E - F . COS7 cells co - transfected with EMTB - CFP - FRB , dNSpastin3Q - mCh - FKBP , and Tom20 - Neon were treated with rapamycin ( 100 nM ) for acute microtubule disruption . The morphology of mitochondria at different levels of microtubule disruption is shown . Scale bar , 10 µm . The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined . n = 4 different cells , three independent experiments . Data are shown as the mean ± S . E . M . Data information : Student ' s t - tests were performed with p values indicated .

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

E-F. COS7 cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon were treated with rapamycin (100 nM) for acute microtubule disruption. The morphology of mitochondria at different levels of microtubule disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


G - H . The normalized intensity of MitoTracker Red in non - transfected cells ( Mock ) or cells co - transfected with EMTB - CFRP - FRB and dNSpastin3Q - YFP - FKBP or dNSpastin3QED - YFP - FKBP after rapamycin ( Rapa , 100 nM ) treatment for the indicated time . Scale bar , 10 µm . n = 41 , 6 , and 18 cells from three independent experiments . Data are shown as the mean ± S . E . M . Data information : Student ' s t - tests were performed with p values indicated .

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

G-H. The normalized intensity of MitoTracker Red in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB and dNSpastin3Q-YFP-FKBP or dNSpastin3QED-YFP-FKBP after rapamycin (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


COS7 cells co - transfected with EMTB - CFP - FRB ( blue ) , dNSpastin3Q - mCh - FKBP ( red ) , and Lifeact - Neon ( heatmap and green ) were treated with rapamycin ( Rapa , 100 nM ) to induce microtubule disassembly . Arrows and arrowheads indicate lamellipodia and stress fibers , respectively . Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals . Scale bar , 10 µm . The normalized level of microtubule area , lamellipodia length , and filopodia density of COS - 7 cells treated as in ( A ) . n = 4 cells from three independent experiments . Data are shown as the mean ± S . E . M .

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

COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Lifeact-Neon (heatmap and green) were treated with rapamycin (Rapa, 100 nM) to induce microtubule disassembly. Arrows and arrowheads indicate lamellipodia and stress fibers, respectively. Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals. Scale bar, 10 µm. The normalized level of microtubule area, lamellipodia length, and filopodia density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.


B . Carfilzomib , Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il - 1β - luciferase transgenic mice . BMDMs from IL - 1β - luciferase transgenic mice were treated by IL - 4 ( 20 ng / mL ) for 24 hours to differentiate into mature M2 macrophages , and then used for screening of FDA compounds ( 5 μM ) . The X axis stands for the code of the drug .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.


F , G . Carfilzomib , Bortezomib and MLN9708 promote the mRNA expression of Il - 1β in M2 macrophages . BMDMs ( F ) or Raw264 . 7 cells ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then treated by DMSO , Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , or MLN9708 ( 2 μM ) . RNA was extracted from BMDMs or Raw264 . 7 cells 6 hours after stimulation and the expression of Il - 1β 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 . 001 ( student ' s t - test ) . H , I . Carfilzomib , Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL - 1β in M2 macrophages . BMDMs ( H ) or Raw264 . 7 cells ( I ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO , Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . Secretion of IL - 1β were measured through ELISA 24 hours later . Data from three experiments are presented as the mean ± SD . T test was used for statistical analysis of differences between groups . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - test ) .

O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 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 B-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 O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O 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, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β 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 < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).


A . The most effective one among the candidate compounds , Carfilzomib , significantly promotes the expression of M1 macrophage markers , as well as reduces the expression of M2 macrophage markers . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . RNA was extracted from cells 6 hours after stimulation and the expression of M1 ( Il - 6 / Inos ) or M2 ( Cd206 / Arg1 ) macrophage markers 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 . 001 ( student ' s t - test ) .

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 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 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 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 O O

A. The most effective one among the candidate compounds, Carfilzomib, significantly promotes the expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage markers. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the expression of M1 (Il-6/Inos) or M2 (Cd206/Arg1) macrophage markers 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 < 0.001 (student's t-test).


D . Statistics for proportion of CD86 , CD80 , MHC - II or CD206 positive cells in BMDMs . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - test ) .

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

D. Statistics for proportion of CD86, CD80, MHC-II or CD206 positive cells in BMDMs. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).


E . Carfilzomib promotes the phagocytosis of macrophages . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 500 nM ) for 12 hours . After starving for 2 hours and stained with Red membrane dye , BMDMs ( red ) were incubated with L1210 - GFP cells ( green ) in serum - free medium for another 2 hours . Phagocytosis effect was observed and photographed under fluorescence microscope . Scale bars , 50 μm ( up ) , 20 μm ( down ) . The yellow arrows indicated L1210 that is phagocytosed by macrophages . F . Statistics represent the number of phagocytosis L1210 in 100 macrophages . Data are means ± SD of three independent experiments . * * p < 0 . 01 ( student ' s t - test ) .

O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 promotes the phagocytosis of macrophages. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (500 nM) for 12 hours. After starving for 2 hours and stained with Red membrane dye, BMDMs (red) were incubated with L1210-GFP cells (green) in serum-free medium for another 2 hours. Phagocytosis effect was observed and photographed under fluorescence microscope. Scale bars, 50 μm (up), 20 μm (down). The yellow arrows indicated L1210 that is phagocytosed by macrophages. F. Statistics represent the number of phagocytosis L1210 in 100 macrophages. Data are means ± SD of three independent experiments. **p < 0.01 (student's t-test).


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 ) .

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 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 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

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 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 ) .

O O O O B-CONTROLLED_VAR O 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 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 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 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 ) .

O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 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 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 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 ) .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-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 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 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 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 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 ) .

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

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 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 ) .

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 B-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 O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-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 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 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 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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 + 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 . *

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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 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 .

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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 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 .

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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.


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 .

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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.


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 .

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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.


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 .

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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 . 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 .

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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 . 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 .

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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 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 . * *

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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 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 . 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 patient ILCs ( n = 5 ) stained with anti - LOXL1 probes ( red ) and 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 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 .

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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 - 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 .

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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 - 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 .

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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 - 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 .

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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 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 .

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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 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 .

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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 ) and 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 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 .

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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 weeks post - colonization , E1 fecal recovery was compared between the N1 WT , ΔtssC , and 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 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 ) .

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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 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 .

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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 . 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 ) .

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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 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 .

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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 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 .

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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 , 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 ) .

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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 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 -

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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 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 .

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

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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 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 .

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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 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 .

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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 individual organoid lines .

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


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 ) .

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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 and parental tissue . Data represent the median , quartiles , maximum , and minimum of the 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 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 .

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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 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

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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 analysis .

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


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 .

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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 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 .

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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 and premature growth arrest compared to the controls as indicated by the 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 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 .

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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.


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 .

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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 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 .

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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 - 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 .

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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 the mean ± SEM 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 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 .

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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.