A Necrostatin - 1 ( Nec ) does not affect LBW - mediated sensitization to VCR - induced apoptosis . Apoptosis induction in LOUCY was determined by flow cytometry 48h after treatment with SM / or VCR ( left ) / or TNFα ( right ) in presence or absence of RIPK1 - inhibitor Nec . Mean ± SD of 3 independent experiments is shown ; * p < 0 . 05 * * p < 0 . 01 by unpaired Student ' s t - .

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

A 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 VCR . LOUCY were transfected with a non - targeting siRNA ( neg - siRNA ) or a RIPK1 - specific siRNA . 6h post transfection , were treated with indicated drugs apoptosis was measured 40h thereafter . Mean ± SD of 3 independent experiments is shown ; * * * p < 0 . 001 by unpaired Student ' s t - . Knockdown ( KD ) was verified by Western blot at time of apoptosis measurement KD efficiency calculated compared to control . One representative immunoblot out of 3 biological replicates is shown .

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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 SM - drug combination does not activate canonical NF - κB pathway . Phosphorylation degradation of IκBα upon treatment with VCR or TNFα / or LBW242 was determined in LOUCY by Western blot following treatment with indicated drugs for 5 , 15 or 60 minutes using Actin as loading control . One representative immunoblot out of 3 independent biological replicates is shown .

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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 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 - .

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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 SM - drug combination activates non - canonical NF - κB pathway . Cleavage of p100 NF - κB2 to p52 fragment was determined by Western blot 24h after treatment of LOUCY with chemotherapy or TNFα in combination with SM . Asterisk indicates an unspecific signal . One representative immunoblot out of 3 biological replicates is shown .

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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 - .

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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.


A Expression of XIAP mRNA in samples obtained from BM of healthy individuals ( Ctrl , n = 10 ) or with AML ( n = 517 ) or ( n = 306 ) . Data are expressed as median , 25th 75th percentile , whiskers indicate min / max ; * * p < 0 . 01 ; * * * * p < 0 . 0001 by Games - Howell post - hoc .

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A 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 of XIAP normalized to loading control in PDX samples from pediatric adult , analyzed by immunoassay ( Simple Western ) depicted as mean ( n = 2 , biological replicates ) ; PBMC = peripheral blood mononuclear

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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 cells


Parental XIAP over - expressing NALM - 6 ( C ) were treated with VCR apoptosis ( Annexin - V / PI ) was measured at indicated time points ( C ) by flow cytometry . Mean ± of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - . XIAP level was analyzed in parental XIAP - overexpressing NALM - 6 by immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown .

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Parental 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 XIAP over - expressing REH ( D ) were treated with VCR or LBW242 ; apoptosis ( Annexin - V / PI ) was measured after 72h ( D ) by flow cytometry . Mean ± of 3 independent experiments is shown ; * * * * p ≤ 0 . 0001 by unpaired Student ' s t - . XIAP level was analyzed by immunoassay ( Simple Western ) ; Actin was used as loading control . One representative immunoassay out of 3 independent experiments is shown .

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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.


B shRNA expression control . Equal expression of ds - RED in sh - CTRL / mTagBFP sh - XIAP / eGFP subpopulation indicates identical expression of control XIAP shRNA . One representative histogram out of 3 independent experiments is shown .

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B 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 of XIAP in NALM - 6 expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP under SFFV promoter ; GAPDH was used as loading control . One representative immunoblot out of 3 independent experiments is shown .

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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 of NALM - 6 with moderate ( 69 % , EF1α promoter ) or strong ( 92 % , SFFV promoter ) KD of XIAP ; GAPDH was used as loading control . XIAP KD ( % KD ) were quantified relative to sh - CTRL . One representative immunoblot out of 3 independent experiments is shown .

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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 expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 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 . 7 days after last treatment , mice were sacrificed , relative proportion of sh - CTRL sh - XIAP subpopulations re - isolated from BM was quantified by flow cytometry . Data are depicted as mean ± of mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - with Welch ' s correction .

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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 expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were mixed at a 1 : 1 injected into groups of mice ( n = 8 ) . 38 or 46 days after injection , mice were sacrificed , relative proportion of sh - CTRL sh - XIAP subpopulations re - isolated from BM was quantified by flow cytometry . Data are depicted as mean ± of mice analyzed with * * * * p ≤ 0 . 0001 by unpaired Student ' s t - .

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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 with strong XIAP inhibition . NALM - 6 were lentivirally transduces with sh - CTRL or sh - XIAP ( SFFV ) . 4 days later , were lysed relative cleavage level of PARP ( % ) was analyzed by immunoassay ( Simple Western ) . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - .

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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 . NALM - 6 were transduced with shCTRL , shXIAP . 1 alone or shXIAP . 1 together with XIAP overexpression vector . At day 0 , transduced were mixed in a 1 : 1 with parental ( untransduced ) NALM - 6 . Distribution of fluorochrome marker was used as a readout to determine growth variations of respective subpopulation over time . Mean ± SD of 3 independent experiments is shown ; * * p < 0 . 01 by unpaired Student ' s t - .

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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.


XIAP plays an essential role for - 265 growing in mice . Experimental scheme stability of XIAP knockdown over passaging . - 265 PDX produced as described in A were mixed at a 1 : 1 injected into mice ( P1 , n = 4 ) . At clinical signs of illness , were isolated re - injected into next recipient mice ( P2 , n = 4 ) . XIAP expression of P1 P2 PDX expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP were analyzed by Western blot at end of each passage ; GAPDH was used as loading control ( B ) .

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XIAP 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 - 265 growing in mice . - 265 PDX produced as described in A were mixed at a 1 : 1 injected into mice ( P1 , n = 4 ) . At clinical signs of illness , were isolated re - injected into next recipient mice ( P2 , n = 4 ) . Relative proportion of - 265 GEPDX expressing sh - CTRL / mTagBFP or sh - XIAP / eGFP re - isolated from BM at end of each passage was quantified by flow cytometry ( C ) . Data are depicted as mean ± of mice analyzed ( n = 4 , each passage ) with * * * * p < 0 . 0001 by unpaired Student ' s t - .

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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 growth disadvantage ; - 265 PDX of a representative mouse were analyzed separately for dsRED / shRNA high low expressing .

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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.


Orthotopic MPNST xenografts at passages 1 ( OT P1 ) 4 ( OT P4 ) were histopathologically similar to their corresponding MPNST ( PT ) in hematoxylin - eosin of paraffin - embedded sections from MPNST - NF1 - 001 MPNST - SP - 002 . Main panels show a general view of tumors at low magnification ( 40 × ) ; inset pictures were taken at higher magnification ( 400 × ) .

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Orthotopic 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 MPNSTs exhibited similar immunohistochemical features . A representative immunostained section of vimentin , CD34 , S100 , Ki - 67 is shown for tumors ( PT ) orthotopic tumors ( OT ) from patientsMPNST - NF1 - 001 MPNST - SP - 002 . Positive antibody signals are shown in brown , hematoxylin counterstain in blue . Main panels show pictures at high magnification ( 400 × ) ; inset pictures show mitotic present in these tumors .

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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.


Stromal elements of tumors were labeled with anti - humanCD34 but not anti - mouseCD34 ; - derived xenografts are labeled with anti - mouseCD34 only no anti - human marker . Representative sections ( at 40 × 400 × magnification ) of tumors ( PT ) orthoxenograft tumors at passages 1 ( OT P1 ) 4 ( OT P4 ) were labeled with anti - humanCD34 ( H ) anti - mouseCD34 ( M ) .

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Stromal 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 germline NF1 mutation c . 350T > A present in that developed two different tumors ( MPNST - NF1 - 001 MPNST - NF1 - 002 ) . Sequencing results from normal tissue , ( PT ) , orthotopic xenograft tumors ( OT ) are shown . of NF1 region revealed WT NF1 alleles in samples , not in corresponding derived orthoxenografts , indicating probable loss of human stromal .

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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 ( PT ) orthotopic xenograft passages 1 ( OT P1 ) 4 ( OT P4 ) for MPNST - NF1 - 001 MPNST - NF1 - 002 tumors . Results correspond to chromosome 17 . show B - allele frequency ( BAF ) for different samples as scatter plots copy number callings below them represented by thick horizontal lines : 2n regions are shown in gray , gained regions in orange , lost regions in green , LOH regions are represented in blue . vertical red indicates location of NF1 locus . In addition , of versus stromal for each sample is represented in a pie chart ( blue , stromal ; red , ) .

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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).


growth effects of treatment with doxorubicin , sorafenib , rapamycin combinations thereof in five MPNST xenograft models . Results are plotted as an average of log2 of volume at different days relative to initial value . Statistically significant differences are shown as * P < 0 . 05 * * P < 0 . 001 versus control group by Bonferroni .

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Tumor 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 sacrificed over time when relapsed masses grew as large solid masses ( usually 1 , 500 - 2 , 000 mm3 ) . graph illustrates differences in time delay ( in days ) of relapsed masses for different treatments .

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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.


A Immunoblot of DDX3 expression in lung cancer lines .

A Immunoblot of DDX3 expression in lung cancer cell lines.


B , C Colony - forming assays in H1299 ( B ) A549 ( C ) lung cancer after knockdown by shRNA lentiviral constructs designed against DDX3 or vector control . Corresponding immunoblots displaying knockdown of DDX3 . Mean from 3 replicates with SD .

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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 H1299 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 - ) .

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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 in parental A549 A549 DDX3 knockdown displaying senescent identified by blue color .

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 carcinoma . H DDX3 expression in adenocarcinoma . I DDX3 expression in small carcinoma . J Expression of DDX3 in different histological types of lung cancer . data sets were compared against each other ( chi - square , P = 0 . 481 ) .

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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 of lung cancer in low high DDX3 expressing tumors ( Kaplan - Meier curve log - rank , P = 0 . 016 ) .

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K Survival analysis of lung cancer patients in low and high DDX3 expressing tumors (Kaplan-Meier curve and log-rank test, P = 0.016).


F Immunoblots of pull - down of DDX3 with biotin , AK - 298 , SK - 153 . Lower panels display results using DDX5 DDX17 antibodies .

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F 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 ) .

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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 of various lung cancer lines treated with RK - 33 for 72 h . Mean from 3 replicates with SD .

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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 of A549 H3255 treated with RK - 33 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 extra sum of squares F - .

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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.


Following injection of 20 mg / kg of RK - 33 , twice a week for 7 , extensive histopathological examination was carried out following necropsy . Identical patterns were observed both in control in treated mice ( n = 2 ) . Samples were stained with H & amp ; amp ; E . Scale bar is 50 μm .

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Following 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 of RK - 33 in mouse plasma tissue .

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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 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 ) , 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 representative of independent experiments performed with two different HAPI passages . OCR is baseline - normalized to point prior to drug or vehicle addition .

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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 were incubated for 1 h in glucose - free XF24 medium that was supplemented with 2 - deoxyglucose ( 50 mM ) 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 - .

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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.


Confirmation of high expression of Twist1 DDX3 in lung tumors of transgenic Twist1 / KrasG12D mouse . Scale bar is 100 μm .

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Confirmation of high expression of Twist1 and DDX3 in the lung tumors of the transgenic Twist1/KrasG12D mouse. Scale bar is 100 μm.


Micro - CT of transgenic Twist1 / KrasG12D mice treated as in ( C ) , before treatment 1 week after treatment . Tumors are indicated by arrows confirmed by H & amp ; amp ; E of lung sections ( lower panel ) . Scale bar is 250 μm . of volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( D ) . Significance was assessed by two - sided , unpaired t - . Error bars represent SD .

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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 model was generated using A549 human lung cancer treated as in ( C ) . Figure displays H & amp ; amp ; E of lung sections from radiation - treated ( upper panel ) RK - 33 - radiation - treated mice ( lower panel ) . Scale bar is 2 mm . of burden ( as 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 - . Error bars represent SD .

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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 of transgenic Twist1 / KrasG12D mice treated as in ( H ) , before treatment 1 week after treatment . of volume measured by micro - CT in Twist1 / KrasG12D mice , as shown in ( I ) expressed as relative . Significance was assessed by two - sided , unpaired t - . Error bars represent .

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.


A cycle of H1299 treated with shDDX3 processed by flow cytometry . Knockdown of DDX3 led to a decrease of in S - phase an increase of in G1 - phase , indicative of a G1 arrest . Significance was assessed by two - sided , unpaired t - . Error bars represent SD .

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A 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 cycle of A549 H1299 by flow cytometry after treatment with RK - 33 ( 0 , 2 , 4 , 6 μM ) . RK - 33 induced a G1 cycle arrest in both lines . Significance was assessed by two - sided , unpaired t - . Error bars represent SD .

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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 cycle - related ( Cyclin D1 Cyclin E1 ) - related ( caspase 7 , caspase 9 ) in A549 H1299 after treatment with RK - 33 ( 10 μM ) . Initially , a strong decrease of Cyclin D1 was observed . After 8 24 h , caspases 9 7 were apparent .

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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 ERK1 / 2 phosphorylated ERK1 / 2 in A549 , H1299 , H3255 ( RK - 33 resistant ) 24 h after treatment with RK - 33 ( 7 . 5 μM or 10 μM ) . ERK2 especially ERK1 become dephosphorylated after treatment with RK - 33 in A549 H1299 but not in H3255 . Outlined boxes indicate spliced lanes .

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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.


A β - catenin ( red ) DDX3 ( green ) expression in H1299 . After overexpressing β - catenin , both DDX3 β - catenin accumulate in nucleus . Scale bar is 10 μm .

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A 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 ) immunoblotted with β - catenin in A549 H1299 . Outlined boxes indicate spliced lanes .

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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 ) immunoblotted with DDX3 in A549 H1299 . 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 TOP / FOP reporter . Co - transfection with β - catenin is indicated below .

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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 A549 were treated with RK - 33 ( 0 , 1 , 2 , 3 μM ) co - transfected with β - catenin in ( F , H ) . Treatment with RK - 33 decreased TCF4 activity in both lines .

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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 ( Axin - 2 , c - Myc , Cyclin D1 ) DDX3 were measured by qRT - PCR in H1299 after knockdown of DDX3 ( J ) treatment with RK - 33 ( K ) . experiments were repeated three times .

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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.


A Immunofluorescence showing 53BP1 γH2AX foci in A549 after 2 - Gy radiation A549 pre - treated with 6 μM RK - 33 , 12 h before radiation . Overlap of 53BP1 γH2AX is seen in merged picture of co - immunofluorescence . Scale bar is 2 μm .

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A 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 were pre - treated with RK - 33 radiated with 2 Gy , 53BP1 γH2AX foci were counted as a measure of damage . with more than 10 foci 53BP1 or γH2AX were counted . More than 400 per sample were evaluated .

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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 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 .

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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 , containing a stable non - homologous end - joining ( NHEJ ) reporter construct , were treated with RK - 33 knockdown of DDX3 . Reporter construct expressed GFP , which was quantified by flow cytometry . experiments were repeated three times .

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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 treated with RK - 33 shDDX3 were validated by qRT - PCR using NHEJ Mechanisms of DSBs Repair PrimePCR plates ( Bio - Rad ) performed in biological triplicates .

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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 repair - related ( ATR XRCC4 ) , DDX3 , actin were assessed by immunoblotting in A549 ( F ) H1299 ( G ) . were pretreated for 4 h with vehicle control or 6 μM RK - 33 then radiated with 0 or 5 Gy . Outlined boxes indicate spliced lanes .

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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.


ATPase of SOL of WT DKO mice ( left ) . Corresponding of type I were determined ( right ) ( n = 5 ) . Scale bars : 100 μm .

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ATPase 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 PLIN5 in of WT DKO mice ( n = 3 for PGC1α ; n = 5 for PLIN5 ) .

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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 PLIN5 in C2C12 transfected with miR - 183 miR - 96 agomirs ( Ago - 183 / 96 ) or antagomirs ( Ant - 183 / 96 ) ( n = 3 ) .

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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 transfected with Ago - 183 / 96 ( J ) or Ant - 183 / 96 ( K ) . oxygen consumption rate ( OCR ) was measured in real time corresponding basal maximal respiration were analyzed by overall OCR ( n = 3 or 4 per group ) .

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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).


Western blot of FoxO1 , PDK4 , p - PDHA1 PDHA1 in of WT DKO mice . Western blot of FoxO1 , PDK4 , p - PDHA1 PDHA1 in C2C12 transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) .

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Western 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 of HSL ATGL in of WT DKO mice . Western blot of HSL ATGL in C2C12 transfected with Ago - 183 / 96 ( left ) or Ant - 183 / 96 ( right ) .

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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 per was quantified at 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 of lipid droplets in C2C12 transfected with agomiR - 183 / 96 ( O ) or antagomir - 183 / 96 ( Q ) . Scale bars : 5 μm .

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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.


Relative expression of miR - 183 miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 agomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 PDK4 in C2C12 treated with Ago - 183 or Ago - 96 ( right ) ( n = 3 ) . Western blot of FoxO1 PDK4 in C2C12 treated with Ago - 183 or Ago - 96 . Relative expression of miR - 183 miR - 96 in C2C12 myocytes transfected with miR - 183 or miR - 96 antagomir ( left ) ( n = 3 ) . Relative mRNA expression of FoxO1 PDK4 in C2C12 treated with Ant - 183 or Ant - 96 ( right ) ( n = 3 ) . Western blot of FoxO1 PDK4 in C2C12 treated with Ant - 183 or Ant - 96 .

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Relative 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 ) western blot ( J , top ) of ATGL in C2C12 transfected with agomir for miR - 183 ( Ago - 183 ) or miR - 96 ( Ago - 96 ) . Relative mRNA expression ( I , right ) western blot ( J , bottom ) of ATGL in C2C12 transfected with antagomir for miR - 183 ( Ant - 183 ) or miR - 96 ( Ant - 96 ) . ( n = 5 per group for RT - PCR )

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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)


Relative mRNA expression of ATGL , PGC1α , PLIN5 , HSL in C2C12 transfected with ATGL ( n = 5 ) .

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Relative mRNA expression of ATGL, PGC1α, PLIN5, and HSL in C2C12 cells transfected with ATGL (n=5).


Relative mRNA expression of PGC1α , PLIN5 , HSL in C2C12 transfected with Ant - 183 / 96 siRNA of PPARδ ( siPPARδ ) .

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


Relative mRNA expression of ATGL , HSL , PLIN5 in SOL of C57 BL / 6 mice ( n = 3 ) . Western blot of ATGL , HSL , PLIN5 in SOL of C57 BL / 6 mice .

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Relative 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 , PLIN5 in of C57 BL / 6 mice fed with HFD for 2 months ( n = 5 ) . Western blot of ATGL , HSL , PLIN5 in of C57 BL / 6 mice fed with HFD for 2 months .

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.


of serum triglyceride ( TAG ) ( left ) non - esterified fatty acid ( NEFA ) ( right ) in WT DKO mice after HFD feeding for 2 months ( n = 5 ) .

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Concentration 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 of FoxO1 , PDK4 , p - PDHA1 , PDHA1 ATGL in of WT DKO mice fed with HFD for 2 months .

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 ( GTT ) under curve ( AUC ) data for GTT in WT DKO mice after HFD feeding ( n = 10 - 11 ) .

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Glucose tolerance test (GTT) and area under the curve (AUC) data for GTT in WT and DKO mice after HFD feeding (n=10-11).


( B ) Correlation between replicate mock infections . Path - seq reads were recovered from samples of macrophage spiked with 0 . 1 % MTB . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 . Inset summarizes mock infection replicates their fraction of MTB reads ( of aligned reads ) from Path - seq standard - seq methods .

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(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 sequenced by - seq ( Illumina TruSeq library prep ) same MTB sample combined with macrophage at 1 : 1000 processed using Path - seq method . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 .

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(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.


( A ) Path - seq profiles of desA1 , desA2 in MTB infected BMDMs . Error bars show standard deviation from three biological samples . Representative results from two experiments are presented .

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(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 ) controlled reaeration ( 3 ) . Points represent average of three biological replicates were measured via fiber optic technology that non - invasively probes oxygen in culture ( PreSens Precision Sensing GmbH ) . Lower panel : expression profiles ( - seq ) of desA1 desA2 over time course oxygen . Error bars show standard deviation from three biological samples .

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(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.


( B ) of TFs with decreased ( purple ) or increased ( green ) activity at specific time points during in vitro or in vivo infection .

(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 evidence for predicted high Rv0681 activity ( induced expression of target genes ) . ( D ) Zur regulon genes differentially expressed in vitro evidence for decreased Zur activity ( de - represses target genes ) . ( E ) Rv0691c regulon genes differentially expressed in vitro in vivo ; evidence for increased Rv0691c activity ( increased up - down - regulation of target genes ) at 24 h from both in vitro in vivo infection .

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(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.


( B ) Plot of read pile - ups from MSM with inducible overexpression of MSMEG _ 0916 shows ChIP - binding in promoters of desA1 desA2 .

(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 - seq of MSM with inducible overexpression of MSMEG _ 0916 . Significant log2 fold change ( FC ) between uninduced ( - ATc ) induced ( + ATc ) samples for MSMEG _ 0916 ( log2 FC = 4 . 99 ) , desA1 ( log2 FC = - 1 . 32 ) desA2 ( log2 FC = - 1 . 8 ) with multiple hypothesis adjusted P - values < 0 . 0001 . Data is from three biological samples ( induced ) nine biological samples ( uninduced ) .

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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).


( A ) Serial ten - fold dilutions of MTB H37Rv wild type ( wt ) MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc .

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(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 ( MAMEs ) obtained from apolar lipids delipidated wall fractions of MSM wt MSM with inducible overexpression of MSMEG _ 0916 . α , αʹ , epoxy ( e ) cyclopropanated α - ( X1 ) MAMES species are labeled . Faster - migrating species that co - migrated with α - MAMES accumulate with induced MSMEG _ 0916 overexpression are indicated as X2 X3 .

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(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 BCG with inducible overexpression of Rv0472c cultures , labeled with 14C - acetate , were induced ( + ATc ) or uninduced ( - ATc ) for 4 h or 8 h . total FAMEs MAMEs were extracted analyzed by autoradiography - TLC using equal ( 15 , 000 cpm ) for each lane .

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(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.


A , Volcano plot of differentially modulated in 22rv1 ( CRPC ) versus CWR22res ( HN ) tumours . Red blue dots represent that are significantly up - down - regulated , respectively ( p - value < 0 . 05 , FC = 1 . 5 ) . Data information : Data reproducibility : n = 3 tumours per group .

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A, 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 of THEM6 expression in CRPC ( 22rv1 LNCaP AI ) HN ( CWR22res 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 .

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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 THEM6 KO 22rv1 - derived orthografts developed in surgically castrated mice . H , Tumour volume of CTL THEM6 KO LNCaP AI - derived xenografts developed in surgically castrated mice . I , Tumour volume ( measured by ultrasound ) of CTL THEM6 KO CWR22res - derived orthografts . Orchidectomy was performed 3 - after implantation . Data information : Data are presented as mean values + / - SD . Statistical : G , H : two - Mann - Whitney U . I : Kruskal - Wallis . Data reproducibility : G , I : n = 5 mice per group . Panel H : n = 7 mice per group .

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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.


B - C , Changes in lipid content ( total amount ) observed in THEM6 KO CRPC when compared to CTL . Data information : Data are presented as mean values + / - SD . Statistical : * p - value < 0 . 05 using a 1 - way ANOVA with a Dunnett ' s multiple comparisons . 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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B-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.


C , Representative electron microscopy ( EM ) pictures of CTL THEM6 KO 22rv1 taken at low ( left ) high ( right ) magnification . Red arrows point towards abnormal ER structure . Scale bar represents 500 nm .

C, 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 of THEM6 , AMFR , SEC61b XPO1 expression in CRPC following THEM6 immunoprecipitation in CRPC . Data information : Data reproducibility : representative image from 2 independent biological experiments .

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 of CALX CALR expression in PCa following THEM6 silencing . Data information : Data reproducibility : representative image from 3 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.


B , RT - qPCR of MVD , FDPS DHCR7 expression in CTL THEM6 KO 22rv1 . CASC3 was used as a normalising control . Data information : Data are presented as mean values + / - SD . Statistical : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons . Data reproducibility : : n = 3 independent biological experiments .

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B, 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 13C - glutamine in CTL THEM6 KO CWR22res after 72 hours of incubation . G , Labelled cholesterol fraction derived from 13C - glucose 13C - glutamine in CTL THEM6 KO MCF - 7 after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical : , F , : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons . G : * p - value < 0 . 05 using a two - Student t - . Data reproducibility : : n = 3 independent wells from same culture .

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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 stearic acid fractions derived from 13C - glucose 13C - glutamine in CTL THEM6 KO 22rv1 after 72 hours of incubation . Data information : Data are presented as mean values + / - SD . Statistical : : * p - value < 0 . 05 using 1 - way ANOVA with a Dunnett ' s multiple comparisons . Data reproducibility : n = 3 independent wells from same culture .

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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.


B , Proteomic highlighting associated with ER stress response significantly down - regulated in THEM6 KO 22rv1 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 .

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B, 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 highlighting ATF4 targets significantly down - regulated in THEM6 KO LNCaP AI 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 .

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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 of ATF4 CHOP expression in CTL THEM6 KO LNCaP AI 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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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.


C , Kaplan - Meier progression - free survival of PCa stratified according high low THEM6 expression using PRAD TCGA dataset . D , Kaplan - Meier recurrence - free survival of PCa stratified according to median THEM6 expression using GSE21034 dataset ( n = 179 ) . Data information : Statistical : logrank . Data reproducibility : C : n = 489 tumours . D : n = 179 .

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C, Kaplan-Meier progression-free survival analysis of PCa patients stratified according high and low THEM6 expression using the PRAD TCGA dataset. D, Kaplan-Meier recurrence-free survival analysis of PCa patients stratified according to median THEM6 expression using the GSE21034 dataset (n = 179). Data information: Statistical analysis: logrank test. Data reproducibility: C: n = 489 tumours. D: n = 179.


E , of THEM6 expression in treatment naïve , NHT - treated , CRPC NEPC tumours . Scale bar represents 100 µm . Data information : Data reproducibility : : n = 132 ; 90 ; 66 ; 137 ; 44 ; 30 tumours for Untreated ; NHT 1 - 3 ; NHT4 - 6 ; NHT > 7 ; CRPC ; NEPC respectively .

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E, IHC staining of THEM6 expression in treatment naïve, NHT-treated, CRPC and NEPC tumours. Scale bar represents 100 µm. Data information: Data reproducibility: : n = 132; 90; 66; 137; 44; 30 tumours for Untreated; NHT 1-3; NHT4-6; NHT>7; CRPC; NEPC respectively.


I - J , Kaplan - Meier recurrence - free ( I ) overall ( J ) survival of PCa stratified according to median THEM6 expression . Data information : Statistical : logrank . Data reproducibility : n = 35 tumours for THEM6 low n = 34 tumours for THEM6 high .

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I-J, Kaplan-Meier recurrence-free (I) and overall (J) survival analysis of PCa patients stratified according to median THEM6 expression. Data information: Statistical analysis: logrank test. Data reproducibility: n = 35 tumours for THEM6 low and n = 34 tumours for THEM6 high.


K , of THEM6 expression in PCa tissue samples according to Ki67 expression in analysed in F . L , of THEM6 expression in PCa tissue samples according to Ki67 expression in analysed in I - J . Data information : Centre corresponds to median of data , top bottom of box correspond to 90th 10th percentile , respectively . Whiskers extend to adjacent values ( minimum maximum data points not considered outliers ) . Statistical : two - Mann - Whitney U . Data reproducibility : K : n = 269 tumours for Ki67 low n = 270 tumours for Ki67 high . L : n = 35 tumours for Ki67 low n = 29 tumours for Ki67 high .

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K, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in F. L, Quantification of THEM6 expression in PCa tissue samples according to Ki67 expression in the patients analysed in I-J. Data information: Centre line corresponds to median of data, top and bottom of box correspond to 90th and 10th percentile, respectively. Whiskers extend to adjacent values (minimum and maximum data points not considered outliers). Statistical analysis: two-tailed Mann-Whitney U test. Data reproducibility: K: n = 269 tumours for Ki67 low and n = 270 tumours for Ki67 high. L: n = 35 tumours for Ki67 low and n = 29 tumours for Ki67 high.


M , Volcano plot of differentially modulated genes in PCa from PRAD TCGA stratified according to THEM6 expression . Red dots represent UPR - related genes extracted from UPR - gene signature Data information : Data reproducibility : n = 489 tumours .

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M, Volcano plot of the differentially modulated genes in PCa patients from the PRAD TCGA stratified according to THEM6 expression. Red dots represent the UPR-related genes extracted from the UPR-gene signature Data information: Data reproducibility: n = 489 tumours.


( A ) Atg5fl / fl Cre − Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 .

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(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n≥3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM release of active caspase ‐ 1 IL ‐ 1β was determined by immunoblotting .

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(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting.


( C ) As in ( A ) , assayed for IL ‐ 18 . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 .

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(C) As in (A), assayed for IL‐18. Data represent mean values±s.d. (n≥3); *P0.05.


( D ) LPS ‐ pretreated BMMs were exposed to alum ( 250 μg / ml ) for 1 h with or without autophagic induction by starvation . Secreted IL ‐ 1β was measured as in ( A ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.


( E ) LPS ‐ pretreated BMMs were exposed to silica ( 250 μg / ml ) for 1 h with or without autophagic induction by starvation . Secreted IL ‐ 1β was measured as in ( A ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

(E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.


( F ) BMMs were transfected with scramble ( Scr ) control siRNA or siRNAs against ASC NLRP3 . After 48 h following transfection , were treated overnight with LPS subjected to nigericin ( 20 μM ) starvation for 1 h . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.


( G ) Immunoblot of ASC NLRP3 knockdowns .

(G) Immunoblot analysis of ASC and NLRP3 knockdowns.


( H ) BMMs were transfected with scramble ( Scr ) control siRNA or siRNAs against ASC NLRP3 . After 48 h following transfection , were treated overnight with LPS subjected to silica ( 250 μg / ml ) starvation for 1 h . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.


( I ) Colocalization of IL ‐ 1β with basal autophagic machinery factor LC3 . : LC3 ( green , Alexa488 ) ; IL ‐ 1β ( red , Alexa568 ) . BMMs were from GFP - LC3 knock ‐ in mice , treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP IL ‐ 1β .

(I) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP-LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β.


(J, K) A line fluorescence tracing from images in (I).


( L ) Pearson ' s colocalization coefficient for IL ‐ 1β LC3 . Pearson ' s coefficient was derived from three independent experiments with five fields per experiment , for a total of 15 fields contributing to cumulative result . Figure source data can be found in Supplementary data .

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(L) Pearson's colocalization coefficient for IL‐1β and LC3. Pearson's coefficient was derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.


( A , B ) LPS ‐ pretreated BMMs were treated with 20 μM nigericin ( Nig ) 100 nM bafilomycin A1 ( Baf ) with ( Starvation ) or without ( Full ) autophagic induction for 1 h secreted IL ‐ 1β ( A ) IL ‐ 18 ( B ) were measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( C ) LPS ‐ pretreated BMMs were treated with 250 μg / ml of silica 100 nM bafilomycin A1 ( Baf ) with ( Starvation ) or without ( Full ) autophagic induction for 1 h secreted IL ‐ 1β were measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(C) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( D ) Colocalization of cathepsin B with basal autophagic machinery factor LC3 IL ‐ 1β . ; LC3 ( green , Alexa488 ) , IL ‐ 1β ( red , Alexa568 ) , cathepsin B ( blue , Alexa633 ) . BMMs from GFP - LC3 knock ‐ in mice were treated with LPS then analysed for immunofluorescence .

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(D) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence.


( E ) Colocalization tracing from in ( D ) .

(E) Colocalization line tracing analysis from images in (D).


( F ) LPS ‐ pretreated BMMs were treated with 20 μM nigericin cathepsin B inhibitor CA ‐ 074 Me ( 10 μM ) , with ( Starvation ) or without ( Full ) autophagic induction , for 1 h secreted IL ‐ 1β was measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( G ) LPS ‐ pretreated Atg5fl / fl Cre − Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM release of cathepsin B was determined by immunoblotting . Figure source data can be found in Supplementary data .

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(G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.


( A ) Colocalization of Rab8a with basal autophagic machinery factor LC3 IL ‐ 1β . ; LC3 ( green , Alexa488 ) , IL ‐ 1β ( red , Alexa568 ) , Rab8a ( blue , Alexa633 ) . BMMs from GFP - LC3 knock ‐ in mice were pretreated with LPS analysed by immunofluorescence microscopy . Arrows indicate triple colocalization .

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(A) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization.


(B) Line tracing analysis of fluorescence signal intensity.


( C ) Pearson ' s colocalization coefficient for IL ‐ 1β Rab8a . Pearson ' s coefficients were derived from three completely independent experiments with > 5 fields per experiment , for a total of ⩾ 15 fields contributing to cumulative result .

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(C) Pearson's colocalization coefficient for IL‐1β and Rab8a. Pearson's coefficients were derived from three completely independent experiments with >5 fields per experiment, for a total of ⩾15 fields contributing to the cumulative result.


( D ) BMMs were transfected with siRNAs against Rab8a or scramble ( Scr ) control . At 24 h after first transfection , were transfected again with siRNA , treated with LPS day after subjected to nigericin in full medium for 1 h , IL ‐ 1β secretion measured .

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(D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured.


( E ) Immunoblot of Rab8a knockdown in BMMs .

(E) Immunoblot analysis of Rab8a knockdown in BMMs.


( F ) RAW 264 . 7 macrophages were transfected with GFP ‐ tagged Rab8a constructs ( WT , wild type ; S22N , dominant ‐ negative mutant ) , treated overnight with LPS stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation . IL ‐ 1β secretion was measured by ELISA . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

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(F) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. Figure source data can be found in Supplementary data.


( A ) BMM were transfected with scramble ( Scr ) control siRNA or siRNA against GRASP55 . After 48 h of transfection , were treated with LPS day after subjected to 20 μM nigericin in EBSS , secreted IL ‐ 1β was measured by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Inset : Immunoblot of GRASP55 knockdown .

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(A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown.


( B ) Immunofluorescence confocal microscopy of LC3 GRASP55 distribution . LC3 ( green , Alexa488 ) , GRASP55 ( red , Alexa568 ) . BMMs were pretreated overnight with 100 ng / ml LPS either not stimulated ( Ctrl ) or stimulated ( Nig ) for 30 min with inflammasome agonist nigericin ( 20 μM ) in full medium .

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(B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium.


( C ) tracings , of signal from in ( B ) .

(C) Line tracings, analysis of fluorescence signal intensity from images in (B).


( D ) Pearson ' s coefficients for LC3 GRASP55 were quantified using SlideBook morphometric software as a measure of adjacency between GRASP55 LC3 profiles . Pearson ' s coefficients were derived from three independent experiments with five fields per experiment , for a total of 15 fields contributing to cumulative result . Figure source data can be found in Supplementary data .

(D) Pearson's coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles. Pearson's coefficients were derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.


( A , B ) Effect of GRASP55 on autophagy induction by measuring LC3 ‐ II . BMM were transfected with GRASP55 siRNAs or scramble ( Scr ) control . At 72 h post transfection , were induced for autophagy , treated or not with Bafilomycin A1 ( Baf ) to inhibit autophagic degradation LC3 ‐ II / actin ratios determined by immunoblotting ( A ) followed by densitometry ( B ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05.


( C , D ) RAW 264 . 7 was transfected with GRASP55 siRNAs or scramble ( Scr ) siRNA control . Following 48 h of siRNA treatment , were transfected with RFP - GFP - LC3 plasmid ( GFP is sensitive to acidification , whereas RFP is not ) , after 24 h induced for autophagy in EBSS for 1 h autophagic induction flux quantified ( graph in D ) by determining number of early autophagic organelles ( GFP + RFP + ) autolysosomal organelles ( GFP − RFP + ) per as illustrated in ( yellow arrows , GFP + RFP + ; red arrows , GFP − RFP + ) . Total , yellow + red per . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

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(C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP-GFP-LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.


( A ) Atg5fl / fl Cre − Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM release of HMGB1 was determined by immunoblotting . Figure source data can be found in Supplementary data .

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(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.


A Coolness stimulates intracellular cyclic guanosine monophosphate ( cGMP ) accumulation in HEK ‐ 293T expressing GC ‐ G . Two days after transfection of empty vector or GC ‐ G ‐ encoding plasmid , were exposed to ambient temperatures ( 37 or 15 ° C ) for 20 min cGMP was measured .

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A Coolness stimulates intracellular cyclic guanosine monophosphate (cGMP) accumulation in HEK‐293T cells expressing GC‐G. Two days after transfection of empty vector or GC‐G‐encoding plasmid, cells were exposed to ambient temperatures (37 or 15°C) for 20 min and cellular cGMP concentration was measured.


B dependence of GC ‐ G activity . preparations from HEK ‐ 293T transfected with empty vector or GC ‐ G ‐ encoding plasmid were used to determine cyclase activity at indicated temperatures for 20 min ( asterisks denote values significantly elevated compared to 37 ° C ) .

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B Temperature dependence of GC‐G activity. Membrane preparations from HEK‐293T cells transfected with empty vector or GC‐G‐encoding plasmid were used to determine cyclase activity at the indicated temperatures for 20 min (asterisks denote values significantly elevated compared to 37°C).


C GC ‐ G is only murine transmembrane GC that can be stimulated by cool temperatures . Expression plasmids encoding FLAG ‐ tagged GC subtypes GC ‐ A to GC ‐ G were transfected into HEK ‐ 293T . Two days after transfection , fractions were prepared used for cyclase activity assays at 37 or 15 ° C . expression of each receptor GC was confirmed by Western blot ( WB ) with anti ‐ FLAG antibody .

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C GC‐G is the only murine transmembrane GC that can be stimulated by cool temperatures. Expression plasmids encoding FLAG‐tagged GC subtypes GC‐A to GC‐G were transfected into HEK‐293T cells. Two days after transfection, membrane protein fractions were prepared and used for cyclase activity assays at 37 or 15°C. Protein expression of each receptor GC was confirmed by Western blot (WB) analysis with anti‐FLAG antibody.