( C ) Time course analysis of the relative mRNA levels of EP3 receptors in hESC - derived BAs during BA differentiation detected by RT - qPCR ( n = 7 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates ( C , Statistical significance was evaluated by Welch ANOVA test followed by Tamhane ' s T2 test for post - hoc comparisons ( C ) * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(C) Time course analysis of the relative mRNA levels of EP3 receptors in hESC-derived BAs during BA differentiation detected by RT-qPCR (n = 7-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates (C, Statistical significance was evaluated by Welch ANOVA test followed by Tamhane's T2 test for post-hoc comparisons (C) *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( D ) Representative pictures of hESC - derived BAs treated with L798106 alone or combined with H89 . Scale bar : 50 μm . Data information : Data are representative of two independent experiments with biological

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(D) Representative pictures of hESC-derived BAs treated with L798106 alone or combined with H89. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates


( E ) Effect of H89 on the relative mRNA level of brown marker gene in hESC - derived BAs treated with L798106 detected by RT - qPCR ( n = 6 - 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two - way ANOVA followed by Tukey ' s test for multiple comparisons ( E , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(E) Effect of H89 on the relative mRNA level of brown marker gene in hESC-derived BAs treated with L798106 detected by RT-qPCR (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons (E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( F ) Effect of H89 on WTAP mRNA expression in hESC - derived BAs treated with L798106 ( n = 6 - 7 ) . ( G ) Effect of H89 on ZNF410 mRNA expression in hESC - derived BAs treated with L798106 ( n = 8 ) . Data information : Data represent the mean ± standard error of mean ( SEM ) . Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two - way ANOVA followed by Tukey ' s test for multiple comparisons F , G ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and ns , not significant ( P > 0 . 05 ) .

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(F) Effect of H89 on WTAP mRNA expression in hESC-derived BAs treated with L798106 (n = 6-7). (G) Effect of H89 on ZNF410 mRNA expression in hESC-derived BAs treated with L798106 (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two-way ANOVA followed by Tukey's test for multiple comparisons F, G). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).


( H ) Western blotting assay of ERK1 / 2 , WTAP , ZNF410 , PRDM16 , and UCP1 in hESC - derived BAs treated with L798106 alone or combined with H89 .

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(H) Western blotting assay of ERK1/2, WTAP, ZNF410, PRDM16, and UCP1 in hESC-derived BAs treated with L798106 alone or combined with H89.


A C57BL / 6 WT or Rag2 − / − mice were immunosuppressed with hydrocortisone + / − FK506 and infected with 105 AF CEA10 conidia intranasally . Mortality was assessed using a 20 % weight loss endpoint . Mortality was significantly increased in FK506 immunosuppressed mice reaching 100 % by day 10 p . i . ( P = 0 . 003 for HC versus HC + FK506 in Rag2 − / − using log - rank test , n = 10 per group ) .

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A C57BL/6 WT or Rag2−/− mice were immunosuppressed with hydrocortisone +/− FK506 and infected with 105 AF CEA10 conidia intranasally. Mortality was assessed using a 20% weight loss endpoint. Mortality was significantly increased in FK506 immunosuppressed mice reaching 100% by day 10 p.i. (P = 0.003 for HC versus HC+FK506 in Rag2−/− using log-rank test, n = 10 per group).


B , C PAS - stained lung sections of Rag2 − / − mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ . FK506 - treated animals had increased inflammation at day 2 p . i . compared to control . P = 0 . 0126 ( using Student ' s t - test ) , n = 3 - 5 per group . ( C ) Close - up of an infectious focus showing increased inflammation and fungal growth in the FK506 - treated group . Scale bar : 400 μm .

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B, C PAS-stained lung sections of Rag2−/− mice were scanned and inflammation quantified by pixel intensity threshold analysis in ImageJ. FK506-treated animals had increased inflammation at day 2 p.i. compared to control. P = 0.0126 (using Student's t-test), n = 3-5 per group. (C) Close-up of an infectious focus showing increased inflammation and fungal growth in the FK506-treated group. Scale bar: 400 μm.


D , E Murine macrophages ( J774A . 1 ) were pre - treated with hydrocortisone , and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy . The nuclei are indicated in red , and the GR is indicated in green . Scale bar : 50 μm . FK506 did not cause increased translocation of the glucocorticoid receptor . Data represent mean + SEM calculated from 10 fields of vision . One out of 2 experiments is shown .

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D, E Murine macrophages (J774A.1) were pre-treated with hydrocortisone, and the effects of FK506 on nuclear shuttling of the glucocorticoid receptor were assessed by confocal immunofluorescence microscopy. The nuclei are indicated in red, and the GR is indicated in green. Scale bar: 50 μm. FK506 did not cause increased translocation of the glucocorticoid receptor. Data represent mean + SEM calculated from 10 fields of vision. One out of 2 experiments is shown.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . A Weight was monitored daily for 7 days p . i . Immunosuppressed mice showed increased weight loss through days 1 - 3 p . i . Data points represent mean ± SEM .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.A Weight was monitored daily for 7 days p.i. Immunosuppressed mice showed increased weight loss through days 1-3 p.i. Data points represent mean ± SEM.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . B BALs were performed over a 3 - day period p . i . and fungal killing assessed by enumerating CFUs . Fungal killing was significantly impaired in immunosuppressed mice . Error bars indicate SEM .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. B BALs were performed over a 3-day period p.i. and fungal killing assessed by enumerating CFUs. Fungal killing was significantly impaired in immunosuppressed mice. Error bars indicate SEM.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . C , D Flow cytometry analysis of the CD45 + leucocyte populations in BAL . Representative dot plots for CD45 + cells ( C ) and percentages of Ly - 6G - positive neutrophils infiltrating the infected lungs over a 48 - h p . i . time course ( D ) . The bars indicate the mean percentage ( ± SEM ) of neutrophils of total CD45 + leucocytes ( n = 5 mice per group ) . Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p . i .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n. C, D Flow cytometry analysis of the CD45+leucocyte populations in BAL. Representative dot plots for CD45+ cells (C) and percentages of Ly-6G-positive neutrophils infiltrating the infected lungs over a 48-h p.i. time course (D). The bars indicate the mean percentage (± SEM) of neutrophils of total CD45+ leucocytes (n = 5 mice per group). Neutrophil influx was significantly impaired in immunosuppressed mice at 6 h p.i.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107 conidia / mouse ) i . n . E PAS - stained lung sections of immunocompetent and FK506 - treated animals at 6 h p . i . Scale bar : 50 μm . IC animals showed inflammatory cell infiltrates around conidia ( black arrows ) , whereas in FK506 - treated animals , only uptake of conidia by the resident macrophage population could be seen .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107 conidia/mouse) i.n.E PAS-stained lung sections of immunocompetent and FK506-treated animals at 6 h p.i. Scale bar: 50 μm. IC animals showed inflammatory cell infiltrates around conidia (black arrows), whereas in FK506-treated animals, only uptake of conidia by the resident macrophage population could be seen.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ . Bars represent mean percentages ( ± SEM ) , n = 3 .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. F Tissue density of whole lung sections was quantified by pixel intensity analysis using ImageJ. Bars represent mean percentages (± SEM), n = 3.


Immunocompetent ( IC ) or FK506 - treated ( 5 mg / kg / day ) C57BL / 6 WT mice were infected with AF CEA10 ( 1 × 107conidia / mouse ) i . n . G Luminex multiplex cytokine analysis of BAL SNs at 6 h p . i . Bars represent mean ± SEM .

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Immunocompetent (IC) or FK506-treated (5 mg/kg/day) C57BL/6 WT mice were infected with AF CEA10 (1 × 107conidia/mouse) i.n. G Luminex multiplex cytokine analysis of BAL SNs at 6 h p.i. Bars represent mean ± SEM.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . A , B Absolute neutrophil numbers in treated and untreated mpx : GFPlarvae were quantified by direct counting of fluorescent neutrophils in non - infected whole fish ( n = 11 - 13 per group ) . There were no significant differences .

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At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.A, B Absolute neutrophil numbers in treated and untreated mpx:GFPlarvae were quantified by direct counting of fluorescent neutrophils in non-infected whole fish (n = 11-13 per group). There were no significant differences.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . C For survival analysis , lyz : dsRedlarvae were microinjected with ˜ 10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan - Meier analysis for survival over 6 days post - infection ( n = 16 per group ) . Immunosuppression led to a significant increase in mortality .

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At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed.C For survival analysis, lyz:dsRedlarvae were microinjected with ˜10 conidia and the effects of FK506 on the outcome from invasive aspergillosis was determined by Kaplan-Meier analysis for survival over 6 days post-infection (n = 16 per group). Immunosuppression led to a significant increase in mortality.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFPlarvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . D TNF - α mRNA levels were measured in whole non - infected or infected larvae 48 h p . i . Immunosuppression led to significant and almost complete inhibition of TNF - α expression during infection .

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At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFPlarvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AFeGFPconidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. D TNF-α mRNA levels were measured in whole non-infected or infected larvae 48 h p.i. Immunosuppression led to significant and almost complete inhibition of TNF-α expression during infection.


At 2 days post - fertilization ( dpf ) , Lyz : dsRed or mpx : GFP larvae were transferred into DMSO ctrl or FK506 ( 1 μg / ml ) containing 0 . 5 × E2 . At 3 dpf , zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality , and fungal burden and neutrophil recruitment were assessed . E - G Lyz : dsRedlarvae were infected with ˜ 50 eGFP - expressing conidia of A . fumigatus and were imaged daily from 24 h p . i . ( E ) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ . A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown ( 60 h p . i . ) . The arrows indicate infectious foci . ( F ) Neutrophil recruitment was minimal throughout the first 60 h p . i . in immunosuppressed larvae ( n = 8 per group ) . ( G ) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements ( n = 8 per group ) .

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At 2 days post-fertilization (dpf),Lyz:dsRed or mpx:GFP larvae were transferred into DMSO ctrl or FK506 (1 μg/ml) containing 0.5 × E2. At 3 dpf, zebrafishlarvae were microinjected with AF eGFP conidia into the hindbrain and mortality, and fungal burden and neutrophil recruitment were assessed. E-G Lyz:dsRedlarvae were infected with ˜50 eGFP-expressing conidia of A. fumigatus and were imaged daily from 24 h p.i. (E) The effects of FK506 on neutrophil recruitment were assessed by integrated density analysis of red fluorescent neutrophils at sites of infection in ImageJ. A representative image showing impaired neutrophil recruitment to the site of infection in immunosuppressed larvae is shown (60 h p.i.). The arrows indicate infectious foci. (F) Neutrophil recruitment was minimal throughout the first 60 h p.i. in immunosuppressed larvae (n = 8 per group). (G) Fungal growth was determined by measurement of the length of individual fluorescent hyphal elements (n = 8 per group).


A J774A . 1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . FK506 significantly reduced TNF - α levels at 5 and 10 ng / ml for AF . A significant reduction in TNF - α was also seen for zymosan , but not LPS .

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A J774A.1 macrophages were treated with an increasing concentration of FK506 and stimulated with AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. FK506 significantly reduced TNF-α levels at 5 and 10 ng/ml for AF. A significant reduction in TNF-α was also seen for zymosan, but not LPS.


B J774A . 1 cells were treated with FK506 ( 10 ng / ml ) and either left non - stimulated ( NS ) or stimulated with AF SC ( MOI = 1 ) for 6 h . TNF - α mRNA levels were quantified from whole - cell RNA extracts by RT - PCR . FK506 significantly reduced TNF - α expression .

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B J774A.1 cells were treated with FK506 (10 ng/ml) and either left non-stimulated (NS) or stimulated with AF SC (MOI=1) for 6 h. TNF-α mRNA levels were quantified from whole-cell RNA extracts by RT-PCR. FK506 significantly reduced TNF-α expression.


C J774A . 1 macrophages were treated with control ( Ctrl . ) or calcineurin A ( CnA ) - targeting siRNA ( 50 nM ) and stimulated with either AF SC ( MOI = 1 ) , zymosan ( 50 μg / ml ) or LPS ( 25 ng / ml ) overnight . TNF - α was measured in the culture supernatant by ELISA . CnA siRNA led to significant reductions in both AF - and zymosan - dependent TNF - α levels , but had no significant effect on LPS responses .

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C J774A.1 macrophages were treated with control (Ctrl.) or calcineurin A (CnA)-targeting siRNA (50 nM) and stimulated with either AF SC (MOI = 1), zymosan (50 μg/ml) or LPS (25 ng/ml) overnight. TNF-α was measured in the culture supernatant by ELISA. CnA siRNA led to significant reductions in both AF- and zymosan-dependent TNF-α levels, but had no significant effect on LPS responses.


A J774A . 1macrophages were stimulated with live AF SC ( MOI = 5 ) , and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2 . NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p . i .

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A J774A.1macrophages were stimulated with live AF SC (MOI=5), and macrophage cytoplasmic and nuclear extracts were probed by Western blot for NFATc2. NFATc2 shuttling to the nucleus was observed and was maximal at 2 h p.i.


B J774A . 1 macrophages were stimulated with live or fixed swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFAT translocation was assessed by confocal microscopy . NFAT is indicated in green , the nuclei are indicated in red , and AF conidia can be seen in pink . Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan . Data represent mean ± SEM . Scale bar : 50 μm . Arrows indicate A . fumigatus or zymosan - positive cells .

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B J774A.1 macrophages were stimulated with live or fixed swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFAT translocation was assessed by confocal microscopy. NFAT is indicated in green, the nuclei are indicated in red, and AF conidia can be seen in pink. Live conidia were observed to stimulate sustained nuclear translocation compared to dead conidia or zymosan. Data represent mean ± SEM. Scale bar: 50 μm. Arrows indicate A. fumigatus or zymosan-positive cells.


C J774A . 1 macrophages were stimulated with zymosan after FK506 ( 10 ng / ml ) or control treatment , and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF - κB translocation . NFATc2 and NF - κB nuclear translocation was observed , which was maximal at 1 h p . i . , in stimulated macrophages . FK506 specifically inhibited NFATc2 translocation , but had no effect on NF - κB .

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C J774A.1 macrophages were stimulated with zymosan after FK506 (10 ng/ml) or control treatment, and macrophage nuclear extracts were probed by Western blot for NFATc2 and NF-κB translocation. NFATc2 and NF-κB nuclear translocation was observed, which was maximal at 1 h p.i., in stimulated macrophages. FK506 specifically inhibited NFATc2 translocation, but had no effect on NF-κB.


D J774A . 1 macrophages were pre - treated with FK506 ( 10 ng / ml ) or vehicle and stimulated with live swollen conidia or zymosan . NFATc2 translocation was evaluated 30 min after stimulation . Box and whisker plot shows median ± maximum / minimum . Scale bar : 50 μm . FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan . Statistical analysis was carried out by Student ' s t - test . NS , non - stimulated . Arrows indicate A . fumigatus or zymosan - positive cells .

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D J774A.1 macrophages were pre-treated with FK506 (10 ng/ml) or vehicle and stimulated with live swollen conidia or zymosan. NFATc2 translocation was evaluated 30 min after stimulation. Box and whisker plot shows median ± maximum/minimum. Scale bar: 50 μm. FK506 completely inhibited nuclear translocation of NFATc2 in response to AF or zymosan. Statistical analysis was carried out by Student's t-test. NS, non-stimulated. Arrows indicate A. fumigatus or zymosan-positive cells.


A BMDMs from WT , Dectin - 1 − / − or MyD88 − / − mice were infected with swollen conidia ( MOI = 1 ) , and TNF - α was measured in the SN after overnight incubation . TNF - α levels were significantly reduced in Dectin1 − / − and Myd88 − / − BMDMs .

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A BMDMs from WT, Dectin-1−/− or MyD88−/− mice were infected with swollen conidia (MOI = 1), and TNF-α was measured in the SN after overnight incubation. TNF-α levels were significantly reduced in Dectin1−/− and Myd88−/− BMDMs.


B BMDMs were stimulated with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) , and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation . Whilst almost complete abrogation of NFAT translocation was seen in Dectin1 − / − macrophages stimulated with zymosan , NFAT responses to AF were unaffected .

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B BMDMs were stimulated with swollen conidia (MOI = 5) or zymosan (50 μg/ml), and NFATc2 translocation was quantified by confocal microscopy 30 min after stimulation. Whilst almost complete abrogation of NFAT translocation was seen in Dectin1−/−macrophages stimulated with zymosan, NFAT responses to AF were unaffected.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( C ) TNF - α was measured in the SN after overnight incubation . Syk inhibition significantly reduced swollen conidia - dependent or zymosan - dependent TNF - α levels .

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C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (C) TNF-α was measured in the SN after overnight incubation. Syk inhibition significantly reduced swollen conidia-dependent or zymosan-dependent TNF-α levels.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( D ) NFATc2 translocation was quantified by confocal microscopy . Syk inhibition impaired zymosan - dependent NFAT translocation , but did not affect AF - dependent NFAT translocation .

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C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (D) NFATc2 translocation was quantified by confocal microscopy. Syk inhibition impaired zymosan-dependent NFAT translocation, but did not affect AF-dependent NFAT translocation.


C - E J774A . 1 macrophages were pre - treated with ( C , D ) the Syk inhibitor piceatannol or ( E ) a control or Syk - targeting siRNA ( 10 nM ) before stimulation with swollen conidia ( MOI = 1 ) or zymosan ( 50 μg / ml ) . ( E ) Whole - cell lysates of Syk or Ctrl . siRNA - treated cells were separated by SDS - PAGE , followed by Western blotting , and membranes were probed with anti - Syk and anti - β - actin antibodies .

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C-E J774A.1 macrophages were pre-treated with (C, D) the Syk inhibitor piceatannol or (E) a control or Syk-targeting siRNA (10 nM) before stimulation with swollen conidia (MOI = 1) or zymosan (50 μg/ml). (E) Whole-cell lysates of Syk or Ctrl. siRNA-treated cells were separated by SDS-PAGE, followed by Western blotting, and membranes were probed with anti-Syk and anti-β-actin antibodies.


F J774A . 1 macrophages were pre - treated with piceatannol and infected with swollen conidia ( MOI = 5 ) . Phagocytosis was quantified by FACS 30 min after stimulation . Syk inhibition led to impaired phagocytosis of zymosan , but had no effect on phagocytosis of SC .

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F J774A.1 macrophages were pre-treated with piceatannol and infected with swollen conidia (MOI = 5). Phagocytosis was quantified by FACS 30 min after stimulation. Syk inhibition led to impaired phagocytosis of zymosan, but had no effect on phagocytosis of SC.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( G ) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (G) NFATc2 translocation after 30 min of stimulation was quantified by confocal microscopy.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( H ) For quantification of NF - κB translocation , nuclear extracts were separated by SDS - PAGE , followed by Western blotting . Membranes were probed with anti - NF - κB p65 and anti - HDAC1 antibodies .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (H) For quantification of NF-κB translocation, nuclear extracts were separated by SDS-PAGE, followed by Western blotting. Membranes were probed with anti-NF-κB p65 and anti-HDAC1 antibodies.


G - I J774A . 1 macrophages were pre - treated with cytochalasin D ( CytoD , 10 μM ) and infected with swollen conidia ( MOI = 5 ) or zymosan ( 50 μg / ml ) . ( I ) After overnight stimulation , TNF - α was measured in the SN by ELISA . Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF , but not ionomycin ( G ) . In addition , inhibition of phagocytosis lead to increased translocation of NF - κB ( H ) and increased levels of TNF - α in response to either zymosan or AF .

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G-I J774A.1 macrophages were pre-treated with cytochalasin D (CytoD, 10 μM) and infected with swollen conidia (MOI = 5) or zymosan (50 μg/ml). (I) After overnight stimulation, TNF-α was measured in the SN by ELISA. Inhibition of phagocytosis with cytochalasin D led to impaired NFAT translocation in response to zymosan or AF, but not ionomycin (G). In addition, inhibition of phagocytosis lead to increased translocation of NF-κB (H) and increased levels of TNF-α in response to either zymosan or AF.


A J774A . 1 macrophages were pre - treated with bafilomycin A1 ( 100 nM ) and stimulated with swollen conidia ( MOI = 3 ) for 30 min . NFATc2 translocation was assessed by confocal microscopy . Acidification was required for NFATc2 translocation .

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A J774A.1 macrophages were pre-treated with bafilomycin A1 (100 nM) and stimulated with swollen conidia (MOI = 3) for 30 min. NFATc2 translocation was assessed by confocal microscopy. Acidification was required for NFATc2 translocation.


B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP - expressing swollen conidia by confocal microscopy . Scale bar : 10 μm . The arrow indicates a TLR9 - positive AF - containing phagosome .

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B TLR9 recruitment was assessed in WT BMDMs 30 min after infection with eGFP-expressing swollen conidia by confocal microscopy. Scale bar: 10 μm. The arrow indicates a TLR9-positive AF-containing phagosome.


C BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 1 ) , and after overnight stimulation , TNF - α was measured in the SN by ELISA . TLR9 − / − BMDMs had significantly lower TNF - α levels .

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C BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 1), and after overnight stimulation, TNF-α was measured in the SN by ELISA. TLR9−/− BMDMs had significantly lower TNF-α levels.


D , E BMDMs from WT or TLR9 − / − mice were infected with swollen conidia ( MOI = 5 ) for 30 min , and NFATc2 translocation was quantified by ( D ) confocal microscopy . TLR9 − / − BMDMs exhibited reduced NFATc2 translocation by both approaches .

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D, E BMDMs from WT or TLR9−/−mice were infected with swollen conidia (MOI = 5) for 30 min, and NFATc2 translocation was quantified by (D) confocal microscopy. TLR9−/− BMDMs exhibited reduced NFATc2 translocation by both approaches.


G BMDMs from WT or MyD88 − / − mice were pre - treated with ODN2088 ( 10 μM ) and infected with swollen conidia ( MOI = 5 ) for 30 min . NFATc2 translocation was quantified by confocal microscopy . In both , WT and MyD88 − / − BMDMs , blocking TLR9 significantly impaired NFATc2 translocation .

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G BMDMs from WT or MyD88−/−mice were pre-treated with ODN2088 (10 μM) and infected with swollen conidia (MOI = 5) for 30 min. NFATc2 translocation was quantified by confocal microscopy. In both, WT and MyD88−/− BMDMs, blocking TLR9 significantly impaired NFATc2 translocation.


A , B J774A . 1 macrophages were pre - treated with the BTK inhibitor LFM - A13 ( 12 . 5 μM ) and infected with swollen conidia . ( A ) After overnight stimulation , TNF - α was measured in the SN by ELISA .

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A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (A) After overnight stimulation, TNF-α was measured in the SN by ELISA.


A , B J774A . 1 macrophages were pre - treated with the BTK inhibitor LFM - A13 ( 12 . 5 μM ) and infected with swollen conidia . ( B ) After 30 min of stimulation , NFATc2 translocation was quantified by confocal microscopy . Both TNF - α levels and NFATc2 translocation were significantly reduced by BTK blockade .

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A, B J774A.1 macrophages were pre-treated with the BTK inhibitor LFM-A13 (12.5 μM) and infected with swollen conidia. (B) After 30 min of stimulation, NFATc2 translocation was quantified by confocal microscopy. Both TNF-α levels and NFATc2 translocation were significantly reduced by BTK blockade.


C J774A . 1 macrophages were treated with a control or BTK - targeting siRNA ( 25 nM ) , and NFAT translocation was quantified by confocal microscopy . BTK knock - down led to a significant reduction in NFATc2 translocation in response to AF .

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C J774A.1 macrophages were treated with a control or BTK-targeting siRNA (25 nM), and NFAT translocation was quantified by confocal microscopy. BTK knock-down led to a significant reduction in NFATc2 translocation in response to AF.


D , E J774A . 1 macrophages were treated with a control , BTK ( 25 nM ) - or TLR9 ( 75 nM ) - targeting siRNA and additionally pre - treated with either ODN2088 ( 10 μM ) or the BTK inhibitor LFM - A13 ( 12 . 5 μM ) . The cells were infected with swollen conidia ( MOI = 5 ) , and NFATc2 translocation was measured by confocal microscopy . Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade .

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D, E J774A.1 macrophages were treated with a control, BTK (25 nM)- or TLR9 (75 nM)-targeting siRNA and additionally pre-treated with either ODN2088 (10 μM) or the BTK inhibitor LFM-A13 (12.5 μM). The cells were infected with swollen conidia (MOI = 5), and NFATc2 translocation was measured by confocal microscopy. Blocking both TLR9 and BTK signalling had no additive effect on NFAT translocation blockade.


Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . Cells were pre - treated for 1 h with FK506 ( 10 ng / ml ) , the NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in the cell culture supernatant by ELISA . A Representative FACS - phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples .

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Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.A Representative FACS-phenotyping demonstrative of a normal alveolar macrophage phenotype is shown for three patient samples.


Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage . Cells were pre - treated for 1 h with FK506 ( 10 ng / ml ) , the NFAT inhibitor MCV1 ( 1 μM ) or SC514 ( 10 μM ) before stimulation with A . fumigatus swollen conidia ( MOI 0 . 1 ) . After 24 h of stimulation , TNF - α was measured in the cell culture supernatant by ELISA . B Inhibition of alveolar macrophage responses to AF with FK506 , MCV1 or SC514 led to a significant reduction in TNF - α levels .

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Alveolar macrophages from 13 different lung transplant recipients were isolated from bronchoalveolar lavage. Cells were pre-treated for 1 h with FK506 (10 ng/ml), the NFAT inhibitor MCV1 (1 μM) or SC514 (10 μM) before stimulation with A. fumigatus swollen conidia (MOI 0.1). After 24 h of stimulation, TNF-α was measured in the cell culture supernatant by ELISA.B Inhibition of alveolar macrophage responses to AF with FK506, MCV1 or SC514 led to a significant reduction in TNF-α levels.


A . Representative FACS plot ( left ) showing a mixed - lineage clone with a T - lymphoid ( CD25 + / CD11b - Gr1 - ) , myeloid ( CD11b + Gr1 + / CD25 - ) , and biphenotypic ( CD11b + Gr1 + / CD25 + ) fraction derived from a single Myc / Bcl2 - transformed DN2cell grown on 1 : 1 OP9 : OP9 - Dll1 co - culture with lineage - promiscuous cytokines IL - 2 , IL - 3 , IL - 6 , IL - 7 , SCF , GM - CSF , Flt3 . Morphology of the indicated flow - sorted cell fractions was analyzed by Giemsa staining ( right ) .

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A. Representative FACS plot (left) showing a mixed-lineage clone with a T-lymphoid (CD25+/CD11b-Gr1-), myeloid (CD11b+Gr1+/CD25-), and biphenotypic (CD11b+Gr1+/CD25+) fraction derived from a single Myc/Bcl2-transformed DN2cell grown on 1:1 OP9:OP9-Dll1 co-culture with lineage-promiscuous cytokines IL-2, IL-3, IL-6, IL-7, SCF,GM-CSF, Flt3. Morphology of the indicated flow-sorted cell fractions was analyzed by Giemsa staining (right).


B . Phenotype frequencies of clones from Myc / Bcl2 - transformed single cells ( grown and phenotyped as mentioned in A ) . Analyzed were 31 clones from LSKs , 107 from GMPs and 28 from DN2 cells .

B. Phenotype frequencies of clones from Myc/Bcl2-transformed single cells (grown and phenotyped as mentioned in A). Analyzed were 31 clones from LSKs, 107 from GMPs and 28 from DN2 cells.


C . Cumulative survival of 31 ± 2 and 27 ± 6 days of mice that received 5104 to 5105 T - lymphoid cells or myeloid cells , respectively , that were flow - sorted from mixed Myc / Bcl2 + DN2 - derived clones . The summarized data are from 3 mice injected with the T - lymphoid and 4 mice injected with the myeloid fraction of two different clones .

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C. Cumulative survival of 31±2 and 27±6 days of mice that received 5104 to 5105 T-lymphoid cells or myeloid cells, respectively, that were flow-sorted from mixed Myc/Bcl2+ DN2-derived clones. The summarized data are from 3 mice injected with the T-lymphoid and 4 mice injected with the myeloid fraction of two different clones.


D . Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc / Bcl2 - transformed LSK or GMPcells on OP9 : OP9 - Dll1 co - cultures .

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D. Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc/Bcl2-transformed LSK or GMPcells on OP9:OP9-Dll1 co-cultures.


E . ( top panels ) Serial replating of Myc - Bcl2 + DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro ( gel : left two lanes ) . Upon transplantation , the same clones induced myeloid leukemia that retained the initial rearrangement in vivo ( gel : right two lanes ) as assessed by nested PCR . GL = germline . ( bottom panels ) Methylcellulose - based replating of Myc - Bcl2 + GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration .

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E. (top panels) Serial replating of Myc-Bcl2+DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro (gel: left two lanes). Upon transplantation, the same clones induced myeloid leukemia that retained the initial rearrangement in vivo (gel: right two lanes) as assessed by nested PCR. GL = germline. (bottom panels) Methylcellulose-based replating of Myc-Bcl2+GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration.


F . Cumulative survival of mice that received 106cells of TCRβ - rearranged myeloid DN2 - derived clones leading to death within 33 ± 4 days after transplantation . Two DN2 clones were transplanted into 5 recipient mice .

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F. Cumulative survival of mice that received 106cells of TCRβ-rearranged myeloid DN2-derived clones leading to death within 33±4 days after transplantation. Two DN2 clones were transplanted into 5 recipient mice.


A . Representative FACS plots of spleens from leukemic recipient mice that had received LSK , GMP or DN2 cell grafts immediately after retroviral Myc / Bcl2 transduction . Numbers indicate frequencies ( % ) of CD45 . 2 donor cells within the indicated gates summarizing the mean ± SEM of at least 8 recipients per group from a total of 10 independent experiments .

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A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments.


B . Cumulative survival of mice that received 2104 to 2105 freshly Myc / Bcl2 - transduced LSK , GMP or DN2 cells ; Graph shows n = 11 Myc - Bcl2 + LSK - , n = 14 DN2 - , and n = 8 GMP - transplanted animals .

B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+LSK-, n=14 DN2-, and n=8 GMP-transplanted animals.


C . Histological spleen sections of Myc - Bcl2 + LSK - , GMP - or DN2 - transplanted mice displaying myeloperoxidasestaining . Leukemic cell engraftment in spleens ranged from 75 . 1 % - 95 . 5 % .

C. Histological spleen sections of Myc-Bcl2+LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidasestaining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%.


D . Multiplex quantitative RT - PCR demonstrating strong expression of PU . 1 in the myeloid ( M3L : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) and biphenotypic T / myeloid ( BL : CD11b + Gr1 + / CD3 + CD4 + CD8 + ) fractions but not in the T - lymphoid ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) fraction that were flow - sorted from diseased Myc - Bcl2 + DN2 - transplanted mice . Myeloid progeny of Myc / Bcl2 + GMP - transplanted or T - lymphoid progeny of Myc / Bcl2 + DN3 - transplanted animals as well as healthy thymocytes ( Thy ) and BM cells from WT mice served as controls . Results are shown as fold over actin transcripts .

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D. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3-CD4-CD8-) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b-Gr1-/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts.


E . D - J rearrangement PCR of flow - sorted T - lymphoid ( TL ) , biphenotypic ( BL ) and myeloid ( ML ) fractions of donor splenocytes of a diseased Myc / Bcl2 + DN2 - transplanted mouse . All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus ( Dβ1Jβ1 . 5 ) demonstrating clonality in DN2 leukemia . thy = thymus .

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E. D-J rearrangement PCR of flow-sorted T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus.


F . FACS plot of diseased intrathymic grafts ( CD45 . 2 - gated ) , demonstrating that Myc / Bcl2 + DN2 cells could produce multi - lineage leukemia within a T - cell supporting environment ; Myc / Bcl2 + GMPs remained myeloid restricted under these conditions . Numbers indicate cell frequencies within the gates ( mean ± SEM ) representing six independent experiments .

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F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments.


G . Intrathymic transplantation of mock - infected non - leukemic DN2 cells produced predominantly T - lymphoid but also myeloid progeny 23 days after injection . This differentiation outcome resembles that of freshly isolated DN2 cells ( Bell and Bhandoola , 2008 ; Richie Ehrlich et al . , 2011 ) , and indicates that the short - term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells .

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G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.


A . GSEA demonstrates that Myc / Bcl2 + DN2 - derived myeloid blasts were significantly ( FDR > 0 . 05 ) enriched for expression of a pan - dendritic cell gene set . Gene sets were published in ( Schonheit et al . , 2013 ) .

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A. GSEA demonstrates that Myc/Bcl2+DN2-derived myeloid blasts were significantly (FDR >0.05) enriched for expression of a pan-dendritic cell gene set. Gene sets were published in (Schonheit et al., 2013).


B . Heat map of the top 100 differentially expressed genes ( adjusted p - value of ≤ 0 . 05 ) in each myeloid fraction ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from GMP - , LSK - and DN2 - derived leukemias .

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B. Heat map of the top 100 differentially expressed genes (adjusted p-value of ≤ 0.05) in each myeloid fraction (ML: CD11b+Gr1+/CD3-CD4-CD8-) from GMP-, LSK- and DN2-derived leukemias.


D . GSEA demonstrates significant enrichment of a normal DN2 - specific gene set ( see methods section ) in the Myc / Bcl2 + DN2 - derived myeloid blasts .

D. GSEA demonstrates significant enrichment of a normal DN2-specific gene set (see methods section) in the Myc/Bcl2+DN2-derived myeloid blasts.


A . Western blot illustrating abundant Bcl11b and Gata3protein expression in Myc / Bcl2 + DN2 - but not LSK - or GMP - derived blasts . Thymus ( Thy ) and bone marrow ( BM ) extracts from healthy WT mice served as controls . Detection of α - tubulin or valosin - containing protein ( VCP ) ensured comparable loading .

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A. Western blot illustrating abundant Bcl11b and Gata3protein expression in Myc/Bcl2+DN2- but not LSK- or GMP-derived blasts. Thymus (Thy) and bone marrow (BM) extracts from healthy WT mice served as controls. Detection of α-tubulin or valosin-containing protein (VCP) ensured comparable loading.


B , C . Proliferation assays illustrating growth of Myc / Bcl2 + DN2 - ( B ) or GMP - derived ( C ) blasts after lentiviral transduction with the indicated shRNAs . The shRNA - transduced cells were flow - sorted based on co - expression of the RFP - reporter and seeded on OP9 / OP9Dll1 1 : 1 layers . Growing cells were RFP - sorted for 3 rounds within 21 days of culture and percent growth was calculated over the initially seeded cell number in relation to the Luc control . Shown are results of 4 independent experiments with cells from 4 different DN2 - and 3 different GMP - leukemic animals . Luc = control shRNA targeting luciferase ( Suzuki et al . , 2003 ) . At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated . * = p ≤ 0 . 05 , Student ' s t - test .

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B,C. Proliferation assays illustrating growth of Myc/Bcl2+ DN2- (B) or GMP-derived (C) blasts after lentiviral transduction with the indicated shRNAs. The shRNA-transduced cells were flow-sorted based on co-expression of the RFP-reporter and seeded on OP9/OP9Dll1 1:1 layers. Growing cells were RFP-sorted for 3 rounds within 21 days of culture and percent growth was calculated over the initially seeded cell number in relation to the Luc control. Shown are results of 4 independent experiments with cells from 4 different DN2- and 3 different GMP-leukemic animals. Luc= control shRNA targeting luciferase (Suzuki et al., 2003). At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated. * = p≤0.05, Student's t-test.


D . Representative FACS plots showing CD45 . 2 - donor splenocytes of a secondary ( CD45 . 1 ) recipient 23 weeks after transplantation with 4103RFP + Myc - Bcl2 + DN2 - leukemia blasts carrying the indicated shRNAs . Luc : luciferase control shRNA . The data are representative of 2 Gata3 , 4 Bcl11b , and 2 luciferase shRNA - transplanted animals .

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D. Representative FACS plots showing CD45.2-donor splenocytes of a secondary (CD45.1) recipient 23 weeks after transplantation with 4103RFP+Myc-Bcl2+DN2-leukemia blasts carrying the indicated shRNAs. Luc: luciferase control shRNA. The data are representative of 2 Gata3, 4 Bcl11b, and 2 luciferase shRNA-transplanted animals.


A . Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow - sorted myeloid leukemia blasts ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from sick primary Myc / Bcl2 + LSK - , GMP - or DN2 - transplanted mice . Cell percentages in indicated gates are shown .

A. Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow-sorted myeloid leukemia blasts (ML: CD11b+Gr1+/CD3-CD4-CD8-) from sick primary Myc/Bcl2+LSK-, GMP- or DN2-transplanted mice. Cell percentages in indicated gates are shown.


B . Table summarizing the phenotype frequencies within the spleens of secondary recipients that were retransplanted with Myc / Bcl2 + LSK - , GMP - or DN2 - derived myeloid blasts as described in A . T - lymphoid leukemia : ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) , biphenotypic leukemia : BL ( CD11b + Gr1 + / CD3 + CD4 + CD8 + ) , myeloid leukemia : ML ( CD11b + Gr1 + / CD3 - CD4 - CD8 - ) . Numbers before the dash represent mice holding the indicated leukemia fraction ; numbers after the dash indicate the total mouse count analyzed .

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B. Table summarizing the phenotype frequencies within the spleens of secondary recipients that were retransplanted with Myc/Bcl2+LSK-, GMP- or DN2-derived myeloid blasts as described in A. T-lymphoid leukemia: (TL: CD11b-Gr1-/CD3+CD4+CD8+), biphenotypic leukemia: BL (CD11b+Gr1+/CD3+CD4+CD8+), myeloid leukemia: ML (CD11b+Gr1+/CD3-CD4-CD8-). Numbers before the dash represent mice holding the indicated leukemia fraction; numbers after the dash indicate the total mouse count analyzed.


C . Single myeloid blasts from 4 different Myc / bcl2 + DN2 - derived leukemias were flow - sorted from transplanted mice and grown on irradiated OP9 : OP9DL1 stroma . The clones were analyzed at two different time points . FACS plots of two representative clones , one with strong ( top ) and one with weak ( bottom ) lineage switching potential are shown .

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C. Single myeloid blasts from 4 different Myc/bcl2+ DN2-derived leukemias were flow-sorted from transplanted mice and grown on irradiated OP9:OP9DL1 stroma. The clones were analyzed at two different time points. FACS plots of two representative clones, one with strong (top) and one with weak (bottom) lineage switching potential are shown.


D . Table summarizing the lineage switching outcome of 20 independent clones that were grown from single DN2 - derived myeloid blasts of 4 different leukemias . Percentages at the right indicate the relative amounts of cells with T - lymphoid ( TL ) , biphenotypic ( BL ) or myeloid ( ML ) phenotype as analyzed by FACS at the end of the experiment .

D. Table summarizing the lineage switching outcome of 20 independent clones that were grown from single DN2-derived myeloid blasts of 4 different leukemias. Percentages at the right indicate the relative amounts of cells with T-lymphoid (TL), biphenotypic (BL) or myeloid (ML) phenotype as analyzed by FACS at the end of the experiment.


E . FACS plot of DN2 - derived primary leukemic cells transduced with a retrovirus carrying GFP alone or GFP together with PU . 1 as a bicistronic RNA . The transduced cells were grown on OP9 / OP9Dll1 1 : 1 layer for 8 - 13 days and analyzed using a GFP - gate . The data are representative of 3 different experiments with similar outcome .

E. FACS plot of DN2-derived primary leukemic cells transduced with a retrovirus carrying GFP alone or GFP together with PU.1 as a bicistronic RNA. The transduced cells were grown on OP9/OP9Dll1 1:1 layer for 8-13 days and analyzed using a GFP-gate. The data are representative of 3 different experiments with similar outcome.


A . Heat map depicting all human AML samples from the Haferlach study ( Haferlach et al . , 2010 ) whose transcriptomes resembled DN2 - leukemia by a Spearman ' s rank correlation coefficient ≥ 0 . 5 . The top 100 up - and downregulated gene signature of Myc / Bcl2 + DN2 - derived myeloid blasts was applied ( see also Figure 3B ) .

A. Heat map depicting all human AML samples from the Haferlach study (Haferlach et al., 2010) whose transcriptomes resembled DN2-leukemia by a Spearman's rank correlation coefficient ≥ 0.5. The top 100 up- and downregulated gene signature of Myc/Bcl2+DN2-derived myeloid blasts was applied (see also Figure 3B).


C , E . GSEA confirming enrichment of the Myc / Bcl2 + DN2 - derived myeloid blast top 100 UP ( C ) and pan - DC ( Schonheit et al . , 2013 ) ( E ) gene signatures in the human FAB M2 DN2 - like AML samples as compared to all residual M2 AMLs from the Verhaak study ( Verhaak et al . , 2009 ) . D . Relative transcript levels of the T cell receptor alpha locus ( TRAC ) in human DN2 - like M2 AML compared to residual M2 AML patient samples from the Verhaak study ( Wilcoxon rank - sum test , * * = p ≤ 0 . 01 ) .

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C,E. GSEA confirming enrichment of the Myc/Bcl2+DN2-derived myeloid blast top 100 UP (C) and pan-DC (Schonheit et al., 2013) (E) gene signatures in the human FAB M2 DN2-like AML samples as compared to all residual M2 AMLs from the Verhaak study (Verhaak et al., 2009). D. Relative transcript levels of the T cell receptor alpha locus (TRAC) in human DN2-like M2 AML compared to residual M2 AML patient samples from the Verhaak study (Wilcoxon rank-sum test, **=p≤0.01).


A . Survival assay of Myc / Bcl2 + GMP - and DN2 - derived leukemia cells that were isolated from diseased mice and treated for 24 h with 0 . 15 μM daunorubicine or 1 μM doxorubicine in liquid culture . Cell death was measured by FACS of 7 - AAD + cells and was normalized to the solvent controls . The graphs show the mean + SEM of three different DN2 - and five GMP - leukemias . * * = p ≤ 0 . 01 .

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A. Survival assay of Myc/Bcl2+GMP- and DN2-derived leukemia cells that were isolated from diseased mice and treated for 24 h with 0.15 μM daunorubicine or 1 μM doxorubicine in liquid culture. Cell death was measured by FACS of 7-AAD+cells and was normalized to the solvent controls. The graphs show the mean+SEM of three different DN2- and five GMP-leukemias. **=p≤0.01.


C . Scatterplot depicting the averaged quantitative changes ( log10 ) of each shRNA within the library in the T1 spleen and BM samples in reference to T0 . The changes in both organs were concordant ( R2 = 0 . 72 ) . T1 samples from 11 mice were compared to 3 T0 reference samples . Blue dots represent shRNAs with a significant depletion by ≥ 10 - fold .

C. Scatterplot depicting the averaged quantitative changes (log10) of each shRNA within the library in the T1 spleen and BM samples in reference to T0. The changes in both organs were concordant (R2=0.72). T1 samples from 11 mice were compared to 3 T0 reference samples. Blue dots represent shRNAs with a significant depletion by ≥10-fold.


D . Top 5 genes with ≥ 10 - fold shRNA depletion in BM , spleen and thymus in comparison to the reference .

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D. Top 5 genes with ≥10-fold shRNA depletion in BM, spleen and thymus in comparison to the reference.


E . Percent cell growth of 5 different DN2 - derived leukemic cell lines transduced with 2 different shRNA constructs targeting Jak2 . The cells were sorted for the GFP or YFP reporter and normalized to the control shRNA targeting the lacZ gene ( lacZ ) . Four of five DN2 - derived leukemias show reduced growth with both shRNAs after the second round of sorting within 8 days in culture . The lacZ control was set to 100 % .

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E. Percent cell growth of 5 different DN2-derived leukemic cell lines transduced with 2 different shRNA constructs targeting Jak2. The cells were sorted for the GFP or YFP reporter and normalized to the control shRNA targeting the lacZ gene (lacZ). Four of five DN2-derived leukemias show reduced growth with both shRNAs after the second round of sorting within 8 days in culture. The lacZ control was set to 100%.


F . Proliferation curves of 3 Myc / Bcl2 + DN2 - and 3 GMP - derived leukemia lines during treatment with 0 . 5 μM ruxolitinib . 1 . 5105 cells were seeded in culture and counted at the indicated time points . Results are shown as fold growth of the starting cell number which was set to 1 . * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , Student ' s t - test .

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F. Proliferation curves of 3 Myc/Bcl2+ DN2- and 3 GMP-derived leukemia lines during treatment with 0.5 μM ruxolitinib. 1.5105 cells were seeded in culture and counted at the indicated time points. Results are shown as fold growth of the starting cell number which was set to 1. *=p≤0.05, **=p≤0.01, Student's t-test.


G . Western blot indicating active Jak / Stat signaling in DN2 - derived leukemia with Stat3 as well as pStat3 protein expression , that diminishes after treatment with the Jak - inhibitor ruxolitinib .

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G. Western blot indicating active Jak/Stat signaling in DN2-derived leukemia with Stat3 as well as pStat3 protein expression, that diminishes after treatment with the Jak-inhibitor ruxolitinib.


H . GSEA indicating significant enrichment of genes of the JAK / STAT signaling pathway of the KEGG pathway database in the DN2 - like M2 AML samples as compared to the residual M2 AML samples using the Verhaak AML data set ( Verhaak et al . , 2009 ) .

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H. GSEA indicating significant enrichment of genes of the JAK/STAT signaling pathway of the KEGG pathway database in the DN2-like M2 AML samples as compared to the residual M2 AML samples using the Verhaak AML data set (Verhaak et al., 2009).


( A ) LncRNA CRNDE expression level was measured by qRT - PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer ( GC ) patients . Data information : Data are expressed as mean ± SD . Student ' s t - test * * P < 0 . 01 .

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(A) LncRNA CRNDE expression level was measured by qRT-PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer (GC) patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.


( C ) LncRNA CRNDE expression level was measured by qRT - PCR in adjacent normal tissue - derived macrophages ( NTMs ) and tumor - associated macrophages ( TAMs ) obtained from 7 cases of GC patients . Data information : Data are expressed as mean ± SD . Student ' s t - test * * P < 0 . 01 .

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(C) LncRNA CRNDE expression level was measured by qRT-PCR in adjacent normal tissue-derived macrophages (NTMs) and tumor-associated macrophages (TAMs) obtained from 7 cases of GC patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.


( C ) and ( D ) Left : representative electron micrograph of exosomes isolated from the medium of BMDMs , BMDM - polarized M2 macrophages , monocytes and monocyte - polarized M2 macrophages are depicted , showing the typical morphology of exosomes . Upper right : the protein levels of CD63 and CD81 were measured by Western blot . Lower left : Nanoparticle tracking analysis showed that most of the particles had a similar size of 50 - 150 nm in diameter with a peak at around 100 nm . Lower right : LncRNA CRNDE expression levels in different exosomes . Data information : Scale bar = 100 nm . The results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - test . * * P < 0 . 01 vs . BMDMs or monocytes or BMDMs - exo or monocytes - exo .

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(C) and (D) Left: representative electron micrograph of exosomes isolated from the medium of BMDMs, BMDM-polarized M2 macrophages, monocytes and monocyte-polarized M2 macrophages are depicted, showing the typical morphology of exosomes. Upper right: the protein levels of CD63 and CD81 were measured by Western blot. Lower left: Nanoparticle tracking analysis showed that most of the particles had a similar size of 50-150 nm in diameter with a peak at around 100 nm. Lower right: LncRNA CRNDE expression levels in different exosomes. Data information: Scale bar= 100 nm. The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. **P<0.01 vs. BMDMs or monocytes or BMDMs-exo or monocytes-exo.


( B ) BMDM - polarized M2 macrophages were transfected with Cy3 - labelled CRNDE and exosomes were isolated from the medium ( Cy3 - CRNDE - EXO ) . Then , MFC cells were incubated with Cy3 - CRNDE - EXO . Left : Confocal microscopy was used to detect the Cy3 - labelled CRNDE in MFC cells . The nuclei were stained with DAPI . Scale bar = 20 µm . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . * * P < 0 . 01 vs . MFC cells incubated with the control EXO . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - test .

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(B) BMDM-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, MFC cells were incubated with Cy3-CRNDE-EXO. Left: Confocal microscopy was used to detect the Cy3-labelled CRNDE in MFC cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. MFC cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.


( D ) Monocyte - polarized M2 macrophages were transfected with Cy3 - labelled CRNDE and exosomes were isolated from the medium ( Cy3 - CRNDE - EXO ) . Then , SGC7901 cells were incubated with Cy3 - CRNDE - EXO . Confocal microscopy was used to detect the Cy3 - labelled CRNDE in SGC7901 cells . The nuclei were stained with DAPI . Scale bar = 20 µm . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . * * P < 0 . 01 vs . SGC7901 cells incubated with the control EXO . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - test .

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(D) Monocyte-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, SGC7901 cells were incubated with Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. SGC7901 cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.


( E ) SGC7901 cells were incubated with chlorpromazine ( CPZ , 50 μM ) , genistein ( GEN , 200 μM ) , 5 - ( n - ethyl - n - isopropyl ) - amiloride ( EIPA , 100 μM ) , or methyl - β - cyclodextrin ( MβCD , 10 mM ) , following by the incubation of Cy3 - CRNDE - EXO . Confocal microscopy was used to detect the Cy3 - labelled CRNDE in SGC7901 cells . The nuclei were stained with DAPI . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA .

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(E) SGC7901 cells were incubated with chlorpromazine (CPZ, 50 μM), genistein (GEN, 200 μM), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA, 100 μM), or methyl-β-cyclodextrin (MβCD, 10 mM), following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.


( F ) SGC7901 cells were incubated with 0 , 25 , 50 , 100 μM CPZ , following by the incubation of Cy3 - CRNDE - EXO . Confocal microscopy was used to detect the Cy3 - labelled CRNDE in SGC7901 cells . The nuclei were stained with DAPI . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA .

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(F) SGC7901 cells were incubated with 0, 25, 50, 100 μM CPZ, following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.


Exosomes were isolated from the media of BMDMs ( BMDMs - exo ) , BMDM - polarized M2 macrophages ( M2 - exo ) , BMDM - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and BMDM - polarized M2 macrophages transfected with the negative control of si - CRNDE ( M2 - si - control - exo ) . ( A ) LncRNA CRNDE expression in indicated exosomes was detected by qRT - PCR . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . : one - way ANOVA . * * P < 0 . 01 .

Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). (A) LncRNA CRNDE expression in indicated exosomes was detected by qRT-PCR. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.


Exosomes were isolated from the media of BMDMs ( BMDMs - exo ) , BMDM - polarized M2 macrophages ( M2 - exo ) , BMDM - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and BMDM - polarized M2 macrophages transfected with the negative control of si - CRNDE ( M2 - si - control - exo ) . Then , MFC cells were treated with indicated exosomes and cisplatin ( CDDP ; 1μg / ml ) for 48 h . ( C ) Representative images of colony formation assay performed on MFC cells .

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

Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). Then, MFC cells were treated with indicated exosomes and cisplatin (CDDP; 1μg/ml) for 48 h. (C) Representative images of colony formation assay performed on MFC cells.


Exosomes were isolated from the medium of monocytes ( monocytes - exo ) , monocyte - polarized M2 macrophages ( M2 - exo ) , monocyte - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and monocyte - polarized M2 macrophages transfected with si - control ( M2 - si - control - exo ) . ( E ) LncRNA CRNDE expression were measured . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

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

Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). (E) LncRNA CRNDE expression were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


Exosomes were isolated from the medium of monocytes ( monocytes - exo ) , monocyte - polarized M2 macrophages ( M2 - exo ) , monocyte - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and monocyte - polarized M2 macrophages transfected with si - control ( M2 - si - control - exo ) . Then , SGC7901 cells were treated with indicated exosomes and CDDP ( 1μg / ml ) . ( G ) cell proliferation were measured .

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

Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). Then, SGC7901 cells were treated with indicated exosomes and CDDP (1μg/ml). (G) cell proliferation were measured.


3 × 105 MFC cells were subcutaneously injected into nude mice . Ten days after injection , BMDMs - exo , M2 - exo , M2 - si - control - exo , or M2 - si - CRNDE - exo ( 10 µg ) was injected into the center of the homograft tumors of mice ( n = 7 each group ) , followed by intraperitoneal injection of CDDP ( 10 mg / kg ) . Six days after CDDP treatment , all mice were sacrificed and the homograft tumors were collected . ( A ) Growth curves of MFC subcutaneous homograft tumors ( The homograft tumor sizes were recorded from the day of CDDP treatment ) . * P < 0 . 05 , * * P < 0 . 01 vs . BMDMs - exo ; # P < 0 . 05 , # # P < 0 . 01 vs . M2 - si - control - exo . Data information : Data are expressed as mean ± SD . one - way ANOVA .

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

3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (A) Growth curves of MFC subcutaneous homograft tumors (The homograft tumor sizes were recorded from the day of CDDP treatment). *P<0.05, **P<0.01 vs. BMDMs-exo; #P<0.05, ##P<0.01 vs. M2-si-control-exo. Data information: Data are expressed as mean ± SD. one-way ANOVA.


3 × 105 MFC cells were subcutaneously injected into nude mice . Ten days after injection , BMDMs - exo , M2 - exo , M2 - si - control - exo , or M2 - si - CRNDE - exo ( 10 µg ) was injected into the center of the homograft tumors of mice ( n = 7 each group ) , followed by intraperitoneal injection of CDDP ( 10 mg / kg ) . Six days after CDDP treatment , all mice were sacrificed and the homograft tumors were collected . ( D ) LncRNA CRNDE expression in homograft tumors of mice . * * P < 0 . 01 . Data information : Data are expressed as mean ± SD . : one - way ANOVA .

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

3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (D) LncRNA CRNDE expression in homograft tumors of mice. **P<0.01. Data information: Data are expressed as mean ± SD. : one-way ANOVA.


( C ) SGC7901 cells were transfected with pcDNA - CRNDE or its negative control ( Mock ) before MG132 treatment ( 10 µM , 6 h ) . The Co - immunoprecipitation ( Co - IP ) assay was performed to examine the combination of PTEN and NEDD4 - 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1.


( E ) The expressions of PTEN and NEDD4 - 1 were measured in SGC7901 cells transfected with pcDNA - CRNDE or Mock at four time - points after cycloheximide ( CHX , 100 μg / mL ) treatment . GAPDH was served as an internal control . Left : Representative bands . Right : Quantitative results . The results shown are from three biological replicates and are expressed as mean ± SD . Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 vs . Mock .

O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The expressions of PTEN and NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock.


G ) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors .

G) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors.


Normal , si - control - transfected , or si - PTEN - transfected SGC7901 cells were treated with CDDP ( 1 µg / ml ) and M2 - si - control - exo or M2 - si - CRNDE - exo . Si - control , the negative control of si - PTEN . ( A ) The protein levels of PTEN , phosphorylated phosphatidylinositol 3 - kinase ( p - PI3K ) , phosphorylated protein kinase B ( p - AKT ) , p - glycoprotein ( p - gp ) , B - cell lymphoma 2 ( Bcl - 2 ) were measured . GAPDH was served as an internal control .

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

Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. (A) The protein levels of PTEN, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), p-glycoprotein (p-gp), B-cell lymphoma 2 (Bcl-2) were measured. GAPDH was served as an internal control.


Normal , si - control - transfected , or si - PTEN - transfected SGC7901 cells were treated with CDDP ( 1 µg / ml ) and M2 - si - control - exo or M2 - si - CRNDE - exo . Si - control , the negative control of si - PTEN . The cell apoptosis in SGC7901 cells . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

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

Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The cell apoptosis in SGC7901 cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


SGC7901 cells were divided into four groups : M2 - si - control - exo , M2 - si - CRNDE - exo , M2 - si - CRNDE - exo + PTEN inhibitor ( SF1670 ; 50 μmol / l ) , M2 - si - control - exo + PTEN inhibitor . All cells were treated with CDDP . The cell survival rate were measured . Data information : The results shown are from seven biological replicates . Data are expressed as mean ± SD . : one - way ANOVA . * * P < 0 . 01 .

O O O O O O O O O O O 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

SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell survival rate were measured. Data information: The results shown are from seven biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.


SGC7901 cells were divided into four groups : M2 - si - control - exo , M2 - si - CRNDE - exo , M2 - si - CRNDE - exo + PTEN inhibitor ( SF1670 ; 50 μmol / l ) , M2 - si - control - exo + PTEN inhibitor . All cells were treated with CDDP . The cell apoptosis were measured . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

O O O O O O O O O O O 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

SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell apoptosis were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


A . cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 and the wildtype , from left to right on control plates without genotoxic agent 10 days after germination . B . The wildtype , single and double mutants of cycb1 were grown on control plates without genotoxic agent . Root lengths were measured 10 days after germination . C . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 1 mM hydroxy urea ( HU ) 10 days after germination . The rightmost plant is the wee1 mutant that shows high sensitivity to HU . D . The wildtype , single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU . Root lengths were measured 10 days after germination . E . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 0 , 6 μg / mL bleomycin ( BLM ) 10 days after germination . The rightmost plant is the ku70 mutant that shows high sensitivity to BLM . F . The wildtype , single and double mutants of cycb1 were grown on plates supplemented with 0 , 6 μg / mL BLM . Root lengths were measured 10 days after germination .

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

A. cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 and the wildtype, from left to right on control plates without genotoxic agent 10 days after germination.B. The wildtype, single and double mutants of cycb1 were grown on control plates without genotoxic agent. Root lengths were measured 10 days after germination.C. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 1 mM hydroxy urea (HU) 10 days after germination. The rightmost plant is the wee1 mutant that shows high sensitivity to HU.D. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU. Root lengths were measured 10 days after germination.E. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 0,6 μg/mL bleomycin (BLM) 10 days after germination. The rightmost plant is the ku70 mutant that shows high sensitivity to BLM.F. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.


G . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 15 μM cisplatin 6 days after germination , i . e . 3 days after transfer from control plates . H . cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination . Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . I . cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . J . cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs .

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

G. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 15 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.H. cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination. Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.I. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.J. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.