M . Carfilzomib promotes M1 - polarization of associated macrophages in vitro . BMDMs were cultured with culture supernatant ( TSN ) produced by L1210 , followed by activation with DMSO or Carfilzomib ( 1 μM ) . was extracted from expression of Il - 1β , Il - 6 , Inos were quantified through RT - qPCR 6 hours after stimulation . data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

M. Carfilzomib promotes the M1-polarization of Tumor associated macrophages in vitro. BMDMs were cultured with tumor culture supernatant (TSN) produced by L1210 cells, followed by activation with DMSO or Carfilzomib (1 μM). RNA was extracted from cells and the expression of Il-1β, Il-6, Inos were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).


A , B . Carfilzomib proteasome inhibitor MG132 activate Il - 1β luciferase in macrophages . ( A ) BMDMs were treated with IL - 4 ( 20 ng / mL ) for 24 hours then stimulated by DMSO , Carfilzomib ( 1 μM ) , or MG132 ( 5 μM ) . ( B ) BMDMs were stimulated by DMSO , Carfilzomib ( 1 μM ) or MG132 ( 5 μM ) ) for 1 hour , then induced with IL - 4 ( 20 ng / mL ) . Luciferase assays were monitored 12 hours after stimulation . data are means ± SD of three independent experiments . * * * p < 0 . 001 ( student ' s t - ) .

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

A, B. Carfilzomib and proteasome inhibitor MG132 activate Il-1β luciferase in macrophages. (A) BMDMs were treated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, Carfilzomib (1 μM), or MG132 (5 μM). (B) BMDMs were stimulated by DMSO, Carfilzomib (1 μM) or MG132 (5 μM)) for 1 hour, then induced with IL-4 (20 ng/mL). Luciferase assays were monitored 12 hours after stimulation. The data are means ± SD of three independent experiments. ***p < 0.001 (student's t-test).


E . Carfilzomib , Bortezomib , MLN9708 MG132 induce ER stress response . BMDMs were pretreated with IL - 4 ( 20 ng / mL ) for 24 hours then stimulated by DMSO or Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . was extracted from 3 hours or 6 hours after stimulation expression of ER stress related genes ( Bip , Chop ) were detected through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

E. Carfilzomib, Bortezomib, MLN9708 and MG132 induce ER stress response. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO or Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 3 hours or 6 hours after stimulation and the expression of ER stress related genes (Bip, Chop) were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


F . ER stress agonists , TUN TG , promote expression of M1 macrophage markers , as well as reduce expression of M2 macrophage markers . BMDMs were pretreated with IL - 4 ( 20 ng / mL ) for 24 hours then stimulated by DMSO , TUN ( 500 nM ) or TG ( 500 nM ) . was extracted from 6 hours after stimulation indicated genes were quantified through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

F. ER stress agonists, TUN and TG, promote the expression of M1 macrophage markers, as well as reduce the expression of M2 macrophage markers. BMDMs were pretreated with IL-4 (20 ng/mL) for 24 hours and then stimulated by DMSO, TUN (500 nM) or TG (500 nM). RNA was extracted from cells 6 hours after stimulation and indicated genes were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


G . Inhibition of ER stress impairs ability of Carfilzomib , Bortezomib , MLN9708 MG132 to reprogram M2 towards M1 - like macrophages . BMDMs were induced by IL - 4 ( 20 ng / mL ) for 24 hours , then pretreated with 4 - PBA ( 5 mM ) for 1 hour stimulated by Carfilzomib ( 1μM ) , Bortezomib ( 1μM ) , MLN9708 ( 2 μM ) , MG132 ( 5 μM ) . was extracted from 6 hours after stimulation expression of Il - 1β , Il - 6 Inos were detected through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

G. Inhibition of ER stress impairs the ability of Carfilzomib, Bortezomib, MLN9708 and MG132 to reprogram M2 towards M1-like macrophages. BMDMs were induced by IL-4 (20 ng/mL) for 24 hours, then pretreated with 4-PBA (5 mM) for 1 hour and stimulated by Carfilzomib (1μM), Bortezomib (1μM), MLN9708 (2 μM), MG132 (5 μM). RNA was extracted from cells 6 hours after stimulation and the expression of Il-1β, Il-6 and Inos were detected through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


A , B Deficiency of Ern1 reduces expression of inflammatory related genes . Wild - type Ern1 - / - Raw264 . 7 ( A ) or BMDMs ( B ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . mRNA of Il - 1β Il - 6 were quantified through RT - qPCR 6 hours after stimulation . data are means ± SD of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

A, B Deficiency of Ern1 reduces the expression of inflammatory related genes. Wild-type and Ern1-/- Raw264.7 cells (A) or BMDMs (B) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). The mRNA of Il-1β and Il-6 were quantified through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 (student's t-test).


E . Kira6 inhibits expression of ER stress related genes inflammatory related genes . BMDMs was treated by IL - 4 ( 20 ng / mL ) for 24 hours , treated with Kira6 ( 100 nM ) for 1 hour , then stimulated by Carfilzomib ( 1 μM ) for 6 hours . was extracted from expression of Bip , Chop , Il - 1β Il - 6 were quantified through RT - qPCR . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - ) . F , G . Carfilzomib has no effect on splicing of XBP1 in M2 macrophages . BMDMs ( F ) or Raw264 . 7 ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated with DMSO , Carfilzomib ( 1 μM ) or TUN ( 500 nM ) . mRNA expression of sXBP1 unXBP1 were detected through RT - qPCR 6 hours after stimulation . data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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

E. Kira6 inhibits the expression of ER stress related genes and inflammatory related genes. BMDMs was treated by IL-4 (20 ng/mL) for 24 hours, treated with Kira6 (100 nM) for 1 hour, and then stimulated by Carfilzomib (1 μM) for 6 hours. RNA was extracted from cells and the expression of Bip, Chop, Il-1β and Il-6 were quantified through RT-qPCR. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test). F, G. Carfilzomib has no effect on splicing of XBP1 in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated with DMSO, Carfilzomib (1 μM) or TUN (500 nM). The mRNA expression of sXBP1 and unXBP1 were detected through RT-qPCR 6 hours after stimulation. The data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


J . Carfilzomib promotes association between IRE1α TRAF2 in M2 macrophages . Raw264 . 7 were pretreated by IL - 4 ( 20ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for 2 or 4 hours . Coimmunoprecipitation ( Co - IP ) IB were performed with indicated antibodies .

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J. Carfilzomib promotes the association between IRE1α and TRAF2 in M2 macrophages. Raw264.7 cells were pretreated by IL-4 (20ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for 2 or 4 hours. Coimmunoprecipitation (Co-IP) and IB were performed with indicated antibodies.


L . Carfilzomib activates NF - κB signaling pathway through IRE1α in M2 macrophages . Wild - type Ern1 - / - Raw264 . 7 were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by Carfilzomib ( 1 μM ) for indicated time points . WCL were analyzed by IB with indicated antibodies .

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

L. Carfilzomib activates NF-κB signaling pathway through IRE1α in M2 macrophages. Wild-type and Ern1-/- Raw264.7 cells were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by Carfilzomib (1 μM) for indicated time points. WCL were analyzed by IB with indicated antibodies.


C . Representative of Hematoxylin eosin ( H & E ) of lung tissue obtained from different treatment groups . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . D . Statistics of number of Ki67 positive in sections of lung tissue . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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C. Representative images of Hematoxylin and eosin (H&E) staining of the lung tissue obtained from different treatment groups. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. D. Statistics of the tumor area and the number of Ki67 positive cells in sections of lung tissue. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


E . Carfilzomib treatment promotes infiltration of M1 macrophages . bearing TD mice were treated as indicated in ( A ) euthanized . Lung tissues were dissected for evaluating M1 / M2 populations through analyze expression of CD80 CD206 by flow cytometry . F . Statistics represent proportion of CD80 or CD206 positive in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - ) . G . Carfilzomib treatment promotes infiltration activation of CD8 + T . Lung tissues of ( E ) were used for evaluating infiltration of CD8 + T their expression of CD69 by flow cytometry . H . Statistics about proportion of CD8 + or CD69 + positive in lung tissue . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - ) .

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E. Carfilzomib treatment promotes the infiltration of M1 macrophages. Tumor bearing TD mice were treated as indicated in (A) and euthanized. Lung tissues were dissected for evaluating M1/M2 populations through analyze the expression of CD80 and CD206 by flow cytometry. F. Statistics represent the proportion of CD80 or CD206 positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test). G. Carfilzomib treatment promotes the infiltration and activation of CD8+ T cells. Lung tissues of (E) were used for evaluating the infiltration of CD8+ T cells and their expression of CD69 by flow cytometry. H. Statistics about the proportion of CD8+ or CD69+ positive cells in lung tissue. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).


K , L . Carfilzomib shrinks through modulating microenvironment . EG7 were inoculated in wild - type or LyzM - cre ; lsl - dTA to allow xenograft to reach a volume of around 80 mm3 . Mice were randomized for treatment ( n = 5 ) by saline or Carfilzomib for two . xenografts were dissected to photograph ( K ) or weigh ( L ) after treatment . Data are shown as mean ± SD n indicates number of biological replicates . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - ) .

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K, L. Carfilzomib shrinks tumor through modulating tumor microenvironment. EG7 cells were inoculated in wild-type or LyzM-cre;lsl-dTA to allow tumor xenograft to reach a volume of around 80 mm3. Mice were randomized for treatment (n=5) by saline or Carfilzomib for two weeks. The xenografts were dissected to photograph (K) or weigh (L) after treatment. Data are shown as mean ±SD and n indicates the number of biological replicates. *p < 0.05, ***p < 0.001 (student's t-test).


D . CD4 + CD8 + T play a role in mediating cancer - shrinking effect of Carfilzomib . Lung - cancer - bearing TD mice were treated by saline , Carfilzomib , anti - mouse CD4 antibody ( ip . ) , anti - mouse CD8 antibody ( ip . ) , or Carfilzomib plus anti - mouse CD4 / 8 antibody . CT scanning was performed after two - week treatment . E . Representative of H & E of treated lung tissues . Expression of Ki - 67 in lung tissues was detected by immunohistochemistry method . F . Carfilzomib synergized with PD1 antibody in shrinking lung cancers . Lung cancer bearing TD mice were treated by saline , anti - PD1 antibody ( ip ) , Carfilzomib , or Carfilzomib plus anti - PD1 ( ip ) . CT scanning was performed after two - week treatment . G . Representative of H & E of lung tissues . Expression of Ki - 67 in lung tissue was detected by immunohistochemistry method .

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

D. CD4+ and CD8+ T cells play a role in mediating cancer-shrinking effect of Carfilzomib. Lung-cancer-bearing TD mice were treated by saline, Carfilzomib, anti-mouse CD4 antibody (ip.), anti-mouse CD8 antibody (ip.), or Carfilzomib plus anti-mouse CD4/8 antibody. CT scanning was performed after two-week treatment. E. Representative images of H&E staining of the treated lung tissues. Expression of Ki-67 in lung tissues was detected by immunohistochemistry method. F. Carfilzomib synergized with PD1 antibody in shrinking lung cancers. Lung cancer bearing TD mice were treated by saline, anti-PD1 antibody (ip), Carfilzomib, or Carfilzomib plus anti-PD1 (ip). CT scanning was performed after two-week treatment. G. Representative images of H&E staining of the lung tissues. Expression of Ki-67 in lung tissue was detected by immunohistochemistry method.


A . Carfilzomib , Bortezomib MLN9708 promote expression of M1 macrophage markers ( Il - 1β , Il - 6 , Tnfα ) reduce expression of M2 macrophage markers ( Il - 10 , Tgfβ ) in macrophages derived from PBMCs . After induced by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) or MLN9708 ( 2 μM ) . was extracted from 6 hours after stimulation expression of mRNA were quantified through RT - qPCR . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

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A. Carfilzomib, Bortezomib and MLN9708 promote the expression of M1 macrophage markers (Il-1β, Il-6, Tnfα) and reduce the expression of M2 macrophage markers (Il-10, Tgfβ) in macrophages derived from PBMCs. After induced by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (1 μM), Bortezomib (1 μM) or MLN9708 (2 μM). RNA was extracted from cells 6 hours after stimulation and the expression of mRNA were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01, ***p < 0.001 (student's t-test).


C - F . Carfilzomib , Bortezomib MLN9708 increase expression of CD80 ( C ) reduce expression of CD206 ( E ) in M2 macrophages derived from PBMCs . After activated by IL - 4 ( 20 ng / ml ) for 24 hours to differentiate into mature M2 macrophages , PBMCs derived macrophages were stimulated by Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . representative histogram of CD80 CD206 expression was analyzed by flow cytometry 12 hours after stimulation . ( D , F ) Statistics represent proportion of CD80 ( D ) or CD206 ( F ) positive in macrophages derived from PBMCs . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - ) .

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

C-F. Carfilzomib, Bortezomib and MLN9708 increase the expression of CD80 (C) and reduce the expression of CD206 (E) in M2 macrophages derived from PBMCs. After activated by IL-4 (20 ng/ml) for 24 hours to differentiate into mature M2 macrophages, PBMCs derived macrophages were stimulated by Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). The representative histogram of CD80 and CD206 expression was analyzed by flow cytometry 12 hours after stimulation. (D, F) Statistics represent the proportion of CD80 (D) or CD206 (F) positive cells in macrophages derived from PBMCs. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. *p < 0.05, **p < 0.01 (student's t-test).


( A , B ) Effect of MPC inhibition on lipid droplet content . ( A ) Live - super - resolution confocal imaging of brown adipocytes pre - stained overnight with fatty acid tracer BODIPY C12 558 / 568 ( Bodipy C12 , red ) . were stained with mitotracker green ( MTG , green ) prior to imaging . were imaged before 120 minutes after treatment with either vehicle ( DMSO ) or 100 nM UK5099 . As a positive control , were imaged 30 min after treatment with 1 µM norepinephrine . LD , lipid droplet ; N , nucleus . Scale bar = 10 µm . ( B ) of changes in lipid droplet cross - sectional with indicated treatments from shown in ( A ) . Data are represented as change in LD at time = 120 min compared to time = 0 min .

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(A,B) Effect of MPC inhibition on cellular lipid droplet content. (A) Live-cell super-resolution confocal imaging of primary brown adipocytes pre-stained overnight with the fatty acid tracer BODIPY C12 558/568 (Bodipy C12, red). Cells were stained with mitotracker green (MTG, green) prior to imaging. Cells were imaged before and 120 minutes after treatment with either vehicle (DMSO) or 100 nM UK5099. As a positive control, cells were imaged 30 min after treatment with 1 µM norepinephrine. LD, lipid droplet; N, nucleus. Scale bar=10 µm. (B) Quantification of changes in lipid droplet cross-sectional area with indicated treatments from images shown in (A). Data are represented as percentage change in LD area at time=120 min compared to time=0 min.


( C , D ) Effect of MPC inhibition on fatty acid uptake incorporation into triacylglycerides ( TAGs ) ( C ) Representative thin - layer chromatography ( TLC ) plate of lipids extracted from brown adipocytes culture media . were incubated with Bodipy C12 in presence of vehicle ( V ) , 100 nM UK5099 ( UK ) or 1 µM norepinephrine ( NE ) for 24 h triacylglyceride ( TAG ) synthesis ( Lipids in ) fatty acid uptake ( Lipids in media ) were detected . relative polarity of lipid species determines motility , with nonpolar TAG migrating furthest . ( D ) of C12 uptake from media , TAG in TAG per amount of free fatty acid uptake shown in ( C ) , n = 5 experiments . UK5099 treatment decreases amount of synthesized TAG per free fatty acid uptake , similarly to NE .

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(C,D) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides (TAGs) (C) Representative thin-layer chromatography (TLC) plate of lipids extracted from primary brown adipocytes and cell culture media. Cells were incubated with Bodipy C12 in presence of vehicle (V), 100 nM UK5099 (UK) or 1 µM norepinephrine (NE) for 24 h and triacylglyceride (TAG) synthesis (Lipids in cells) and fatty acid uptake (Lipids in media) were detected. The relative polarity of the lipid species determines the motility, with nonpolar TAG migrating the furthest. (D) Quantification of C12 uptake from media, TAG in cells and TAG per amount of free fatty acid uptake shown in (C), n=5 individual experiments. UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake, similarly to NE.


( E - G ) Effect of UK5099 treatment on energy expenditure . Fully differentiated brown adipocytes were pre - treated with vehicle ( DMSO ) or UK5099 at indicated concentrations for 2 h . Oxygen consumption rates ( OCR ) were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle or UK - 5099 . ( E ) of non - stimulated OCR from n = 3 - 7 experiments . Data were normalized to vehicle for each experiment . UK5099 increases OCR in non - stimulated brown adipocytes . vehicle . ( F ) Brown adipocytes were treated with 100 nM UK5099 or vehicle . Etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . Representative OCR traces averaging 6 technical replicates . ( G ) of non - stimulated OCR , etomoxir sensitive , etomoxir insensitive OCR from n = 4 experiments . Data were normalized to vehicle for each experiment . *

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(E-G) Effect of UK5099 treatment on cellular energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or UK5099 at indicated concentrations for 2 h. Oxygen consumption rates (OCR) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK-5099. (E) Quantification of non-stimulated OCR from n=3-7 individual experiments. Data were normalized to vehicle for each individual experiment. UK5099 increases OCR in non-stimulated brown adipocytes. vehicle. (F) Brown adipocytes were treated with 100 nM UK5099 or vehicle. Etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. Representative OCR traces averaging 6 technical replicates. (G) Quantification of non-stimulated OCR, etomoxir sensitive, and etomoxir insensitive OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. *


( H , I ) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid . ( H ) Schematic representation of metabolite tracing using [ U - 13C16 ] palmitate . ( I ) [ U - 13C16 ] palmitate tracing in fully differentiated brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h . Data shows mole enrichment ( MPE ) of isotope labeled substrate into respective metabolite .

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(H,I) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid. (H) Schematic representation of metabolite tracing using [U-13C16] palmitate. (I) [U-13C16] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h. Data shows mole percent enrichment (MPE) of isotope labeled substrate into respective metabolite.


( A , B ) Effect of UK5099 treatment on norepinephrine - stimulated energy expenditure . Fully differentiated brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 4 technical replicates . ( B ) of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak Eto - sensitive OCR as shown in ( A ) from n = 9 experiments . effects of UK5099 treatment were normalized to vehicle for each experiment .

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(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment.


brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble ( Scramble ) . ( C ) Representative Western Blot of MPC1 , MPC2 actin . ( D ) of MPC1 MPC2 expression normalized to actin from Western Blots in ( C ) from n = 3 experiments .

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Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (C) Representative Western Blot analysis of MPC1, MPC2 and actin. (D) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in (C) from n=3 individual experiments.


brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble ( Scramble ) . ( E ) mRNA of MPC1 MPC2 from n = 3 experiments .

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Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (E) mRNA levels of MPC1 and MPC2 from n=3 individual experiments.


brown adipocytes were transfected with MPC1 siRNA ( MPC1 KD ) or Scramble ( Scramble ) . ( F ) Representative OCR traces of differentiated brown adipocytes averaging 6 technical replicates . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( G ) of basal , NE - stimulated Eto - sensitive OCR as shown in ( F ) from n = 7 experiments . Data were normalized to Scramble for each experiment . ( H ) of basal OCR in response to 100 nM UK5099 treatment in Scramble of MPC1 siRNA transfected from n = 5 experiments . Data were normalized to vehicle for each experiment .

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Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment.


( A , B ) Requirement for glutamine in UK5099 - induced energy expenditure . brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 h . OCR were measured in presence of either 5 mM glucose 3 mM glutamine or 5 mM glucose alone . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak etomoxir - sensitive OCR after vehicle or UK5099 treatment as shown in ( A ) from n = 4 experiments .

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(A,B) Requirement for glutamine in UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and etomoxir-sensitive OCR after vehicle or UK5099 treatment as shown in (A) from n=4 individual experiments.


( C ) Effect of glutaminase inhibitor on UK5099 - induced energy expenditure . brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , or 100 nM UK5099 + 2 . 5 µM CB839 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle , UK5099 or UK5099 + CB839 from n = 5 experiments . Data were normalized to vehicle for each experiment .

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(C) Effect of glutaminase inhibitor on UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, or 100 nM UK5099 + 2.5 µM CB839 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099 or UK5099 + CB839 from n=5 individual experiments. Data were normalized to vehicle for each experiment.


( D ) Effect of MPC inhibition on glutamate / aspartate measurement . Glutamate / aspartate was quantified using GC - MS as a measure of glutamine catabolism . brown adipocytes were treated for 24 h with vehicle ( DMSO ) or 10 µM UK5099 from n = 6 experiments . * *

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(D) Effect of MPC inhibition on glutamate/aspartate ratio measurement. Glutamate/aspartate ratio was quantified using GC-MS as a measure of glutamine catabolism. Primary brown adipocytes were treated for 24 h with vehicle (DMSO) or 10 µM UK5099 from n=6 individual experiments. **


( B ) Effect of transaminase inhibition on UK5099 - induced energy expenditure . Brown adipocytes were pre - treated with vehicle ( DMSO ) , 50 - 100 nM UK5099 , 1 mM aminooxyacetic acid ( AOA ) , or a combination of UK5099 AOA for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of tested compounds . Data show of non - stimulated OCR from n = 5 experiments .

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(B) Effect of transaminase inhibition on UK5099-induced energy expenditure. Brown adipocytes were pre-treated with vehicle (DMSO), 50-100 nM UK5099, 1 mM aminooxyacetic acid (AOA), or a combination of UK5099 and AOA for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of the tested compounds. Data show quantification of non-stimulated OCR from n=5 individual experiments.


( C - E ) Effect OGC1 downregulation on UK5099 - induced energy expenditure . brown adipocytes were transduced with Scramble ( Scramble ) or shOGC1 ( OGC1 KD ) adenovirus . were pre - treated for 2 h with vehicle ( DMSO ) or 100 nM UK5099 before OCR measurements . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM , ) oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( C ) Representative OCR traces averaging 4 technical replicates . ( D ) of non - stimulated OCR as measured in ( C ) from n = 6 experiments . ( E ) of Eto - sensitive OCR as measured in ( C ) from n = 6 experiments . Data were normalized to vehicle for each experiment .

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(C-E) Effect OGC1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transduced with Scramble RNA (Scramble) or shOGC1 (OGC1 KD) adenovirus. Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM,) oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (C) Representative OCR traces averaging 4 technical replicates. (D) Quantification of non-stimulated OCR as measured in (C) from n=6 individual experiments. (E) Quantification of Eto-sensitive OCR as measured in (C) from n=6 individual experiments. Data were normalized to vehicle for each experiment.


( F - H ) Effect of Aralar1 downregulation on UK5099 - induced energy expenditure . brown adipocytes were transfected with Scramble ( Scramble ) or siRNA for Aralar1 ( Aralar1 KD ) . were pre - treated for 2 h with vehicle ( DMSO ) or 100 nM UK5099 before OCR measurements . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin a ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) antimycin a ( AA ; 4 µM ) were injected where indicated . ( F ) Representative OCR traces averaging 4 technical replicates . ( G ) of non - stimulated OCR as measured ( G ) from n = 4 experiments . Data were normalized to vehicle for each experiment . ( H ) of Eto - sensitive OCR as measured in ( F ) from n = 4 experiments .

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(F-H) Effect of Aralar1 downregulation on UK5099-induced energy expenditure. Primary brown adipocytes were transfected with Scramble RNA (Scramble) or siRNA for Aralar1 (Aralar1 KD). Cells were pre-treated for 2 h with vehicle (DMSO) or 100 nM UK5099 before OCR measurements. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin a (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin a (AA; 4 µM) were injected where indicated. (F) Representative OCR traces averaging 4 technical replicates. (G) Quantification of non-stimulated OCR as measured (G) from n=4 individual experiments. Data were normalized to vehicle for each experiment. (H) Quantification of Eto-sensitive OCR as measured in (F) from n=4 individual experiments.


( I ) Brown adipocytes transfected with Scramble or Aralar1 siRNA were treated for 24 hours with vehicle ( DMSO ) or 10 µM UK5099 . Data shows of of aspartate to glutamate abundance as measured by GC - MS from n = 3 experiments .

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(I) Brown adipocytes transfected with Scramble RNA or Aralar1 siRNA were treated for 24 hours with vehicle (DMSO) or 10 µM UK5099. Data shows quantification of the ratio of aspartate to glutamate abundance as measured by GC-MS from n=3 individual experiments.


( A , B ) Effect of UK5099 on energy expenditure contributed by ATP demand by mitochondrial proton leak in non - stimulated brown adipocytes . brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 hours . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle or UK5099 . Oligomycin A ( Oligo ; 4 µM ) , mitochondrial uncoupler FCCP ( 2 µM ) antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 6 technical replicates . ( B ) of mitochondrial proton leak ATP - linked OCR ( oligomycin sensitive ) as measured in ( A ) from n = 9 experiments . Data were normalized to vehicle for each experiment .

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(A,B) Effect of UK5099 on energy expenditure contributed by ATP demand and by mitochondrial proton leak in non-stimulated brown adipocytes. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 hours. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Oligomycin A (Oligo; 4 µM), mitochondrial uncoupler FCCP (2 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of mitochondrial proton leak and ATP-linked OCR (oligomycin sensitive) as measured in (A) from n=9 individual experiments. Data were normalized to vehicle for each individual experiment.


( C ) To determine contribution of ATP demand to increased energy expenditure under UK5099 were permeabilized ADP concentrations were clamped . OCR were measured in presence of 5 mM pyruvate , 3 mM malate , 0 . 1 mM palmitoyl - CoA , 0 . 5 mM carnitine 5 mM ADP . were treated with either 100 nM , 1 µM or 10 µM UK5099 . Data shows maximal state 3 OCR normalized to maximal 4 activity from n = 3 experiments .

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(C) To determine the contribution of ATP demand to increased energy expenditure under UK5099 cells were permeabilized and cytosolic ADP concentrations were clamped. OCR were measured in the presence of 5 mM pyruvate, 3 mM malate, 0.1 mM palmitoyl-CoA, 0.5 mM carnitine and 5 mM ADP. Cells were treated with either 100 nM, 1 µM or 10 µM UK5099. Data shows maximal state 3 OCR normalized to maximal complex 4 activity from n=3 individual experiments.


( E ) Effect of MPC inhibition on glycerol 3 - phosphate ( G3P ) dihydroxyacetone phosphate ( DHAP ) generation . G3P / DHAP is used as a measure of acylglycerol synthesis . Brown adipocytes were treated with either vehicle , 100 nM UK5099 or 1 µM norepinephrine ( NE ) for 24 h from n = 4 experiments .

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(E) Effect of MPC inhibition on glycerol 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) generation. G3P/DHAP is used as a measure of acylglycerol synthesis. Brown adipocytes were treated with either vehicle, 100 nM UK5099 or 1 µM norepinephrine (NE) for 24 h from n=4 individual experiments.


( F , G ) Contribution of lipolysis to UK5099 induced energy demand . brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle , UK5099 , or Atglistatin . ( F ) of non - stimulated OCR from n = 4 experiments . ( G ) of ATP - linked respiration from n = 4 experiments . Data were normalized to vehicle for each experiment .

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(F,G) Contribution of lipolysis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 40 µM Atglistatin or a combination of Atglistatin with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, or Atglistatin. (F) Quantification of non-stimulated OCR from n=4 individual experiments. (G) Quantification of ATP-linked respiration from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.


( H , I ) Contribution of TAG synthesis to UK5099 induced energy demand . brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 1 µM DGAT1 1 µM DGAT2 or a combination of DGAT1 / 2 inhibitors with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle , UK5099 , DGAT1 / 2 inhibitors , or UK5099 + DGAT1 / 2 inhibitors . ( H ) of non - stimulated OCR from n = 6 experiments . ( I ) of ATP - linked OCR from n = 6 experiments . Data were normalized to vehicle for each experiment .

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(H,I) Contribution of TAG synthesis to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 1 µM DGAT1 and 1 µM DGAT2 or a combination of DGAT1/2 inhibitors with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, DGAT1/2 inhibitors, or UK5099 + DGAT1/2 inhibitors. (H) Quantification of non-stimulated OCR from n=6 individual experiments. (I) Quantification of ATP-linked OCR from n=6 individual experiments. Data were normalized to vehicle for each individual experiment.


( J , K ) Contribution of ACS to UK5099 induced energy demand . brown adipocytes were pre - treated with either vehicle ( DMSO ) , 100 nM UK5099 , 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose 3 mM glutamine in presence of vehicle , UK5099 , Triacsin C , or UK5099 + Triacsin C . ( J ) of non - stimulated OCR from n = 4 experiments . ( K ) of ATP - linked OCR from n = 4 experiments . Data were normalized to vehicle for each experiment .

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(J,K) Contribution of ACS to UK5099 induced energy demand. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, 5 µM Triacsin C or a combination of 5 µM Triacsin C with UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099, Triacsin C, or UK5099 + Triacsin C. (J) Quantification of non-stimulated OCR from n=4 individual experiments. (K) Quantification of ATP-linked OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment.


E - H Whole mount stereomicrographs of carmine alum stained mammary either uninjected ( E ) , 2 months after intraductal injection with MCF7 , arrows point to undilated tips ( F ) , MM134 , dotted highlight subtending ( G ) , or SUM44 ( H ) , ( n > 6 ) scale bar , 1 mm .

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E-H Whole mount stereomicrographs of carmine alum stained mammary glands either uninjected (E), 2 months after intraductal injection with MCF7, arrows point to undilated ductal tips (F), MM134, dotted line highlight the subtending duct (G), or SUM44 cells (H), (n>6) scale bar, 1 mm.


I Ex vivo luminescence of bones , brain , lungs liver from MM134 engrafted mice 1 month post injection ( top ) ovaries ( bottom ) 12 months post injection . Scale bar , 1 cm . J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 SUM44 plotted over organ time of . Number of mice analyzed MM134 SUM44 for 30 - 180 : N = 5 , 10 , 180 - 270 days : N = 9 , 5 , 270 - 360 days : N = 14 , 20 , respectively . Dashed indicates background .

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I Ex vivo luminescence images of bones, brain, lungs and liver from MM134 engrafted mice 1 month post injection (top) and ovaries (bottom) 12 months post injection. Scale bar, 1 cm. J Dot plot showing ex vivo bioluminescence of different organs from mice xenografted with MM134 and SUM44 cells plotted over organ and time of analysis. Number of mice analyzed MM134 and SUM44 for 30-180: N=5, 10, 180-270 days: N=9, 5, 270-360 days: N=14, 20, respectively. Dashed line indicates background levels.


E , F Representative of for Ki67 , ER , PR on histological sections of MM134 ( E ) SUM44 ( F ) xenografts 5 months after intraductal injection counter stained with hematoxylin . Scale bars , 200 μm . Bar plots represent means ± of measurements indicate of positive for > 1000 counted on 3 .

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E, F Representative micrographs of IHC for Ki67, ER, and PR on histological sections of MM134 (E) and SUM44 (F) xenografts 5 months after intraductal injection counter stained with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands.


G Representative of PR - on from 5 - month - old MM134 xenografts from hosts treated for 2 months with vehicle ( left ) or E2 ( right ) pellets . Scale bars , 200 μm . Right , bar plot showing of PR + in MM134 SUM44 xenografts with n > 1000 per condition on from 3 different mice . Data represent mean ± unpaired , Student ' s t - , two - ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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G Representative micrographs of PR-IHC on glands from 5-month-old MM134 xenografts from hosts treated for 2 months with vehicle (left) or E2 (right) pellets. Scale bars, 200 μm. Right, bar plot showing percentage of PR+ cells in MM134 and SUM44 xenografts with n>1000 cells per condition on glands from 3 different mice. Data represent mean ± SEM unpaired, Student's t-test, two-tailed; *** p < 0.001, **** p < 0.0001.


D Representative RNAscope image of sections from ILCs ( n = 5 ) stained with anti - LOXL1 probes ( red ) counterstained with DAPI ( blue ) . Scale bars ; 50 μm .

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D Representative RNAscope image of sections from patient ILCs (n=5) stained with anti-LOXL1 probes (red) and counterstained with DAPI (blue). Scale bars; 50 μm.


E Representative immunofluorescence image of ILC ( n = 3 ) stained with anti - LOXL1 ( green ) anti - ER ( red ) antibodies counterstained with DAPI ( blue ) . Scale bars ; 50 μm right inset 30 μm .

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E Representative immunofluorescence image of primary ILC (n=3) stained with anti-LOXL1 (green) and anti-ER (red) antibodies counterstained with DAPI (blue). Scale bars; 50 μm and right inset 30 μm.


E Radiance - growth curves ( mean ± SE ) for T47D treated with vehicle ( n = 8 ) or BAPN ( n = 12 ) 8 days after intraductal injections . n . s not significant .

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E Radiance-based tumor growth curves (mean ± SE) for T47D treated with vehicle (n=8) or BAPN (n=12) 8 days after intraductal injections. n.s not significant.


F Radiance - growth curves for ( mean ± SE ) ILC ER + PDXs T125 T137 treated with vehicle ( n = 6 , 7 ) or BAPN ( n = 7 , 8 ) 66 days after engraftment . * * p < 0 . 01 .

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F Radiance-based tumor growth curves for (mean ± SE) ILC ER+ PDXs T125 and T137 treated with vehicle (n=6, 7) or BAPN (n=7, 8) 66 days after engraftment. ** p < 0.01.


G Radiance - growth curves ( mean ± SE ) for non - ILC ER + PDXs T99 T157 treated with vehicle ( n = 7 , 7 ) or BAPN ( n = 8 , 12 ) , 8 days after intraductal injection 3 after intraductal injections . n . s not significant .

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G Radiance-based tumor growth curves (mean ± SE) for non-ILC ER+ PDXs T99 and T157 treated with vehicle (n=7, 7) or BAPN (n=8, 12), 8 days after intraductal injection 3 weeks after intraductal injections. n.s not significant.


E Representative stereo - of injected with MM134 : RFP - luc2 ( left , top bottom ) infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 ( bottom ) , n = 5 3 . Scale bar , 1000 μm . F Barplot showing of GFP / RFP signal from stereo - of engrafted with MM134 , n = 8 . Statistical by paired Student t - , two - . * p < 0 . 05 .

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E Representative fluorescent stereo-micrographs of glands injected with MM134:RFP-luc2 cells (left, top and bottom) infected with either GFP:sh-scramble (top) or GFP+ shLOXL1 (bottom), n=5 and 3. Scale bar, 1000 μm. F Barplot showing the ratio of GFP/RFP signal from fluorescent stereo-micrographs of glands engrafted with MM134 cells, n=8. Statistical analysis by paired Student t-test, two-tailed. *p < 0.05.


H Representative stereo - of engrafted with SUM44 : RFPluc2 infected with either GFP : sh - scramble ( top ) or GFP + shLOXL1 , n = 6 . Scale bar , 1000 μm . I Barplot ( mean ± ) showing of GFP / RFP from stereo - of SUM44 . Statistical by unpaired Student ' s t - , two - , n = 7 sh - scramble n = 6 for sh - LOXL1 , * * * * p < 0 . 0001 .

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H Representative fluorescent stereo-micrographs of glands engrafted with SUM44:RFPluc2 cells infected with either GFP:sh-scramble (top) or GFP+ shLOXL1, n=6. Scale bar, 1000 μm. I Barplot (mean ± SEM) showing quantification of the ratio GFP/RFP from fluorescent stereo-micrographs of SUM44. Statistical analysis by unpaired Student's t-test, two-tailed, n=7 sh-scramble and n=6 for sh-LOXL1, ****p < 0.0001.


J , K Representative photomicrographs of picrosirius red stained histological sections for sh - scramble or sh - LOXL1 MM134 ( J ) SUM44 ( K ) xenografts . Scale bars , 100 μm .

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J, K Representative photomicrographs of picrosirius red stained histological sections for sh-scramble or sh-LOXL1 MM134 (J) and SUM44 (K) xenografts. Scale bars, 100 μm.


A - C . SPF C57BL / 6Jmice were co - colonized with E1 N1 wild type , ( WT , A , n = 5 mice ) N1 T6SS mutant , ( ΔtssC , B , n = 4 ) or N1 complemented ( ΔtssC pTssC , C , n = 5 ) . FecalCFU was quantified for E1 ( closed squares ) N1 ( open squares ) weekly .

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A-C. SPF C57BL/6Jmice were co-colonized with E1 and N1 wild type, (WT, A, n=5 mice) N1 T6SS mutant, (ΔtssC, B, n=4) or N1 complemented (ΔtssC pTssC, C, n=5). FecalCFU was quantified for E1 (closed squares) and N1 (open squares) weekly.


D . Four post - colonization , E1 fecal recovery was compared between N1 WT , ΔtssC , ΔtssC pTssC groups .

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D. Four weeks post-colonization, E1 fecal recovery was compared between the N1 WT, ΔtssC, and ΔtssC pTssC groups.


B - E . Co - colonization of N1 WT ( B D , n = 4 mice ) or N1 Δbte2 ( C , n = 4 ) with E1 WT ( B C ) or E1 overexpressing Bti2a ( E1 pBti2a , D ) . FecalCFU was monitored over time ( B - D ) E1 CFU compared to N1 WT - E1 WT group at four - post co - colonization ( E ) .

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B-E. Co-colonization of N1 WT (B and D, n=4 mice) or N1 Δbte2 (C, n=4) with E1 WT (B and C) or E1 overexpressing Bti2a (E1 pBti2a, D). FecalCFU was monitored over time (B-D) and E1 CFU compared to N1 WT-E1 WT group at four-weeks post co-colonization (E).


A B . Initial colonization of gnotobiotic ( A ) or SPF ( B ) mice ( n = 4 mice per group ) with N1 followed by secondary challenge with N1 ( closed squares ) or E1 ( open squares ) . FecalCFU was determined for secondary colonization strains through four post - secondary challenges .

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A and B. Initial colonization of gnotobiotic (A) or SPF (B) mice (n=4 mice per group) with N1 followed by secondary challenge with N1 (closed squares) or E1 (open squares). FecalCFU was determined for the primary and secondary colonization strains through four weeks post-secondary challenges.


A - F . colonization of SPF mice with N2 WT , T6SS mutant ( ΔtssC ) complemented ( ΔtssC pTssC ) followed by secondary challenge with N1 WT ( A B , n = 5 mice ) N2 WT ( C D , n = 5 ) or E1 WT ( E F , n = 4 ) was performed . A , C E . FecalCFU for secondary strains was determined for four post - secondary challenge . B , D F . Selected time points were tested for statistical difference of secondary challenge between groups . This includes four post - secondary challenge ( B ) three days post - challenge ( D F ) .

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A-F. Primary colonization of SPF mice with N2 WT, T6SS mutant (ΔtssC) and complemented (ΔtssC pTssC) followed by secondary challenge with N1 WT (A and B, n=5 mice) N2 WT (C and D, n=5) or E1 WT (E and F, n=4) was performed.A, C and E. FecalCFU for primary and secondary strains was determined for four weeks post-secondary challenge.B, D and F. Selected time points were tested for statistical difference of secondary challenge between groups. This includes four weeks post-secondary challenge (B) and three days post-challenge (D and F).


A . Mice were co - colonized with E1 either N1 WT ( n = 4 ) or N1 ΔtssC ( n = 3 ) . Five days post - inoculation , fecal was extracted tested for BFT expression via qRT - PCR .

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A. Mice were co-colonized with E1 and either N1 WT (n=4) or N1 ΔtssC (n=3). Five days post-inoculation, fecal RNA was extracted and tested for BFT expression via qRT-PCR.


B . Four after co - colonization with E1 either N1 WT or N1 ΔtssC ( n = 4 mice per group ) , sera were collected , tested via ELISA for anti - BFT IgG end point titer calculated .

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B. Four weeks after co-colonization with E1 and either N1 WT or N1 ΔtssC (n= 4 mice per group), the sera were collected, tested via ELISA for anti-BFT IgG and end point titer calculated.


C - F . Mice pre - treated with DSS were inoculated with no organisms ( sham ) , E1 only , or E1 competed with N1 WT or N1 ΔtssC . Five days post - inoculation , ceca were weighed ( C ) fixed for histopathological examination after sham ( D ) , E1 only ( E ) E1 - N1 WT ( F ) colonizations . Scale bars denote 100μm ( main image ) 200μm ( inset ) .

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C-F. Mice pre-treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post-inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1-N1 WT (F) colonizations. Scale bars denote 100μm (main image) and 200μm (inset).


In vitro niche dependency of HGSOC . Phase - contrast pictures illustrate that isolated ovarian cancer rely on EGF supplementation for growth , while they do not grow at in Wnt3a supplemented medium . Also , inhibition of BMP signaling through Noggin has strong negative effect on initial growth . Scale bar : 500 µm . E - EGF , F - FGF10 , N - Noggin , R - R - spondin1 , B - Basic medium , P -

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In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - Passage


Cancer organoids express HGSOC markers Pax8 EpCAM have lost cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal from two representative organoid lines . Scale bar : 20 µm .

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Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm.


HE of organoids respective tissue confirm high similarity in structure tissue organization . Scale bar : 100 µm .

HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm.


HGSOC organoids show differential response to carboplatin treatment , confirming - specific sensitivity of cultures . viability was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors . Data represent mean ± SD of technical triplicates .

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HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.


Overview of mutations found in organoid cultures matching tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue in vitro long - term organoid culture . Color code indicates type of mutations which were detected .

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Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected.


Representative western blot showing loss or overexpression of p53 in organoid lines .

Representative western blot showing loss or overexpression of p53 in individual organoid lines.


Strong p53 signal in immunostainings of HGSOC organoids is indicative of a gain - of - function TP53 mutation . Scale bars : 100 µm ( Tissue ) 50 µm ( Organoid ) .

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Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).


Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids parental tissue . Data represent median , quartiles , maximum , minimum of normalized expression from 8 different donors .

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Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors.


Heat map of differentially expressed genes between cancer healthy tissue / organoids reveals upregulation of several HGSOC biomarkers reduction of FT differentiation markers in cancer samples . Differential expression determined by single - color microarray for 8 different samples was significant for genes with p < 0 . 05 except for OVGP1 TOP2A in organoids .

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Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.


Experimental approach for genetic manipulation of FT epithelial ( FTECs ) . FTECs were sequentially transduced in 2D culture with replication - deficient containing specific shRNAs different selection markers . After sorting were seeded in Matrigel for organoid formation . Scale bars : 2 cm ( tissue ) , 200 µm ( 2D culture ) 500 µm ( 3D culture ) . Confirmation of robust knockdowns of p53 , PTEN RB in FT organoids on level . Relative mRNA were normalized to WT mRNA level are given as mean ± from 3 different donors . A representative western blot from one donor is depicted . as well as samples were taken at passage 1 or 2 of each organoid . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

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Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture). Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type


Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot .

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Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis.


Confocal of triple KD organoids reveal increased damage ( yH2AX , marked by asterisks ) , atypic nuclei ( DRAQ5 ) loss of apicobasal polarity . Scale bar : 20µm .

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Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.


Confirmation of robust knockdowns of p53 , PTEN RB in FT organoids on level . Relative mRNA were normalized to WT mRNA level are given as mean ± from 3 different donors . A representative western blot from one donor is depicted . as well as samples were taken at passage 1 or 2 of each organoid . KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot . Presence of ciliated ( arrowheads ) , as revealed by immunostaining against detyrosinated Tubulin ( dtyrTub ) in triple KD organoids , proves capacity for terminal differentiation . Scale bar : 50µm .

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Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis. Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.


In long - term 3D culture triple KD organoids show loss of growth capacity premature growth arrest compared to controls as indicated by representative growth curves of FT234 . Scale bar : 500 µm .

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In long-term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234. Scale bar: 500 µm.


Growth curves phase - contrast of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long - term growth capacity . Scale bar : 500 µm .

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Growth curves and phase-contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long-term growth capacity. Scale bar: 500 µm.


of damage ( yH2AX ) apoptosis ( caspase - 3 , PARP1 ) do not differ among different media as shown by representative western blots comparing triple KD organoids their controls .

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Levels of DNA damage (yH2AX) and apoptosis (cleaved caspase-3, PARP1) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls.


Triple KD ( p53 / PTEN / RB ) organoids are characterized by a HGSOC gene expression signature as revealed by microarray comparing WT KD organoids grown in either OCM or FTM . Differential expression determined by dual - color microarray for 2 biological replicates was significant for genes with p < 0 . 00005 .

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Triple KD (p53/PTEN/RB) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.00005.


Differential expression of stemness - differentiation - related genes of triple KD organoids grown in OCM vs . FTM . Fold - changes were derived from microarray data indicate an increase in stemness ( CD133 , SOX2 , MYCN ) a drop in differentiation ( OVGP1 , PGR , FOXJ1 ) under OCM conditions . Differential expression determined by dual - color microarray for 2 biological replicates was significant for genes with p < 0 . 05 .

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Differential expression of stemness- and differentiation-related genes of triple KD organoids grown in OCM vs. FTM. Fold-changes were derived from microarray data and indicate and an increase in stemness (CD133, SOX2, MYCN) and a drop in differentiation (OVGP1, PGR, FOXJ1) under OCM conditions. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.05.


FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM , suggesting a decrease in differentiation capacity . Data represent mean ± from technical triplicates for two independent knockdown cultures .

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FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM, suggesting a decrease in differentiation capacity. Data represent the mean ±SEM from technical triplicates for two independent knockdown cultures.


Proportion of ciliated ( detyrosinated Tubulin ; dtyrTub ) is significantly reduced in KD organoids under OCM growth conditions , confirming inhibition of differentiation as illustrated by confocal ( Scale bar : 50 µm ) . plot depicts mean ± from 3 independent biological replicates , on of average number of ciliated per counted nuclei . 1000 were counted per experiment . * p < 0 . 05 , two - sided Student ' s t - .

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Proportion of ciliated cells (detyrosinated Tubulin; dtyrTub) is significantly reduced in KD organoids under OCM growth conditions, confirming inhibition of differentiation as illustrated by confocal images (Scale bar: 50 µm). The quantification plot depicts the mean ±SEM from 3 independent biological replicates, based on quantification of the average number of ciliated cells per counted nuclei. 1000 cells were counted per individual experiment. *p < 0.05, two-sided Student's t-test.


proportion of CD133 + was determined by FACS after growing triple KD control in FTM vs . OCM for two passages . graph is representative of two biological replicates .

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The proportion of CD133+ cells was determined by FACS analysis after growing triple KD and control cells in FTM vs. OCM for two passages. The graph is representative of two biological replicates.


Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable number ( representative graph of 3 independent experiments ) . luminescent viability was performed in triplicate bar plot depicts mean ± . * p < 0 . 05 , two - sided Student ` s t - ; KD - Knockdown , Vec Ctrl - Vector Control , WT - Wild

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Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number (representative graph of 3 independent experiments). The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ±SEM. * p < 0.05, two-sided Student`s t-test; KD - Knockdown, Vec Ctrl - Vector Control, WT - Wild type


Phase - contrast of cancer organoids which entered growth arrest upon treatment with Wnt agonists ( Wnt3a / RSPO1 ) over 2 passages ( Scale bar : 500µm ) . Differences in number were confirmed by respective viability assays ( performed in technical triplicates ) . Data represent mean ± for 5 different OC organoid lines ( n = 5 ) . * * * p < 0 . 001 , two - sided Student ` s t - .

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Phase-contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists (Wnt3a/RSPO1) over 2 passages (Scale bar: 500µm). Differences in cell number were confirmed by the respective cell viability assays (performed in technical triplicates). Data represent the mean ±SEM for 5 different OC organoid lines (n=5). *** p < 0.001, two-sided Student`s t-test.


number of CD133 + determined by FACS for OC7 OC4 organoids grown in different media with or without Wnt3a / RSPO1 with or without Noggin over 2 passages , confirms a sharp drop in stemness in presence of Wnt agonists .

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The number of CD133+ cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a/RSPO1 and with or without Noggin over 2 passages, confirms a sharp drop in stemness in the presence of Wnt agonists.


Addition of conditioned medium collected from parental Wnt3a did not suppress growth of cancer organoids , proving that growth inhibitory effect is due to presence of Wnt agonists . bar plot represents mean ± of triplicates performed with a luminescent viability . * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ` s t - .

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Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids, proving that the growth inhibitory effect is due to the presence of Wnt agonists. The bar plot represents the mean ±SEM of triplicates performed with a luminescent cell viability assay. * p < 0.05, *** p < 0.001, two-sided Student`s t-test.


Comparative of microarray data revealed consistent up - regulation of MYCN , canonical Wnt inhibitors KREMEN2 DKK3 as well as differentiation marker FOXJ1 , AR PGR in HGSOC cancer organoids as well as triple KD FT organoids . Differential expression was determined either by single - color microarray for cancer samples ( 8 replicates ) or dual - color microarray for knockdowns ( 2 replicates ) is significant for genes with p < 0 . 05 .

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Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05.


Transcriptional induction of MYCN is confirmed by qPCR for three between cancer organoids normal FT organoids as well as triple KD organoids grown in OCM vs . FTM conditions . Depicted is mean ± of normalized MYCN mRNA expression level of three biological replicates ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 , two - sided Student ` s t - .

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Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test.


Microarray data revealed that expression of Wnt target genes are downregulated in triple KD compared to WT control FT organoids . Differential expression determined by dual - color microarray for 2 biological replicates was significant for genes with p ≤ 0 . 005 .

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Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005.


FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR of 3 different samples . Data represent mean ± of technical triplicates .

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FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.


A Co ‐ localization of endogenous TBC1D5 VPS35 with mCherry ‐ ATG9A , in steady state autophagy induced by mTOR inhibitor KU0063794 ( 6 h ) .

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A Co‐localization of endogenous TBC1D5 and VPS35 with mCherry‐ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h).


B , C of co ‐ localization experiments presented in ( A ) .

B, C Quantification of co‐localization experiments presented in (A).


D Co ‐ immunoprecipitation of HA ‐ Flag ‐ TBC1D5 with myc ‐ VPS29 , transiently overexpressed in 293T , DMSO or KU0063794 ( 6 h ) treated .

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D Co‐immunoprecipitation of HA‐Flag‐TBC1D5 with myc‐VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated.


E , F Co ‐ immunoprecipitation of endogenous TBC1D5 ATG9A with myc ‐ VPS29 ( E ) transiently overexpressed in 293T or with HA ‐ Flag ‐ VPS35 ( F ) transiently overexpressed in 293T , treated with DMSO or KU0063794 ( 6 h ) .

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E, F Co‐immunoprecipitation of endogenous TBC1D5 and ATG9A with myc‐VPS29(E) transiently overexpressed in 293T cells or with HA‐Flag‐VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h).


G Co ‐ immunoprecipitation of endogenous VPS29 ATG9A with HA ‐ Flag ‐ TBC1D5stably expressed in 293T , treated with DMSO or KU0063794 ( 6 h ) .

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G Co‐immunoprecipitation of endogenous VPS29 and ATG9A with HA‐Flag‐TBC1D5stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h).


H Co ‐ immunoprecipitation of endogenous ULK1 pATG13 ( Ser318 ) with HA ‐ Flag ‐ TBC1D5 , stably expressed in T ‐ REx 293T , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

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H Co‐immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA‐Flag‐TBC1D5, stably expressed in T‐REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


I Magnified still extracted from Supplementary Movie S2 . mCherry ‐ TBC1D5 co ‐ localizes with GFP ‐ ATG9 upon autophagy induction by mTOR inhibitor , KU0063794 .

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I Magnified still images extracted from Supplementary Movie S2. mCherry‐TBC1D5 co‐localizes with GFP‐ATG9 upon autophagy induction by mTOR inhibitor, KU0063794.


H Co - immunoprecipitation of endogenous ULK1 pATG13 ( Ser318 ) with HA - Flag - TBC1D5 , stably expressed in T - REx 293T , induced by doxycycline ( 50 ng / ml ) for 20 h prior to DMSO or KU0063794 ( 6 h ) treatment .

H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment.


A Lysates of U2OS stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5 . were treated with DMSO or KU0063794 ( 6 h ) , lysed in RIPA buffer ( 1 % SDS ) lysates were subjected to SDS - PAGE .

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A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS-PAGE.


B U2OS stably expressing mCherry ‐ ATG9 were depleted of VPS29 TBC1D5 using shRNAs , treated with DMSO or KU0063794 ( 6 h ) , fixed with 2 % PFA , immunostained with anti ‐ LAMP1 antibody .

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B U2OS cells stably expressing mCherry‐ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti‐LAMP1 antibody.


C of ATG9 vesicular structures in from ( B ) under non ‐ stimulated conditions .

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C Quantification of ATG9 vesicular structures in cells from (B) under non‐stimulated conditions.


D of ATG9 LAMP1 co ‐ localization in presented in ( B ) .

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D Quantification of ATG9 and LAMP1 co‐localization in cells presented in (B).


E of ATG9 LAMP1 co − localization in stably depleted for TBC1D5 ( shRNA # 1 ) or VPS29 ( shRNA # 2 ) , transiently transfected with myc − TBC1D5 plasmid resistant to TBC1D5 shRNA # 1 . were treated with KU0063794 ( 6 h ) , 20 h post − transfection , fixed , immunostained with anti − LAMP1 anti − myc antibodies .

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E Quantification of ATG9 and LAMP1 co−localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc−TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post−transfection, fixed, and immunostained with anti−LAMP1 and anti−myc antibodies.


F Control shRNA U2OS lysates depleted for TBC1D5 ( shRNA # 1 ) , transfected with empty plasmid or with shRNA ‐ resistant myc ‐ TBC1D5 .

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F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA‐resistant myc‐TBC1D5.


G Immunofluorescence of TBC1D5 ( shRNA # 1 ) transiently transfected with shRNA ‐ resistant myc ‐ TBC1D5 , treated with KU0063794 ( 6 h ) . 20 h post ‐ transfection were treated , subsequently fixed , immunostained with anti ‐ myc anti ‐ LAMP1 antibodies .

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G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA‐resistant myc‐TBC1D5, treated with KU0063794 (6 h). 20 h post‐transfection cells were treated, subsequently fixed, and immunostained with anti‐myc and anti‐LAMP1 antibodies.


A HA ‐ Flag ‐ TBC1D5 expression was induced in T ‐ REx HeLa for 20 h , lysates were subjected to GSTpull ‐ down , − AP2M1 . Co ‐ precipitatedTBC1D5 was detected with anti − Flag antibody .

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A HA‐Flag‐TBC1D5 expression was induced in T‐REx HeLa cells for 20 h, lysates were subjected to GSTpull‐down, −AP2M1. Co‐precipitatedTBC1D5 was detected with anti−Flag antibody.


B Control TBC1D5 ( shRNA # 1 ) U2OS lysates subjected to SDS - PAGE , subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 AP2M1 .

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B Control cells and TBC1D5 (shRNA#1) U2OS cell lysates subjected to SDS-PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1.


C Immunofluorescent of AP2A1 in U2OS shRNA control , or shRNA # 1 TBC1D5 treated with KU0063974 or DMSO ( 6 h ) .

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C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h).


D U2OS stably expressing HA ‐ Flag ‐ ATG9A were fixed stained for endogenous AP2A1 TBC1D5 . Regions of co ‐ localization are indicated with arrows .

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D U2OS cells stably expressing HA‐Flag‐ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co‐localization are indicated with arrows.


E U2OS stably expressing HA ‐ Flag ‐ ATG9A were starved in EBSS ( 4 h ) or treated with KU0063794 ( 6 h ) , subsequently fixed stained with anti ‐ AP2A1 antibody .

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E U2OS cells stably expressing HA‐Flag‐ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti‐AP2A1 antibody.


F U2OS stably expressing mCherry ‐ ATG9A were treated with KU0063794 ( 6 h ) , lysed in co ‐ immunoprecipitation buffer . Lysates from DMSO ‐ or KU0063794 ‐ treated were split equal volumes were incubated with RFP ‐ Trap beads or GFP ‐ Trap beads , or agarose as a negative control , overnight at 4 ° C . Precipitated were analyzed by SDS - PAGE .

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F U2OS cells stably expressing mCherry‐ATG9A were treated with KU0063794 (6 h), lysed in co‐immunoprecipitation buffer. Lysates from DMSO‐ or KU0063794‐treated cells were split and equal volumes were incubated with RFP‐Trap beads or GFP‐Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS-PAGE.


A U2OS stably expressing HA ‐ Flag ‐ ATG9A transfected with siRNA targeting AP2A1 ( 40 nM ) , or siRNA AllStar control ( 40 nM ) were treated with KU0063794 or DMSO ( 6 h ) 4 days post ‐ transfection , fixed , stained with anti ‐ HA antibody .

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A U2OS cells stably expressing HA‐Flag‐ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post‐transfection, fixed, and stained with anti‐HA antibody.