstrate the individual cell layers of the zebrafish retina . The photoreceptor layer ( PR , comprising rod and cone photoreceptors ) lies immediately adjacent to the retinal pigment epithelium ( RPE ) at the outermost surface of the eye . Thioflavin - S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer ( marked with yellow dotted lines ) . No labelling was observed in the retina of rho : : GFP at 8 d . p . f . , whereas distinct thioflavin - S - positive tangles ( arrows ) were observed in the photoreceptor layer of rho : : GFP - tau fish . Note , the RPE is highly autofluorescent due to the presence of silver pigment . High power regions are shown in the top right of each panel . ( b ) Unilateral electroporation of CALM into the retina of rho : : GFP - tau zebrafish resulted in a marked increase in thioflavin - S positive tangles in the electroporated retina in the photoreceptor layer ( PR ) compared with the control side . Top panel are lower magnification images to show the retinal cell layers . Thioflavin - S labelling is restricted to the PR layer . Note the RPE is highly autofluorescent due to the presence of silver pigment . Bottom panel are higher magnification images to show individual thioflavin - S tangles the largest of which are indicated by arrows .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f10(a) Histological section to demonstrate the individual cell layers of the zebrafish retina. The photoreceptor layer (PR, comprising rod and cone photoreceptors) lies immediately adjacent to the retinal pigment epithelium (RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer (marked with yellow dotted lines). No labelling was observed in the retina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positive tangles (arrows) were observed in the photoreceptor layer of rho::GFP-tau fish. Note, the RPE is highly autofluorescent due to the presence of silver pigment. High power regions are shown in the top right of each panel.(b) Unilateral electroporation of CALM into the retina of rho::GFP-tau zebrafish resulted in a marked increase in thioflavin-S positive tangles in the electroporated retina in the photoreceptor layer (PR) compared with the control side. Top panel are lower magnification images to show the retinal cell layers. Thioflavin-S labelling is restricted to the PR layer. Note the RPE is highly autofluorescent due to the presence of silver pigment. Bottom panel are higher magnification images to show individual thioflavin-S tangles the largest of which are indicated by arrows.


Figure 1A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine ( VCR ) for 48h . Apoptosis was determined by flow cytometry upon staining with Annexin - V and Propidium Iodide ( PI ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test . B Fresh primary leukemic cells from two pediatric patients , one with common B - ALL at relapse ( left ) and one with T - ALL at diagnosis ( right ) , both with poor response to second line chemotherapy , were treated with LBW242 and VCR at the indicated concentrations . Apoptosis was measured after 24 h as described in A . The SM - drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cells were stained with respective fluorescent substrates for caspase - 8 ( C ) and - 9 ( D ) , and were subsequently analyzed by flow cytometry . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test . The SM - drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cells were stained with an antibody detecting the active caspase - 3 subunit ( E ) , and were subsequently analyzed by flow cytometry . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 by unpaired Student ' s t - test . The SM - drug combination induces PARP cleavage . LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h . Cleavage of PARP was measured by Western blot using Actin as loading control ( F ) . One representative immunoblot out 3 independent experiments is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A LOUCY cells were treated in vitro with the indicated SMs with or without vincristine (VCR) for 48h. Apoptosis was determined by flow cytometry upon staining with Annexin-V and Propidium Iodide (PI). Mean ± SD of 3 independent experiments is shown; ** p <0.01, *** p <0.001 and **** p <0.0001 by unpaired Student's t-test.B Fresh primary leukemic cells from two pediatric patients, one with common B-ALL at relapse (left) and one with T-ALL at diagnosis (right), both with poor response to second line chemotherapy, were treated with LBW242 and VCR at the indicated concentrations. Apoptosis was measured after 24 h as described in A.The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with respective fluorescent substrates for caspase-8 (C) and -9 (D) , and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.The SM-drug combination activates caspases LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cells were stained with an antibody detecting the active caspase-3 subunit (E), and were subsequently analyzed by flow cytometry. Mean ± SD of 3 independent experiments is shown; * p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 by unpaired Student's t-test.The SM-drug combination induces PARP cleavage. LOUCY cells were incubated with the indicated chemotherapeutic drugs with or without LBW242 for 48 h. Cleavage of PARP was measured by Western blot using Actin as loading control (F). One representative immunoblot out 3 independent experiments is shown.


Figure 2A Necrostatin - 1 ( Nec ) does not affect LBW - mediated sensitization to VCR - induced apoptosis . Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and / or VCR ( left ) and / or TNFα ( right ) in the presence or absence of the RIPK1 - inhibitor Nec . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 and * * p < 0 . 01 by unpaired Student ' s t - test . B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR . LOUCY cells were transfected with a non - targeting siRNA ( neg - siRNA ) or a RIPK1 - specific siRNA . 6h post transfection , cells were treated with the indicated drugs and apoptosis was measured 40h thereafter . Mean ± SD of 3 independent experiments is shown ; * * * p < 0 . 001 by unpaired Student ' s t - test . Knockdown ( KD ) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control . One representative immunoblot out of 3 biological replicates is shown . C The SM - drug combination does not activate the canonical NF - κB pathway . Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and / or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5 , 15 or 60 minutes using Actin as loading control . One representative immunoblot out of 3 independent biological replicates is shown . D KD of NEMO does not affect the SM - mediated sensitization to chemotherapy - induced apoptosis . Experiment was performed as described in B , except that an siRNA targeting NEMO was used . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 by unpaired Student ' s t - test . E The SM - drug combination activates the non - canonical NF - κB pathway . Cleavage of p100 NF - κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM . Asterisk indicates an unspecific signal . One representative immunoblot out of 3 biological replicates is shown . F KD of RelB does not inhibit apoptosis . Experiment was performed as described in B , except that an siRNA targeting RelB was used . Mean ± SD of 3 independent experiments is shown ; differences are not significant , by unpaired Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Necrostatin-1 (Nec) does not affect LBW-mediated sensitization to VCR-induced apoptosis. Apoptosis induction in LOUCY cells was determined by flow cytometry 48h after treatment with SM and/or VCR (left) and/or TNFα (right) in the presence or absence of the RIPK1-inhibitor Nec. Mean ± SD of 3 independent experiments is shown; * p <0.05 and ** p<0.01 by unpaired Student's t-test.B RIPK1 is dispensable for apoptosis induction by LBW242 and VCR. LOUCY cells were transfected with a non-targeting siRNA (neg-siRNA) or a RIPK1-specific siRNA. 6h post transfection, cells were treated with the indicated drugs and apoptosis was measured 40h thereafter. Mean ± SD of 3 independent experiments is shown; *** p<0.001 by unpaired Student's t-test. Knockdown (KD) was verified by Western blot at the time of apoptosis measurement and KD efficiency calculated compared to the control. One representative immunoblot out of 3 biological replicates is shown.C The SM-drug combination does not activate the canonical NF-κB pathway. Phosphorylation and degradation of IκBα upon treatment with VCR or TNFα and/or LBW242 was determined in LOUCY cells by Western blot following treatment with the indicated drugs for 5, 15 or 60 minutes using Actin as loading control. One representative immunoblot out of 3 independent biological replicates is shown.D KD of NEMO does not affect the SM-mediated sensitization to chemotherapy-induced apoptosis. Experiment was performed as described in B, except that an siRNA targeting NEMO was used. Mean ± SD of 3 independent experiments is shown; * p<0.05 by unpaired Student's t-test.E The SM-drug combination activates the non-canonical NF-κB pathway. Cleavage of p100 NF-κB2 to the p52 fragment was determined by Western blot 24h after treatment of LOUCY cells with chemotherapy or TNFα in combination with SM. Asterisk indicates an unspecific signal. One representative immunoblot out of 3 biological replicates is shown.F KD of RelB does not inhibit apoptosis. Experiment was performed as described in B, except that an siRNA targeting RelB was used. Mean ± SD of 3 independent experiments is shown; differences are not significant, by unpaired Student's t-test.


Figure 3A Expression of XIAP mRNA in samples obtained from BM of healthy individuals ( Ctrl , n = 10 ) or patients with AML ( n = 517 ) or ALL ( n = 306 ) . Data are expressed as median , 25th and 75th percentile , whiskers indicate min / max ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by Games - Howell post - hoc test . B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients , analyzed by protein immunoassay ( Simple Western ) and depicted as mean ( n = 2 , biological replicates ) ; PBMC = peripheral blood mononuclear cellsParental and XIAP over - expressing NALM - 6 cells ( C ) were treated with VCR apoptosis ( Annexin - V / PI ) was measured at the indicated time points ( C ) by flow cytometry . Mean ± SEM of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . XIAP protein level was analyzed in parental and XIAP - overexpressing NALM - 6 cells by protein immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown . D Parental and XIAP over - expressing REH cells ( D ) were treated with VCR or LBW242 ; apoptosis ( Annexin - V / PI ) was measured after 72h ( D ) by flow cytometry . Mean ± SEM of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . XIAP protein level was analyzed by protein immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown .

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Figure 3A Expression of XIAP mRNA in samples obtained from BM of healthy individuals (Ctrl, n=10) or patients with AML (n=517) or ALL (n=306). Data are expressed as median, 25th and 75th percentile, whiskers indicate min/max; ** p<0.01; **** p<0.0001 by Games-Howell post-hoc test.B Protein levels of XIAP normalized to loading control in PDX ALL samples from pediatric and adult patients, analyzed by protein immunoassay (Simple Western) and depicted as mean (n=2, biological replicates); PBMC = peripheral blood mononuclear cellsParental and XIAP over-expressing NALM-6 cells (C) were treated with VCR apoptosis (Annexin-V / PI) was measured at the indicated time points (C) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed in parental and XIAP-overexpressing NALM-6 cells by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.D Parental and XIAP over-expressing REH cells (D) were treated with VCR or LBW242; apoptosis (Annexin-V / PI) was measured after 72h (D) by flow cytometry. Mean ± SEM of 3 independent experiments is shown; **** p ≤0.0001 by unpaired Student's t-test. XIAP protein level was analyzed by protein immunoassay (Simple Western); Actin was used as loading control. One representative immunoassay out of 3 independent experiments is shown.


Figure 4B shRNA expression control . Equal expression levels of ds - RED in the sh - CTRL / mTagBFP and sh - XIAP / eGFP subpopulation indicates identical expression levels of control and XIAP shRNA . One representative histogram out of 3 independent experiments is shown . C Expression of IAP . Western blot analysis of XIAP in NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP under the SFFV promoter ; GAPDH was used as loading control . One representative immunoblot out of 3 independent experiments is shown . D Western blot analysis of NALM - 6 cells with moderate ( 69 % , EF1α promoter ) or strong ( 92 % , SFFV promoter ) KD of XIAP ; GAPDH was used as loading control . The XIAP KD levels ( % KD ) were quantified relative to sh - CTRL . One representative immunoblot out of 3 independent experiments is shown . F Moderate knockdown of XIAP sensitizes towards chemotherapy . NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 ratio and injected into groups of mice . 4 days after injection , mice were either treated with PBS ( n = 4 ) , or with Vincristin ( VCR , n = 8 ) , 0 . 3mg / kg i . v . once a week for 3 consecutive weeks . 7 days after last treatment , mice were sacrificed , and relative proportion of sh - CTRL and sh - XIAP subpopulations re - isolated from the BM was quantified by flow cytometry . Data are depicted as mean ± SEM of all mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test with Welch ' s correction . G Strong knockdown of XIAP impairs NALM - 6 growth in vivo . NALM - 6 cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 ratio and injected into groups of mice ( n = 8 ) . 38 or 46 days after injection , mice were sacrificed , and relative proportion of sh - CTRL and sh - XIAP subpopulations re - isolated from the BM was quantified by flow cytometry . Data are depicted as mean ± SEM of all mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - test . H PARP cleavage in NALM - 6 cells with strong XIAP inhibition . NALM - 6 cells were lentivirally transduces with sh - CTRL or sh - XIAP ( SFFV ) . 4 days later , cells were lysed and relative cleavage level of PARP ( % ) was analyzed by protein immunoassay ( Simple Western ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - test . I XIAP reconstitution in NALM - 6 cells . NALM - 6 cells were transduced with shCTRL , shXIAP . 1 alone or shXIAP . 1 together with the XIAP overexpression vector . At day 0 , transduced cells were mixed in a 1 : 1 ratio with parental ( untransduced ) NALM - 6 cells . Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B shRNA expression control. Equal expression levels of ds-RED in the sh-CTRL/mTagBFP and sh-XIAP/eGFP subpopulation indicates identical expression levels of control and XIAP shRNA. One representative histogram out of 3 independent experiments is shown.C Expression of IAP. Western blot analysis of XIAP in NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of 3 independent experiments is shown.D Western blot analysis of NALM-6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh-CTRL. One representative immunoblot out of 3 independent experiments is shown.F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. 4 days after injection, mice were either treated with PBS (n=4), or with Vincristin (VCR, n=8), 0.3mg/kg i.v. once a week for 3 consecutive weeks. 7 days after last treatment, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test with Welch's correction.G Strong knockdown of XIAP impairs NALM-6 growth in vivo. NALM-6 cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice (n=8). 38 or 46 days after injection, mice were sacrificed, and relative proportion of sh-CTRL and sh-XIAP subpopulations re-isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** p ≤0.0001 by unpaired Student's t-test.H PARP cleavage in NALM-6 cells with strong XIAP inhibition. NALM-6 cells were lentivirally transduces with sh-CTRL or sh-XIAP (SFFV). 4 days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.I XIAP reconstitution in NALM-6 cells. NALM-6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM-6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of 3 independent experiments is shown; ** p <0.01 by unpaired Student's t-test.


Figure 5XIAP plays an essential role for ALL - 265 growing in mice . Experimental scheme and stability of XIAP knockdown over passaging . ALL - 265 PDX cells produced as described in A were mixed at a 1 : 1 ratio and injected into mice ( P1 , n = 4 ) . At clinical signs of illness , cells were isolated and re - injected into next recipient mice ( P2 , n = 4 ) . XIAP expression levels of P1 and P2 PDX cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were analyzed by Western blot at the end of each passage ; GAPDH was used as loading control ( B ) . XIAP plays an essential role for ALL - 265 growing in mice . ALL - 265 PDX cells produced as described in A were mixed at a 1 : 1 ratio and injected into mice ( P1 , n = 4 ) . At clinical signs of illness , cells were isolated and re - injected into next recipient mice ( P2 , n = 4 ) . Relative proportion of ALL - 265 GEPDX cells expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP re - isolated from the BM at the end of each passage was quantified by flow cytometry ( C ) . Data are depicted as mean ± SEM of all mice analyzed ( n = 4 , each passage ) with * * * * p < 0 . 0001 by unpaired Student ' s t - test . D Dose - response relationship between strength of XIAP knockdown and growth disadvantage ; ALL - 265 PDX cells of a representative mouse were analyzed separately for dsRED / shRNA high and low expressing cells .

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Figure 5XIAP plays an essential role for ALL-265 growing in mice. Experimental scheme and stability of XIAP knockdown over passaging. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). XIAP expression levels of P1 and P2 PDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP were analyzed by Western blot at the end of each passage; GAPDH was used as loading control (B).XIAP plays an essential role for ALL-265 growing in mice. ALL-265 PDX cells produced as described in A were mixed at a 1:1 ratio and injected into mice (P1, n=4). At clinical signs of illness, cells were isolated and re-injected into next recipient mice (P2, n=4). Relative proportion of ALL-265 GEPDX cells expressing sh-CTRL/mTagBFP or sh-XIAP/eGFP re-isolated from the BM at the end of each passage was quantified by flow cytometry (C). Data are depicted as mean ± SEM of all mice analyzed (n=4, each passage) with **** p<0.0001 by unpaired Student's t-test.D Dose-response relationship between strength of XIAP knockdown and growth disadvantage; ALL-265 PDX cells of a representative mouse were analyzed separately for dsRED/shRNA high and low expressing cells.


Figure 2Orthotopic MPNST xenografts at passages 1 ( OT P1 ) and 4 ( OT P4 ) were histopathologically similar to their corresponding primary MPNST ( PT ) in hematoxylin - eosin staining of paraffin - embedded tumor sections from patients MPNST - NF1 - 001 and MPNST - SP - 002 . Main panels show a general view of the tumors at low magnification ( 40 × ) ; inset pictures were taken at higher magnification ( 400 × ) . Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features . A representative immunostained section of vimentin , CD34 , S100 , and Ki - 67 is shown for primary tumors ( PT ) and orthotopic tumors ( OT ) from patientsMPNST - NF1 - 001 and MPNST - SP - 002 . Positive antibody signals are shown in brown , and the hematoxylin counterstain in blue . Main panels show pictures at high magnification ( 400 × ) ; inset pictures show mitotic cells present in these tumors .

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Figure 2Orthotopic MPNST xenografts at passages 1 (OT P1) and 4 (OT P4) were histopathologically similar to their corresponding primary MPNST (PT) in hematoxylin-eosin staining of paraffin-embedded tumor sections from patients MPNST-NF1-001 and MPNST-SP-002. Main panels show a general view of the tumors at low magnification (40×); inset pictures were taken at higher magnification (400×).Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patientsMPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.


Figure 3Stromal elements of the primary tumors were labeled with anti - humanCD34 but not anti - mouseCD34 ; patient - derived xenografts are labeled with anti - mouseCD34 only and no anti - human marker . Representative sections ( at 40 × and 400 × magnification ) of the primary tumors ( PT ) and the orthoxenograft tumors at passages 1 ( OT P1 ) and 4 ( OT P4 ) were labeled with anti - humanCD34 ( H ) and anti - mouseCD34 ( M ) . Sanger sequencing of the germline NF1 mutation c . 350T > A present in the patient that developed two different tumors ( MPNST - NF1 - 001 and MPNST - NF1 - 002 ) . Sequencing results from normal tissue , primary tumor ( PT ) , and orthotopic xenograft tumors ( OT ) are shown . The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples , and not in the corresponding derived orthoxenografts , indicating probable loss of the human stromal cells . SNP array of primary tumor ( PT ) and orthotopic xenograft passages 1 ( OT P1 ) and 4 ( OT P4 ) for MPNST - NF1 - 001 and MPNST - NF1 - 002 tumors . Results correspond to chromosome 17 . Images show the B - allele frequency ( BAF ) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines : 2n regions are shown in gray , gained regions in orange , lost regions in green , and LOH regions are represented in blue . The vertical red line indicates the location of the NF1 locus . In addition , the percentage of tumor versus stromal cells for each sample is represented in a pie chart ( blue , stromal cells ; red , tumor cells ) .

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Figure 3Stromal elements of the primary tumors were labeled with anti-humanCD34 but not anti-mouseCD34; patient-derived xenografts are labeled with anti-mouseCD34 only and no anti-human marker. Representative sections (at 40× and 400× magnification) of the primary tumors (PT) and the orthoxenograft tumors at passages 1 (OT P1) and 4 (OT P4) were labeled with anti-humanCD34 (H) and anti-mouseCD34 (M).Sanger sequencing of the germline NF1 mutation c.350T>A present in the patient that developed two different tumors (MPNST-NF1-001 and MPNST-NF1-002). Sequencing results from normal tissue, primary tumor (PT), and orthotopic xenograft tumors (OT) are shown. The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples, and not in the corresponding derived orthoxenografts, indicating probable loss of the human stromal cells.SNP array of primary tumor (PT) and orthotopic xenograft passages 1 (OT P1) and 4 (OT P4) for MPNST-NF1-001 and MPNST-NF1-002 tumors. Results correspond to chromosome 17. Images show the B-allele frequency (BAF) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines: 2n regions are shown in gray, gained regions in orange, lost regions in green, and LOH regions are represented in blue. The vertical red line indicates the location of the NF1 locus. In addition, the percentage of tumor versus stromal cells for each sample is represented in a pie chart (blue, stromal cells; red, tumor cells).


Figure 7Tumor growth effects of treatment with doxorubicin , sorafenib , rapamycin and combinations thereof in the five MPNST xenograft models . Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value . Statistically significant differences are shown as * P < 0 . 05 and * * P < 0 . 001 versus control group by the Bonferroni test . For long - term studies , a subgroup of treated mice ( n = 3 - 5 mice / group ) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses ( usually 1 , 500 - 2 , 000 mm3 ) . The graph illustrates differences in the time delay ( in days ) of relapsed tumor masses for the different treatments .

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Figure 7Tumor growth effects of treatment with doxorubicin, sorafenib, rapamycin and combinations thereof in the five MPNST xenograft models. Results are plotted as an average of the log2 ratio of tumor volume at different days relative to the initial value. Statistically significant differences are shown as *P < 0.05 and **P < 0.001 versus control group by the Bonferroni test.For long-term studies, a subgroup of treated mice (n = 3-5 mice/group) was kept alive for a maximum period of 4 months and sacrificed over time when relapsed tumor masses grew as large solid masses (usually 1,500-2,000 mm3). The graph illustrates differences in the time delay (in days) of relapsed tumor masses for the different treatments.


Figure 1A Immunoblot of DDX3 expression in lung cancer cell lines . B , C Colony - forming assays in H1299 ( B ) and A549 ( C ) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control . Corresponding immunoblots displaying knockdown levels of DDX3 . Mean from 3 replicates with SD . D Proliferation of A549 and H1299 cells after knockdown of DDX3 . Mean from 3 replicates with SD . ( A549 P = 0 . 011 , H1299 P = 0 . 014 ; exponential curve fit , extra sum of squares F - test ) . E β - galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color . F Expression of DDX3 by immunohistochemistry in normal lung tissue . G DDX3 expression in squamous cell carcinoma . H DDX3 expression in adenocarcinoma . I DDX3 expression in small cell carcinoma . J Expression of DDX3 in different histological types of lung cancer . All data sets were compared against each other ( chi - square test , P = 0 . 481 ) . K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors ( Kaplan - Meier curve and log - rank test , P = 0 . 016 ) .

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Figure 1A Immunoblot of DDX3 expression in lung cancer cell lines.B, C Colony-forming assays in H1299 (B) and A549 (C) lung cancer cells after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control. Corresponding immunoblots displaying knockdown levels of DDX3. Mean from 3 replicates with SD.D Proliferation of A549 and H1299 cells after knockdown of DDX3. Mean from 3 replicates with SD. (A549 P = 0.011, H1299 P = 0.014; exponential curve fit, extra sum of squares F-test).E β-galactosidase staining in parental A549 cells and A549 DDX3 knockdown cells displaying senescent cells identified by the blue color.F Expression of DDX3 by immunohistochemistry in normal lung tissue.G DDX3 expression in squamous cell carcinoma.H DDX3 expression in adenocarcinoma.I DDX3 expression in small cell carcinoma.J Expression of DDX3 in different histological types of lung cancer. All data sets were compared against each other (chi-square test, P = 0.481).K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors (Kaplan-Meier curve and log-rank test, P = 0.016).


Figure 2F Immunoblots of pull - down assay of DDX3 with biotin , AK - 298 , and SK - 153 . Lower panels display results using DDX5 and DDX17 antibodies . H Immunoblot displaying increasing concentrations of RK - 33 ( 50 , 100 , 200 nM ) resulting in increased inhibition of unwinding of oligomer products ( lanes 4 - 6 ) . I MTS viability assay of various lung cancer cell lines treated with RK - 33 for 72 h . Mean from 3 replicates with SD . J , K Colony - forming assay of A549 and H3255 cells treated with RK - 33 and with various doses of radiation 4 h later . Curves were fitted with a quadratic polynomial equation . Mean from 2 replicates with SD . P - values were determined by the extra sum of squares F - test .

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Figure 2F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4-6).I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.


Figure 5Following injection of 20 mg / kg of RK - 33 , twice a week for 7 weeks , extensive histopathological examination was carried out following necropsy . Identical patterns were observed both in the control and in the treated mice ( n = 2 ) . Samples were stained with H & amp ; amp ; E . Scale bar is 50 μm . Pharmacokinetics of RK - 33 in SCID mice at various time intervals . Results are mean ± SD from 5 mice . LC - MS / MS method was used to determine concentration of RK - 33 in mouse plasma and tissue . HAPI cells were treated with RK - 33 ( 10 μM ) , HA14 - 1 ( 25 μM ) , or DMSO vehicle , followed by oligomycin ( oligo , 0 . 5 μg / ml ) , FCCP ( 3 μM ) , and antimycin A ( AA , 1 μM ) while oxygen consumption rate ( OCR ) was measured . Pyruvate ( 10 mM ) was added in combination with FCCP to ensure that substrate supply was not rate - limiting for maximal OCR . Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages . OCR is baseline - normalized to the point prior to drug or vehicle addition . HAPI microglial cells were incubated for 1 h in glucose - free XF24 assay medium that was supplemented with 2 - deoxyglucose ( 50 mM ) and pyruvate ( 10 mM ) . RK - 33 ( 10 μM ) , oligomycin ( 0 . 5 μg / ml ) , or vehicle control was additionally present as indicated . Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages . Significance was assessed by two - sided , unpaired t - test .

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Figure 5Following injection of 20 mg/kg of RK-33, twice a week for 7 weeks, extensive histopathological examination was carried out following necropsy. Identical patterns were observed both in the control and in the treated mice (n = 2). Samples were stained with H&amp;E. Scale bar is 50 μm.Pharmacokinetics of RK-33 in SCID mice at various time intervals. Results are mean ± SD from 5 mice. LC-MS/MS method was used to determine concentration of RK-33 in mouse plasma and tissue.HAPI cells were treated with RK-33 (10 μM), HA14-1 (25 μM), or DMSO vehicle, followed by oligomycin (oligo, 0.5 μg/ml), FCCP (3 μM), and antimycin A (AA, 1 μM) while oxygen consumption rate (OCR) was measured. Pyruvate (10 mM) was added in combination with FCCP to ensure that substrate supply was not rate-limiting for maximal OCR. Data are mean ± SD from 2 to 3 wells and representative of independent experiments performed with two different HAPI passages. OCR is baseline-normalized to the point prior to drug or vehicle addition.HAPI microglial cells were incubated for 1 h in glucose-free XF24 assay medium that was supplemented with 2-deoxyglucose (50 mM) and pyruvate (10 mM). RK-33 (10 μM), oligomycin (0.5 μg/ml), or vehicle control was additionally present as indicated. Results are mean ± SD from 12 replicates pooled from experiments using two consecutive passages. Significance was assessed by two-sided, unpaired t-test.


Figure 6Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1 / KrasG12D mouse . Scale bar is 100 μm . Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( C ) , before treatment and 1 week after treatment . Tumors are indicated by arrows and confirmed by H & amp ; amp ; E staining of lung sections ( lower panel ) . Scale bar is 250 μm . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( D ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in ( C ) . Figure displays H & amp ; amp ; E staining of lung sections from radiation - treated ( upper panel ) and RK - 33 - and radiation - treated mice ( lower panel ) . Scale bar is 2 mm . Quantification of tumor burden ( as tumor surface divided by total lung surface ) in orthotopic A549 lung cancer mouse model , as shown in ( F ) . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . Micro - CT images of transgenic Twist1 / KrasG12D mice treated as in ( H ) , before treatment and 1 week after treatment . Quantification of tumor volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( I ) and expressed as relative tumor size . Significance was assessed by two - sided , unpaired t - test . Error bars represent SEM .

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Figure 6Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm.Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (C), before treatment and 1 week after treatment. Tumors are indicated by arrows and confirmed by H&amp;E staining of lung sections (lower panel). Scale bar is 250 μm.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (D). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.An orthotopic lung tumor model was generated using A549 human lung cancer cells and treated as in (C). Figure displays H&amp;E staining of lung sections from radiation-treated (upper panel) and RK-33- and radiation-treated mice (lower panel). Scale bar is 2 mm.Quantification of tumor burden (as tumor surface divided by total lung surface) in orthotopic A549 lung cancer mouse model, as shown in (F). Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.Micro-CT images of transgenic Twist1/KrasG12D mice treated as in (H), before treatment and 1 week after treatment.Quantification of tumor volume measured by micro-CT in Twist1/KrasG12D mice, as shown in (I) and expressed as relative tumor size. Significance was assessed by two-sided, unpaired t-test. Error bars represent SEM.


Figure 7A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry . Knockdown of DDX3 led to a decrease of cells in S - phase and an increase of cells in G1 - phase , indicative of a G1 arrest . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . B , C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK - 33 ( 0 , 2 , 4 , and 6 μM ) . RK - 33 induced a G1 cell cycle arrest in both cell lines . Significance was assessed by two - sided , unpaired t - test . Error bars represent SD . D , E Immunoblot of cell cycle - related proteins ( Cyclin D1 and Cyclin E1 ) and cell death - related proteins ( cleaved caspase 7 , cleaved caspase 9 ) in A549 and H1299 cells after treatment with RK - 33 ( 10 μM ) . Initially , a strong decrease of Cyclin D1 was observed . After 8 and 24 h , cleaved caspases 9 and 7 were apparent . F Immunoblot of MAPK pathway - related proteins ERK1 / 2 and phosphorylated ERK1 / 2 in A549 , H1299 , and H3255 ( RK - 33 resistant ) cells 24 h after treatment with RK - 33 ( 7 . 5 μM or 10 μM ) . ERK2 and especially ERK1 become dephosphorylated after treatment with RK - 33 in A549 and H1299 cells but not in H3255 cells . Outlined boxes indicate spliced lanes .

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Figure 7A Cell cycle analysis of H1299 cells treated with shDDX3 and processed by flow cytometry. Knockdown of DDX3 led to a decrease of cells in S-phase and an increase of cells in G1-phase, indicative of a G1 arrest. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.B, C Cell cycle analysis of A549 and H1299 cells by flow cytometry after treatment with RK-33 (0, 2, 4, and 6 μM). RK-33 induced a G1 cell cycle arrest in both cell lines. Significance was assessed by two-sided, unpaired t-test. Error bars represent SD.D, E Immunoblot of cell cycle-related proteins (Cyclin D1 and Cyclin E1) and cell death-related proteins (cleaved caspase 7, cleaved caspase 9) in A549 and H1299 cells after treatment with RK-33 (10 μM). Initially, a strong decrease of Cyclin D1 was observed. After 8 and 24 h, cleaved caspases 9 and 7 were apparent.F Immunoblot of MAPK pathway-related proteins ERK1/2 and phosphorylated ERK1/2 in A549, H1299, and H3255 (RK-33 resistant) cells 24 h after treatment with RK-33 (7.5 μM or 10 μM). ERK2 and especially ERK1 become dephosphorylated after treatment with RK-33 in A549 and H1299 cells but not in H3255 cells. Outlined boxes indicate spliced lanes.


Figure 8A β - catenin ( red ) and DDX3 ( green ) expression in H1299 cells . After overexpressing β - catenin , both DDX3 and β - catenin accumulate in the nucleus . Scale bar is 10 μm . B Immunoprecipitation with DDX3 or H3K4Me3 ( control ) and immunoblotted with β - catenin in A549 and H1299 cells . Outlined boxes indicate spliced lanes . C Immunoprecipitation with β - catenin or H3K4Me3 ( control ) and immunoblotted with DDX3 in A549 and H1299 cells . Outlined boxes indicate spliced lanes . D , E β - catenin / TCF4 activity was determined by the TOP / FOP reporter assay . Co - transfection with β - catenin is indicated below . F - I H1299 and A549 cells were treated with RK - 33 ( 0 , 1 , 2 , and 3 μM ) and co - transfected with β - catenin in ( F , H ) . Treatment with RK - 33 decreased TCF4 activity in both cell lines . J , K Normalized mRNA expression of TCF4 - regulated proteins ( Axin - 2 , c - Myc , Cyclin D1 ) and DDX3 were measured by qRT - PCR in H1299 cells after knockdown of DDX3 ( J ) and treatment with RK - 33 ( K ) . All experiments were repeated three times .

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Figure 8A β-catenin (red) and DDX3 (green) expression in H1299 cells. After overexpressing β-catenin, both DDX3 and β-catenin accumulate in the nucleus. Scale bar is 10 μm.B Immunoprecipitation with DDX3 or H3K4Me3 (control) and immunoblotted with β-catenin in A549 and H1299 cells. Outlined boxes indicate spliced lanes.C Immunoprecipitation with β-catenin or H3K4Me3 (control) and immunoblotted with DDX3 in A549 and H1299 cells. Outlined boxes indicate spliced lanes.D, E β-catenin/TCF4 activity was determined by the TOP/FOP reporter assay. Co-transfection with β-catenin is indicated below.F-I H1299 and A549 cells were treated with RK-33 (0, 1, 2, and 3 μM) and co-transfected with β-catenin in (F, H). Treatment with RK-33 decreased TCF4 activity in both cell lines.J, K Normalized mRNA expression of TCF4-regulated proteins (Axin-2, c-Myc, Cyclin D1) and DDX3 were measured by qRT-PCR in H1299 cells after knockdown of DDX3 (J) and treatment with RK-33 (K). All experiments were repeated three times.


Figure 9A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2 - Gy radiation and A549 cells pre - treated with 6 μM RK - 33 , 12 h before radiation . Overlap of 53BP1 and γH2AX is seen in the merged picture of the co - immunofluorescence staining . Scale bar is 2 μm . B A549 cells were pre - treated with RK - 33 and radiated with 2 Gy , and 53BP1 and γH2AX foci were counted as a measure of DNA damage . Cells with more than 10 foci 53BP1 or γH2AX were counted . More than 400 cells per sample were evaluated . C H1299 cells stably transfected with a homologous recombination ( HR ) reporter construct were treated with RK - 33 . Reporter constructs expressed GFP , which was quantified by flow cytometry . Experiments were repeated three times . D H1299 cells , containing a stable non - homologous end - joining ( NHEJ ) reporter construct , were treated with RK - 33 and knockdown of DDX3 . Reporter construct expressed GFP , which was quantified by flow cytometry . All experiments were repeated three times . E Microarray results from MDA - MB - 231 cells treated with RK - 33 and shDDX3 were validated by qRT - PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates ( Bio - Rad ) and performed in biological triplicates . F , G DNA repair - related proteins ( ATR and XRCC4 ) , DDX3 , and actin were assessed by immunoblotting in A549 ( F ) and H1299 ( G ) cells . Cells were pretreated for 4 h with vehicle control or 6 μM RK - 33 and then radiated with 0 or 5 Gy . Outlined boxes indicate spliced lanes .

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Figure 9A Immunofluorescence images showing 53BP1 and γH2AX foci in A549 cells after 2-Gy radiation and A549 cells pre-treated with 6 μM RK-33, 12 h before radiation. Overlap of 53BP1 and γH2AX is seen in the merged picture of the co-immunofluorescence staining. Scale bar is 2 μm.B A549 cells were pre-treated with RK-33 and radiated with 2 Gy, and 53BP1 and γH2AX foci were counted as a measure of DNA damage. Cells with more than 10 foci 53BP1 or γH2AX were counted. More than 400 cells per sample were evaluated.C H1299 cells stably transfected with a homologous recombination (HR) reporter construct were treated with RK-33. Reporter constructs expressed GFP, which was quantified by flow cytometry. Experiments were repeated three times.D H1299 cells, containing a stable non-homologous end-joining (NHEJ) reporter construct, were treated with RK-33 and knockdown of DDX3. Reporter construct expressed GFP, which was quantified by flow cytometry. All experiments were repeated three times.E Microarray results from MDA-MB-231 cells treated with RK-33 and shDDX3 were validated by qRT-PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates (Bio-Rad) and performed in biological triplicates.F, G DNA repair-related proteins (ATR and XRCC4), DDX3, and actin were assessed by immunoblotting in A549 (F) and H1299 (G) cells. Cells were pretreated for 4 h with vehicle control or 6 μM RK-33 and then radiated with 0 or 5 Gy. Outlined boxes indicate spliced lanes.


Figure 1ATPase staining of GAS and SOL muscles of WT and DKO mice ( left ) . Corresponding percentage of type I fibers were determined ( right ) ( n = 5 ) . Scale bars : 100 μm . Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice ( n = 3 for PGC1α analysis ; n = 5 for PLIN5 analysis ) . Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR - 183 and miR - 96 agomirs ( Ago - 183 / 96 ) or antagomirs ( Ant - 183 / 96 ) ( n = 3 ) . Mitochondrial respiration profile of C2C12 cells transfected with Ago - 183 / 96 ( J ) or Ant - 183 / 96 ( K ) . Cellular oxygen consumption rate ( OCR ) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR ( n = 3 or 4 per group ) .

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Figure 1ATPase staining of GAS and SOL muscles of WT and DKO mice (left). Corresponding percentage of type I fibers were determined (right) (n=5). Scale bars: 100 μm.Relative mRNA expression of PGC1α and PLIN5 in GAS muscles of WT and DKO mice (n=3 for PGC1α analysis; n=5 for PLIN5 analysis).Relative mRNA expression of PGC1α and PLIN5 in C2C12 cells transfected with miR-183 and miR-96 agomirs (Ago-183/96) or antagomirs (Ant-183/96) (n=3).Mitochondrial respiration profile of C2C12 cells transfected with Ago-183/96 (J) or Ant-183/96 (K). Cellular oxygen consumption rate (OCR) was measured in real time and corresponding basal and maximal respiration were analyzed by overall OCR (n=3 or 4 per group).


Figure 2Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in GAS muscles of WT and DKO mice . Western blot analysis of FoxO1 , PDK4 , p - PDHA1 and PDHA1 in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) . Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice . Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) . LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR - 183 / 96 ( N ) or antagomir - 183 / 96 ( P ) stained with Nile red ( n = 5 ) . Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR - 183 / 96 ( O ) or antagomir - 183 / 96 ( Q ) . Scale bars : 5 μm .

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Figure 2Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in GAS muscles of WT and DKO mice. Western blot analysis of FoxO1, PDK4, p-PDHA1 and PDHA1 in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).Western blot analysis of HSL and ATGL in GAS muscles of WT and DKO mice. Western blot analysis of HSL and ATGL in C2C12 cells transfected with Ago-183/96 (left) or Ant-183/96 (right).LD area per cell was quantified at the indicated time points to measure LD turnover in C2C12 myocytes transfected with agomiR-183/96 (N) or antagomir-183/96 (P) stained with Nile red (n=5). Representative immunofluorescence images of lipid droplets in C2C12 cells transfected with agomiR-183/96 (O) or antagomir-183/96 (Q). Scale bars: 5 μm.


Figure 3Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 agomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago - 183 or Ago - 96 . Relative expression of miR - 183 and miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 antagomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 ( right ) ( n = 3 ) . Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant - 183 or Ant - 96 . Relative mRNA expression ( I , left ) and western blot analysis ( J , top ) of ATGL in C2C12 cells transfected with agomir for miR - 183 ( Ago - 183 ) or miR - 96 ( Ago - 96 ) . Relative mRNA expression ( I , right ) and western blot analysis ( J , bottom ) of ATGL in C2C12 cells transfected with antagomir for miR - 183 ( Ant - 183 ) or miR - 96 ( Ant - 96 ) . ( n = 5 per group for RT - PCR analysis )

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Figure 3Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 agomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ago-183 or Ago-96. Relative expression of miR-183 and miR-96 in C2C12 myocytes transfected with miR-183 or miR-96 antagomir (left) (n=3). Relative mRNA expression of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96 (right) (n=3). Western blot analysis of FoxO1 and PDK4 in C2C12 cells treated with Ant-183 or Ant-96.Relative mRNA expression (I, left) and western blot analysis (J, top) of ATGL in C2C12 cells transfected with agomir for miR-183 (Ago-183) or miR-96 (Ago-96). Relative mRNA expression (I, right) and western blot analysis (J, bottom) of ATGL in C2C12 cells transfected with antagomir for miR-183 (Ant-183) or miR-96 (Ant-96). (n=5 per group for RT-PCR analysis)


Figure 4Relative mRNA expression of ATGL , PGC1α , PLIN5 , and HSL in C2C12 cells transfected with ATGL ( n = 5 ) . Relative mRNA expression of PGC1α , PLIN5 , and HSL in C2C12 cells transfected with Ant - 183 / 96 and siRNA of PPARδ ( siPPARδ ) .

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Figure 4Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5).Relative mRNA expression of PGC1α, PLIN5, and HSL in C2C12 cells transfected with Ant-183/96 and siRNA of PPARδ (siPPARδ).


Figure 5Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice ( n = 3 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS and SOL muscles of C57 BL / 6 mice . Relative mRNA expression of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months ( n = 5 ) . Western blot analysis of ATGL , HSL , and PLIN5 in GAS muscles of C57 BL / 6 mice fed with HFD for 2 months .

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Figure 5Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice (n=3). Western blot analysis of ATGL, HSL, and PLIN5 in GAS and SOL muscles of C57 BL/6 mice.Relative mRNA expression of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months (n=5). Western blot analysis of ATGL, HSL, and PLIN5 in GAS muscles of C57 BL/6 mice fed with HFD for 2 months.


Figure 6Concentration of serum triglyceride ( TAG ) ( left ) and non - esterified fatty acid ( NEFA ) ( right ) in WT and DKO mice after HFD feeding for 2 months ( n = 5 ) . Western blot analysis of FoxO1 , PDK4 , p - PDHA1 , PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months . Glucose tolerance test ( GTT ) and area under the curve ( AUC ) data for GTT in WT and DKO mice after HFD feeding ( n = 10 - 11 ) .

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Figure 6Concentration of serum triglyceride (TAG) (left) and non-esterified fatty acid (NEFA) (right) in WT and DKO mice after HFD feeding for 2 months (n=5).Western blot analysis of FoxO1, PDK4, p-PDHA1, PDHA1 and ATGL in GAS muscles of WT and DKO mice fed with HFD for 2 months.Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).


Figure 1 ( B ) Correlation between replicate mock infections . Path - seq reads were recovered from samples of macrophage RNA spiked with 0 . 1 % MTB RNA . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 . Inset summarizes the mock infection replicates and their fraction of MTB reads ( of all aligned reads ) from Path - seq and standard RNA - seq methods . ( C ) Correlation between MTB RNA sequenced by RNA - seq ( Illumina TruSeq library prep ) and the same MTB RNA sample combined with macrophage RNA at 1 : 1000 ratio and processed using Path - seq method . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 .

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Figure 1(B) Correlation between replicate mock infections. Path-seq reads were recovered from samples of macrophage RNA spiked with 0.1% MTB RNA. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001. Inset summarizes the mock infection replicates and their fraction of MTB reads (of all aligned reads) from Path-seq and standard RNA-seq methods.(C) Correlation between MTB RNA sequenced by RNA-seq (Illumina TruSeq library prep) and the same MTB RNA sample combined with macrophage RNA at 1:1000 ratio and processed using Path-seq method. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001.


Figure 2 ( A ) Path - seq profiles of desA1 , desA2 in MTB infected BMDMs . Error bars show the standard deviation from three biological samples . Representative results from two experiments are presented . ( B ) Upper panel : dissolved oxygen curve over 120 h time course showing controlled depletion ( 1 ) , sustained hypoxia ( 2 ) and controlled reaeration ( 3 ) . Points represent the average of three biological replicates and were measured via fiber optic technology that non - invasively probes oxygen levels in the culture ( PreSens Precision Sensing GmbH ) . Lower panel : expression profiles ( RNA - seq ) of desA1 and desA2 over the time course and oxygen levels . Error bars show the standard deviation from three biological samples .

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Figure 2(A) Path-seq profiles of desA1, desA2 in MTB infected BMDMs. Error bars show the standard deviation from three biological samples. Representative results from two experiments are presented.(B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.


Figure 3 ( B ) Heatmap of TFs with decreased ( purple ) or increased ( green ) activity at specific time points during in vitro or in vivo infection . ( C ) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity ( induced expression of target genes ) . ( D ) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity ( de - represses target genes ) . ( E ) Rv0691c regulon genes differentially expressed in vitro and in vivo ; evidence for increased Rv0691c activity ( increased up - and down - regulation of target genes ) at 24 h from both in vitro and in vivo infection .

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Figure 3(B) Heatmap of TFs with decreased (purple) or increased (green) activity at specific time points during in vitro or in vivo infection.(C) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity (induced expression of target genes). (D) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity (de-represses target genes). (E) Rv0691c regulon genes differentially expressed in vitro and in vivo; evidence for increased Rv0691c activity (increased up- and down-regulation of target genes) at 24 h from both in vitro and in vivo infection.


Figure 4 ( B ) Plot of read pile - ups from MSM with inducible overexpression of MSMEG _ 0916 shows ChIP - binding in the promoters of desA1 and desA2 . Boxplots representing RPKM values from RNA - seq of MSM with inducible overexpression of MSMEG _ 0916 . Significant log2 fold change ( FC ) between uninduced ( - ATc ) and induced ( + ATc ) samples for MSMEG _ 0916 ( log2 FC = 4 . 99 ) , desA1 ( log2 FC = - 1 . 32 ) and desA2 ( log2 FC = - 1 . 8 ) with multiple hypothesis adjusted P - values < 0 . 0001 . Data is from three biological samples ( induced ) and nine biological samples ( uninduced ) .

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Figure 4(B) Plot of read pile-ups from MSM with inducible overexpression of MSMEG_0916 shows ChIP-binding in the promoters of desA1 and desA2.Boxplots representing RPKM values from RNA-seq of MSM with inducible overexpression of MSMEG_0916. Significant log2 fold change (FC) between uninduced (-ATc) and induced (+ATc) samples for MSMEG_0916 (log2 FC = 4.99), desA1 (log2 FC = -1.32) and desA2 (log2 FC = -1.8) with multiple hypothesis adjusted P-values < 0.0001. Data is from three biological samples (induced) and nine biological samples (uninduced).


Figure 5 ( A ) Serial ten - fold dilutions of MTB H37Rv wild type ( wt ) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc . ( B ) Argentation TLC of 14C - labeled methyl esters of mycolic acids ( MAMEs ) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG _ 0916 . The α , αʹ , epoxy ( e ) and cyclopropanated α - ( X1 ) MAMES species are labeled . Faster - migrating species that co - migrated with α - MAMES and accumulate with induced MSMEG _ 0916 overexpression are indicated as X2 and X3 . ( C ) BCG wt and BCG with inducible overexpression of Rv0472c cultures , labeled with 14C - acetate , were induced ( + ATc ) or uninduced ( - ATc ) for 4 h or 8 h . The total FAMEs and MAMEs were extracted and analyzed by autoradiography - TLC using equal counts ( 15 , 000 cpm ) for each lane .

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Figure 5(A) Serial ten-fold dilutions of MTB H37Rv wild type (wt) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc.(B) Argentation TLC of 14C-labeled methyl esters of mycolic acids (MAMEs) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG_0916. The α, αʹ, epoxy (e) and cyclopropanated α- (X1) MAMES species are labeled. Faster-migrating species that co-migrated with α-MAMES and accumulate with induced MSMEG_0916 overexpression are indicated as X2 and X3.(C) BCG wt and BCG with inducible overexpression of Rv0472c cultures, labeled with 14C-acetate, were induced (+ATc) or uninduced (-ATc) for 4 h or 8 h. The total FAMEs and MAMEs were extracted and analyzed by autoradiography-TLC using equal counts (15,000 cpm) for each lane.


Figure 1A , Volcano plot of the differentially modulated proteins in 22rv1 ( CRPC ) versus CWR22res ( HN ) tumours . Red and blue dots represent the proteins that are significantly up - and down - regulated , respectively ( p - value < 0 . 05 , FC = 1 . 5 ) . Data information : Data reproducibility : n = 3 tumours per group . B , Western blot analysis of THEM6 expression in CRPC ( 22rv1 and LNCaP AI ) and HN ( CWR22res and LNCaP ) prostate orthografts . VCL was used as a sample loading control . Data information : Data reproducibility : : n = 1 gel loaded with three prostate orthografts per condition . G , Tumour volume ( measured by ultrasound ) of CTL and THEM6 KO 22rv1 - derived orthografts developed in surgically castrated mice . H , Tumour volume of CTL and THEM6 KO LNCaP AI - derived xenografts developed in surgically castrated mice . I , Tumour volume ( measured by ultrasound ) of CTL and THEM6 KO CWR22res - derived orthografts . Orchidectomy was performed 3 - weeks after cell implantation . Data information : Data are presented as mean values + / - SD . Statistical analysis : G , H : two - tailed Mann - Whitney U test . I : Kruskal - Wallis test . Data reproducibility : G , I : n = 5 mice per group . Panel H : n = 7 mice per group .

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Figure 1A, Volcano plot of the differentially modulated proteins in 22rv1 (CRPC) versus CWR22res (HN) tumours. Red and blue dots represent the proteins that are significantly up- and down-regulated, respectively (p-value < 0.05, FC = 1.5). Data information: Data reproducibility: n = 3 tumours per group.B, Western blot analysis of THEM6 expression in CRPC (22rv1 and LNCaP AI) and HN (CWR22res and LNCaP) prostate orthografts. VCL was used as a sample loading control. Data information: Data reproducibility: : n = 1 gel loaded with three prostate orthografts per condition.G, Tumour volume (measured by ultrasound) of CTL and THEM6 KO 22rv1-derived orthografts developed in surgically castrated mice. H, Tumour volume of CTL and THEM6 KO LNCaP AI-derived xenografts developed in surgically castrated mice. I, Tumour volume (measured by ultrasound) of CTL and THEM6 KO CWR22res-derived orthografts. Orchidectomy was performed 3-weeks after cell implantation. Data information: Data are presented as mean values +/- SD. Statistical analysis: G, H: two-tailed Mann-Whitney U test. I: Kruskal-Wallis test. Data reproducibility: G, I: n = 5 mice per group. Panel H: n = 7 mice per group.


Figure 2B - C , Changes in lipid content ( total amount ) observed in THEM6 KO CRPC cells when compared to CTL . Data information : Data are presented as mean values + / - SD . Statistical analysis : * p - value < 0 . 05 using a 1 - way ANOVA with a Dunnett ' s multiple comparisons test . Data reproducibility : n = 3 independent biological experiments . DAG : diacylglycerol ; TG : triglyceride ; PC : phosphatidylcholine ; PE : phosphatidylethanolamine ; PI : phosphatidylinositol ; LysoPC : lysophosphatidylcholine ; LysoPE : lysophosphatidylethanolamine ; Cer : Ceramide ; SM : sphingomyelin ; CE : Cholesteryl ester .

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Figure 2B-C, Changes in lipid content (total amount) observed in THEM6 KO CRPC cells when compared to CTL. Data information: Data are presented as mean values +/- SD. Statistical analysis : *p-value < 0.05 using a 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent biological experiments. DAG: diacylglycerol; TG: triglyceride; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; LysoPC: lysophosphatidylcholine; LysoPE: lysophosphatidylethanolamine; Cer: Ceramide; SM: sphingomyelin; CE: Cholesteryl ester.


Figure 3C , Representative electron microscopy ( EM ) pictures of CTL and THEM6 KO 22rv1 cells taken at low ( left ) and high ( right ) magnification . Red arrows point towards abnormal ER structure . Scale bar represents 500 nm . I , Western blot analysis of THEM6 , AMFR , SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells . Data information : Data reproducibility : representative image from 2 independent biological experiments . J , Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing . Data information : Data reproducibility : representative image from 3 independent biological experiments .

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Figure 3C, Representative electron microscopy (EM) pictures of CTL and THEM6 KO 22rv1 cells taken at low (left) and high (right) magnification. Red arrows point towards abnormal ER structure. Scale bar represents 500 nm.I, Western blot analysis of THEM6, AMFR, SEC61b and XPO1 expression in CRPC cells following THEM6 immunoprecipitation in CRPC cells. Data information: Data reproducibility: representative image from 2 independent biological experiments.J, Western blot analysis of CALX and CALR expression in PCa cells following THEM6 silencing. Data information: Data reproducibility: representative image from 3 independent biological experiments.


Figure 4B , RT - qPCR analysis of MVD , FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells . CASC3 was used as a normalising control . Data information : Data are presented as mean values + / - SD . Statistical analysis : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons test . Data reproducibility : : n = 3 independent biological experiments . F , Labelled cholesterol fraction derived from 13C - glucose and 13C - glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation . G , Labelled cholesterol fraction derived from 13C - glucose and 13C - glutamine in CTL and THEM6 KO MCF - 7 cells after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical analysis : , F , : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons test . G : * p - value < 0 . 05 using a two - tailed Student t - test . Data reproducibility : : n = 3 independent wells from the same cell culture . J , Labelled palmitic , oleic and stearic acid fractions derived from 13C - glucose and 13C - glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical analysis : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons test . Data reproducibility : n = 3 independent wells from the same cell culture .

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Figure 4B, RT-qPCR analysis of MVD, FDPS and DHCR7 expression in CTL and THEM6 KO 22rv1 cells. CASC3 was used as a normalising control. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: : n = 3 independent biological experiments.F, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO CWR22res cells after 72 hours of incubation. G, Labelled cholesterol fraction derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO MCF-7 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: , F, : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. G: *p-value < 0.05 using a two-tailed Student t-test. Data reproducibility: : n = 3 independent wells from the same cell culture.J, Labelled palmitic, oleic and stearic acid fractions derived from 13C-glucose and 13C-glutamine in CTL and THEM6 KO 22rv1 cells after 72 hours of incubation. Data information: Data are presented as mean values +/- SD. Statistical analysis: : *p-value < 0.05 using 1-way ANOVA with a Dunnett's multiple comparisons test. Data reproducibility: n = 3 independent wells from the same cell culture.


Figure 5B , Proteomic analysis highlighting proteins associated with the ER stress response significantly down - regulated in THEM6 KO 22rv1 cells when compared to CTL ( p - value ≤ 0 . 05 , FC = 1 . 3 ) . Data information : Data are presented as mean values + / - SD . Data reproducibility : n = 3 independent biological experiments . E , Proteomic analysis highlighting ATF4 targets significantly down - regulated in THEM6 KO LNCaP AI cells when compared to CTL ( p - value ≤ 0 . 05 , FC = 1 . 3 ) . Data information : Data are presented as mean values + / - SD . Data reproducibility : n = 3 independent biological experiments . J , Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid ( 200 µM ) or hexadecylglycerol ( 50 µM ) for 48 hours . Data information : HSC70 was used as a sample loading control . Data reproducibility : representative image from 3 independent biological experiments .

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Figure 5B, Proteomic analysis highlighting proteins associated with the ER stress response significantly down-regulated in THEM6 KO 22rv1 cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.E, Proteomic analysis highlighting ATF4 targets significantly down-regulated in THEM6 KO LNCaP AI cells when compared to CTL (p-value ≤ 0.05, FC = 1.3). Data information: Data are presented as mean values +/- SD. Data reproducibility: n = 3 independent biological experiments.J, Western blot analysis of ATF4 and CHOP expression in CTL and THEM6 KO LNCaP AI cells treated with palmitic acid (200 µM) or hexadecylglycerol (50 µM) for 48 hours. Data information: HSC70 was used as a sample loading control. Data reproducibility: representative image from 3 independent biological experiments.