Figure 2A , Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV - injection . In mice injected with GFP control virus , virtually all GFP positive cells depict an astrocytic morphology , many GFP positive cells in ALNe - 218 and ALN - treated animals show a neuron - like morphology . Data information : Scale bars indicate 20 µm . B , Immunohistochemical analysis showing GFP + / GFAP + double positive cells 13 wpi . Arrows indicate double positive GFP + / GFAP + cells , arrowheads indicate GFP + / GFAP - cells . Quantification of GFAP + / GFP + cells shows a significant decrease upon ALNe - 218 and ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0064 , GFP vs . ALN P = 0 . 0002 and ALN vs . ALNe - 218 P = 0 . 0127 ) . Multiple comparison ANOVA F ( 2 , 7 ) = 32 . 06 . C , Double immunostaining for GFP and the neuronal marker NeuN . Arrowheads indicate double positive GFP + / NeuN + cell 13 wpi . Quantification demonstrate a significant increase in NeuN + / GFP + cells upon ALNe - 218 and ALN - induction ( GFP vs . ALNe - 218 P < 0 . 0001 , GFP vs . ALN P < 0 . 0001 and ALN vs . ALNe - 218 P = 0 . 0012 ) . Multiple comparison ANOVA F ( 2 , 6 ) = 170 . 3 . Data information : Scale bars indicate 20 µm . Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . n = 3 - 4 mice per condition . Error bars represent mean ± SD . D , Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP - control , ALNe - 218 and ALN - treated animals . Arrowheads indicate GFP + / TH - cells with neuronal morphology . No GFP + / TH + cell could be detected in any of the experimental groups . Scale bars indicate 20 µm . Data information : Scale bars indicate 20 µm .

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

Figure 2A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In mice injected with GFP control virus, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.B, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0064, GFP vs. ALN P=0.0002 and ALN vs. ALNe-218 P=0.0127). Multiple comparison ANOVA F(2,7)=32.06. C, Double immunostaining for GFP and the neuronal marker NeuN. Arrowheads indicate double positive GFP+/NeuN+ cell 13 wpi. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALNe-218 P<0.0001, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0012). Multiple comparison ANOVA F(2,6)=170.3. Data information: Scale bars indicate 20 µm. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. n = 3-4 mice per condition. Error bars represent mean ± SD.D, Double immunostaining for GFP and TH as a marker for dopaminergic neurons in the dorsal striatum of GFP-control, ALNe-218 and ALN-treated animals. Arrowheads indicate GFP+/TH- cells with neuronal morphology. No GFP+/TH+ cell could be detected in any of the experimental groups. Scale bars indicate 20 µm. Data information: Scale bars indicate 20 µm.


Figure 3B , RT - qPCR analysis of Ascl1 induction comparing the activation capacity of full - length versus split - dCas9 in Neuro2A cells ( data in fold change normalized to non - activated control ; dCas9 6116 ± 847 . 3 , split - dCas9 4415 ± 748 . 8 , n = 3 biological replicates , box - and - whiskers plots indicate median , 25th - 75th percentile and min / max whiskers ) . C , Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa . Astrocytes are infected with two lentiviruses expressing dCas9 - VPR in a intein - split version similar to the AAV - dCAS system but driven by a Tet - O promoter . Tet - O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively . Arrows indicate single MAP2 positive background neurons , arrowheads indicate double positive induced neurons . Scale bar indicates 20 µm . Data information : Abbreviations : OE - overexpressionE , Multiplexed activation of Ascl1 / Lmx1a / NeuroD1 , Ascl1 / Lmx1a / Nr4a2 and Ascl1 / Lmx1a / Nr4a2 / Pitx3 / FoxA2 in primary astrocytic cells . n = 2 - 3 biological replicates , one representative run is shown , additional data in supplement . Activation levels are depicted as fold change between cells transfected with and without sgRNAs . All levels were normalized to β - Actin . Error bars represent mean ± SD between technical replicates .

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

Figure 3B, RT-qPCR analysis of Ascl1 induction comparing the activation capacity of full-length versus split-dCas9 in Neuro2A cells (data in fold change normalized to non-activated control; dCas9 6116 ± 847.3, split-dCas9 4415 ± 748.8, n=3 biological replicates, box-and-whiskers plots indicate median, 25th-75th percentile and min/max whiskers).C, Immunocytochemistry analysis of reprogrammed primary astrocytes cells 16 days after lentiviral transduction revealed successful in vitro reprogramming into neurons using CRISPRa. Astrocytes are infected with two lentiviruses expressing dCas9-VPR in a intein-split version similar to the AAV-dCAS system but driven by a Tet-O promoter. Tet-O driven dsRed and Ascl1 cDNA expressing construct were used as negative and positive controls respectively. Arrows indicate single MAP2 positive background neurons, arrowheads indicate double positive induced neurons. Scale bar indicates 20 µm. Data information: Abbreviations: OE - overexpressionE, Multiplexed activation of Ascl1/Lmx1a/NeuroD1, Ascl1/Lmx1a/Nr4a2 and Ascl1/ Lmx1a/ Nr4a2/Pitx3/FoxA2 in primary astrocytic cells. n=2-3 biological replicates, one representative run is shown, additional data in supplement. Activation levels are depicted as fold change between cells transfected with and without sgRNAs. All levels were normalized to β-Actin. Error bars represent mean ± SD between technical replicates.


Figure 4A , Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV - injection . In the GFP control condition , virtually all GFP positive cells depict an astrocytic morphology , many GFP positive cells in ALNe - 218 and ALN - treated animals show a neuron - like morphology . Data information : Scale bars indicate 20 µm . B , Immunohistochemical analysis showing GFP + / GFAP + double positive cells 13 wpi . Arrows indicate double positive GFP + / GFAP + cells , arrowheads indicate GFP + / GFAP - cells . Quantification of GFAP + / GFP + cells shows a significant decrease upon ALNe - 218 and ALN - treatment ( GFP vs . ALNe - 218 P = 0 . 0083 , GFP vs . ALN P < 0 . 0001 and ALN vs . ALNe - 218 P = 0 . 0006 , multiple comparison ANOVA F ( 2 , 6 ) = 79 . 76 ) . C , Photomicrographs showing GFP + / NeuN + neurons 13 wpi . Arrowheads indicating GFP + / NeuN + induced neurons . Quantification demonstrate a significant increase in NeuN + / GFP + cells upon ALNe - 218 and ALN - induction ( GFP vs . ALN P = 0 . 0008 , ALN vs . ALNe - 218 P = 0 . 0092 , multiple comparison ANOVA F ( 2 , 7 ) = 21 . 74 ) . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 3 - 4 mice ( b , c ) per condition . Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . D , In ALN - induced neurons , confocal analysis is demonstrating a co - localization of GFP and glutamic acid decarboxylase ( Gad65 / 67 ) , a marker specific for GABAergic neurons . E , Quantification of GAD6567 + / GFP + cells with neuronal morphology shows that the majority of ALN - induced neurons are colocalizing with this GABAergic marker both in the AAV - dCAS as well as in the dCAM setting . Data information : Scale bars indicate 20 µm . Error bars represent mean ± SD . n = 6 - 8 mice ( e ) per condition . Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

Figure 4A, Representative photomicrographs taken from the dorsal striatum 13 weeks after AAV-injection. In the GFP control condition, virtually all GFP positive cells depict an astrocytic morphology, many GFP positive cells in ALNe-218 and ALN-treated animals show a neuron-like morphology. Data information: Scale bars indicate 20 µm.B, Immunohistochemical analysis showing GFP+/GFAP+ double positive cells 13 wpi. Arrows indicate double positive GFP+/GFAP+ cells, arrowheads indicate GFP+/GFAP- cells. Quantification of GFAP+/GFP+ cells shows a significant decrease upon ALNe-218 and ALN-treatment (GFP vs. ALNe-218 P=0.0083, GFP vs. ALN P<0.0001 and ALN vs. ALNe-218 P=0.0006, multiple comparison ANOVA F(2,6)=79.76). C, Photomicrographs showing GFP+/NeuN+ neurons 13 wpi. Arrowheads indicating GFP+/NeuN+ induced neurons. Quantification demonstrate a significant increase in NeuN+/GFP+ cells upon ALNe-218 and ALN-induction (GFP vs. ALN P=0.0008, ALN vs. ALNe-218 P=0.0092, multiple comparison ANOVA F(2,7)=21.74). Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n = 3-4 mice (b,c) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.D, In ALN-induced neurons, confocal analysis is demonstrating a co-localization of GFP and glutamic acid decarboxylase (Gad65/67), a marker specific for GABAergic neurons. E, Quantification of GAD6567+/GFP+ cells with neuronal morphology shows that the majority of ALN-induced neurons are colocalizing with this GABAergic marker both in the AAV-dCAS as well as in the dCAM setting. Data information: Scale bars indicate 20 µm. Error bars represent mean ± SD. n= 6-8 mice (e) per condition. Tukey's multiple comparisons test * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


Figure 7A , Gait analysis using the CatWalk XT system reveals motor defects in 6 - OHDA lesion model . Both dCAM and AAV - dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread . Naive vs . 6 - OHDA P = 0 . 0063 . AAV - dCAS : GFP vs . ALN P = 0 . 015 multiple comparison ANOVA F ( 2 , 17 ) = 12 . 81 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . the stride length of front paws : B , Naive vs . 6 - OHDA P = 0 . 0164 . AAV - dCAS : GFP vs . ALN P = 0 . 005 , ALN vs . ALNe - 218 P = 0 . 0042 , multiple comparison ANOVA F ( 4 , 22 ) = 9 . 9 ) Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . the duty cycle of left front paws . C , Naive vs . 6 - OHDA P = 0 . 0096 . dCAM : GFP vs . ALN P = 0 . 036 and ALN vs . ALNe - 218 P = 0 . 0252 , multiple comparison ANOVA F ( 2 , 20 ) = 5 . 199 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . D , In contrast to this , dopamine dependent drug - induced behavior does not show rescue effects : amphetamine - induced rotation analysis : change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance . Net rotation = ipsilateral rotation - contralateral rotation ( naïve vs . 6 - OHDA P = 0 . 09 ) . Data information : Statistics : naive vs . 6 - OHDA unpaired t - test ( two - tailed ) * P < 0 . 05 , * * P < 0 . 01 . GFP vs ALN , GFP vs ALNe - 218 and ALN vs ALNe - 218 Tukey ' s multiple comparisons test * P < 0 . 05 , * * P < 0 . 01 . n = 4 - 8 mice per condition . CatWalk error bars represent mean ± SD . Rotation analysis error bars represent mean ± SEM .

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

Figure 7A, Gait analysis using the CatWalk XT system reveals motor defects in 6-OHDA lesion model. Both dCAM and AAV-dCAS animals transduce with AAV containing specific gRNAs show a significant improvement in different aspects of voluntary movement like the average speed of tread. Naive vs. 6-OHDA P=0.0063. AAV-dCAS: GFP vs. ALN P=0.015 multiple comparison ANOVA F(2,17)=12.81) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.the stride length of front paws: B, Naive vs. 6-OHDA P=0.0164. AAV-dCAS: GFP vs. ALN P=0.005, ALN vs. ALNe-218 P=0.0042, multiple comparison ANOVA F(4,22)=9.9) Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.the duty cycle of left front paws. C, Naive vs. 6-OHDA P=0.0096. dCAM: GFP vs. ALN P=0.036 and ALN vs. ALNe-218 P=0.0252, multiple comparison ANOVA F(2,20)=5.199). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition.D, In contrast to this, dopamine dependent drug-induced behavior does not show rescue effects: amphetamine-induced rotation analysis: change in rotational behavior in lesioned animals upon treatment with dopamine releaser substance. Net rotation = ipsilateral rotation-contralateral rotation (naïve vs. 6-OHDA P=0.09). Data information: Statistics: naive vs. 6-OHDA unpaired t-test (two-tailed) * P<0.05, ** P<0.01. GFP vs ALN, GFP vs ALNe-218 and ALN vs ALNe-218 Tukey's multiple comparisons test * P<0.05, ** P<0.01. n = 4-8 mice per condition. CatWalk error bars represent mean ± SD. Rotation analysis error bars represent mean ± SEM.


Figure 1 ( E - G ) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids , incubated for two days in vitro and treated with Tf - 555 for 30 minutes before fixation . Cells were immunostained with the indicated antibodies . The images are obtained with high - resolution microscopy ( Nikon ) . Blue alone channels are shown in black and white images . Lower panels in ( E ) and ( F ) are high magnification images indicated by white or blue rectangles in upper panels . The graph in ( G ) shows the Pearson ' s correlation coefficient of Tf - 555 and Rab11 or APPL1 . Each score represents the mean with the individual points . Control : n = 23 cells , Rab21 - sh115 : n = 18 cells , Rab5 - sh232 ( Tf - Rab11 ) : n = 23 cells , Rab5 - sh232 ( Tf - APPL1 ) : n = 22 cells .

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

Figure 1(E-G) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro and treated with Tf-555 for 30 minutes before fixation. Cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). Blue alone channels are shown in black and white images. Lower panels in (E) and (F) are high magnification images indicated by white or blue rectangles in upper panels. The graph in (G) shows the Pearson's correlation coefficient of Tf-555 and Rab11 or APPL1. Each score represents the mean with the individual points. Control: n = 23 cells, Rab21-sh115: n = 18 cells, Rab5-sh232 (Tf - Rab11): n = 23 cells, Rab5-sh232 (Tf - APPL1): n = 22 cells.


Figure 2Cerebral cortices at E17 , electroporated with the indicated plasmids plus pCAG - EGFP at E14 . ( C ) The ratio of locomoting neurons with more than three primary neurites in the IZ . Each score represents the mean with the individual points . Control : n = 4 brains ( 60 cells ) , Rab21 - sh115 : n = 3 brains ( 65 cells ) , Rab5 - sh232 : n = 4 brains ( 111 cells ) . ( E ) Time - lapse imaging of control and Rab21 - sh115 - electroporated cells in cortical slices from E16 cerebral cortices , electroporated with the indicated plasmids at E14 . After formation of the leading process , control neurons rapidly eliminated their immature neurites ( yellow arrows ) , whereas the Rab21 - knockdown neurons retained the immature neurites ( white arrows ) for long periods . Time interval of each frame is 30 min .

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

Figure 2Cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (C) The ratio of locomoting neurons with more than three primary neurites in the IZ. Each score represents the mean with the individual points. Control: n = 4 brains (60 cells), Rab21-sh115: n = 3 brains (65 cells), Rab5-sh232: n = 4 brains (111 cells).(E) Time-lapse imaging of control and Rab21-sh115-electroporated cells in cortical slices from E16 cerebral cortices, electroporated with the indicated plasmids at E14. After formation of the leading process, control neurons rapidly eliminated their immature neurites (yellow arrows), whereas the Rab21-knockdown neurons retained the immature neurites (white arrows) for long periods. Time interval of each frame is 30 min.


Figure 3Primary cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 ( green ) and incubated for two days in vitro . Cells were immunostained with the indicated antibodies ( red and blue / white ) . The transfected PM - mAG1 is a marker for the plasma membrane . The images are obtained with high - resolution microscopy ( Nikon ) and the lower panels are high magnification images near the plasma membrane . Blue alone channels are shown in black and white images . Each score represents the mean of ratios with the individual points . Rab5 : n = 18 cells ( middle and right ) , Rab21 : n = 31 cells ( left and middle ) or 20 cells ( right ) , Caveolin - 1 : n = 43 cells ( left ) . ( H - I ) HeLa cells were immunostained with the indicated antibodies . The images are obtained with high - resolution microscopy ( Nikon ) . The graph in ( I ) shows the Pearson ' s correlation coefficient between caveolin - 1 and Rab5 or Rab21 . Each score represents the mean of ratios with the individual points . Rab5 : n = 80 cells , Rab21 : n = 62 cells .

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

Figure 3Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1 (green) and incubated for two days in vitro. Cells were immunostained with the indicated antibodies (red and blue/white). The transfected PM-mAG1 is a marker for the plasma membrane. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images near the plasma membrane. Blue alone channels are shown in black and white images. Each score represents the mean of ratios with the individual points. Rab5: n = 18 cells (middle and right), Rab21: n = 31 cells (left and middle) or 20 cells (right), Caveolin-1: n = 43 cells (left).(H-I) HeLa cells were immunostained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon). The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Rab5 or Rab21. Each score represents the mean of ratios with the individual points. Rab5: n = 80 cells, Rab21: n = 62 cells.


Figure 4Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG - EGFP , incubated for two days in vitro and treated with BODIPY - LacCer ( LacCer ) ( green ) ( A - B ) for 30 minutes before fixation . White arrows in ( A ) indicate the perinuclear accumulation of LacCer . Blue alone channels are shown in black and white images . The graphs in ( B ) show the ratio of cells with perinuclear accumulation of LacCer ( B ) which was quantified in a blinded counting . Each score represents the mean with the individual points . B : n = 5 biological replicates ( Control : 115 cells , Cav1 - sh490 : 127 cells , Rab21 - sh115 : 136 cells , Rab5 - sh232 : 216 cells , Rab21 - sh115 + wt - Rab21 : 132 cells )

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Figure 4Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-EGFP , incubated for two days in vitro and treated with BODIPY-LacCer (LacCer) (green) (A-B) for 30 minutes before fixation. White arrows in (A) indicate the perinuclear accumulation of LacCer. Blue alone channels are shown in black and white images. The graphs in (B) show the ratio of cells with perinuclear accumulation of LacCer (B) which was quantified in a blinded counting. Each score represents the mean with the individual points. B: n = 5 biological replicates (Control: 115 cells, Cav1-sh490: 127 cells, Rab21-sh115: 136 cells, Rab5-sh232: 216 cells, Rab21-sh115 + wt-Rab21: 132 cells)


Figure 5Primary cortical neurons from E15 cerebral cortices were transfected with pCAG - PM - mAG1 , incubated for two days in vitro and stained with the indicated antibodies . Blue alone channels are shown in black and white images . Arrowheads indicate accumulation of N - cadherin at the plasma membrane . Lower panels in ( D ) are high magnification images indicated by white or blue rectangles . The images are obtained with high - resolution microscopy ( Nikon ) . Immature neurons in the IZ of the cerebral cortices at E17 , electroporated with the indicated plasmids plus pCAG - PM - mAG1 and pCAG - HA - N - cadherin at E14 . Frozen sections were immunostained with anti - mAG1 and anti - HA antibodies . Arrow and arrowheads indicate the accumulation of HA - N - cadherin at the perinuclear regions or the plasma membrane , respectively . Control : n = 77 cells from 3 biological replicates , Rab21 - sh115 : n = 108 cells from 3 biological replicates .

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Figure 5Primary cortical neurons from E15 cerebral cortices were transfected with pCAG-PM-mAG1, incubated for two days in vitro and stained with the indicated antibodies. Blue alone channels are shown in black and white images. Arrowheads indicate accumulation of N-cadherin at the plasma membrane. Lower panels in (D) are high magnification images indicated by white or blue rectangles. The images are obtained with high-resolution microscopy (Nikon).Immature neurons in the IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-PM-mAG1 and pCAG-HA-N-cadherin at E14. Frozen sections were immunostained with anti-mAG1 and anti-HA antibodies. Arrow and arrowheads indicate the accumulation of HA-N-cadherin at the perinuclear regions or the plasma membrane, respectively. Control: n = 77 cells from 3 biological replicates, Rab21-sh115: n = 108 cells from 3 biological replicates.


Figure 6 ( A - B ) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG - PM - mAG1 ( green ) and incubated for two days in vitro . Immunocytochemical analyses with anti - caveolin - 1 ( red ) antibody were performed . The images are obtained with high - resolution microscopy ( Nikon ) . The graph in ( B ) indicates the ratio of the caveolin - 1 staining signals in the plasma membrane to total fluorescence intensities of caveolin - 1 in each immature neuron . Each score represents the mean of ratios with the individual points . Control : n = 20 cells , Rab21 - sh115 : n = 22 cells , Rab21 - sh115 + wt - Rab21 : n = 23 cells . Overexpression of wt - Rab21 resulted in neuronal cell death , which making it difficult to determine the accurate DNA concentration of wt - Rab21 in the rescue experiments . ( H - I ) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids , incubated for two days in vitro , treated with Bafilomycin A1 ( Baf A1 ) for 6 h and stained with the indicated antibodies . The images are obtained with high - resolution microscopy ( Nikon ) and the lower panels are high magnification images of the lysosomes , indicated by white or blue rectangles . Blue alone channels are shown in black and white images . The graph in ( I ) shows the Pearson ' s correlation coefficient between caveolin - 1 and Lamp1 , a lysosomal marker , in control and Rab21 - sh115 - transfected neurons . Each score represents the mean of ratios with the individual points . Control : n = 31 cells , Rab21 - sh115 : n = 22 cells .

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Figure 6(A-B) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids plus pCAG-PM-mAG1 (green) and incubated for two days in vitro. Immunocytochemical analyses with anti-caveolin-1 (red) antibody were performed. The images are obtained with high-resolution microscopy (Nikon). The graph in (B) indicates the ratio of the caveolin-1 staining signals in the plasma membrane to total fluorescence intensities of caveolin-1 in each immature neuron. Each score represents the mean of ratios with the individual points. Control: n = 20 cells, Rab21-sh115: n = 22 cells, Rab21-sh115 + wt-Rab21: n = 23 cells. Overexpression of wt-Rab21 resulted in neuronal cell death, which making it difficult to determine the accurate DNA concentration of wt-Rab21 in the rescue experiments.(H-I) Primary cortical neurons from E15 cerebral cortices were transfected with the indicated plasmids, incubated for two days in vitro, treated with Bafilomycin A1 (Baf A1) for 6 h and stained with the indicated antibodies. The images are obtained with high-resolution microscopy (Nikon) and the lower panels are high magnification images of the lysosomes, indicated by white or blue rectangles. Blue alone channels are shown in black and white images. The graph in (I) shows the Pearson's correlation coefficient between caveolin-1 and Lamp1, a lysosomal marker, in control and Rab21-sh115-transfected neurons. Each score represents the mean of ratios with the individual points. Control: n = 31 cells, Rab21-sh115: n = 22 cells.


Figure 7 ( A ) Locomoting neurons in the upper IZ of the cerebral cortices at E17 , electroporated with the indicated plasmids plus pCAG - EGFP at E14 . ( B ) The ratio of locomoting neurons with more than three primary neurites . Each score represents the mean of ratios with the individual points . Control : n = 4 brains , Rab21 - sh115 : n = 5 brains , Rab21 - sh115 + Caveolin - 1 : n = 6 brains . ( C ) The box - and - whisker plot shows average leading process length of the locomoting neurons in the IZ . Control : n = 115 cells , Rab21 - sh115 : n = 188 cells , Rab21 - sh115 + Caveolin - 1 : n = 185 cells . ( ( D - E ) Cerebral cortices at P0 , electroporated with the indicated plasmids plus pCAG - EGFP at E14 . The lower graphs in ( D ) and the graph in ( E ) show the estimation of cell migration , as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software . Each bar in the graph in ( E ) represents the mean percentage of relative intensities . Rab21 - sh115 : n = 4 brains , Rab21 - sh115 + CAG - wt - caveolin - 1 : n = 6 brains . II - IV : layers II - IV of the cortical plate , V - VI : layers V - VI of the cortical plate , IZ : intermediate zone , WM : white matter , SVZ / VZ : subventricular zone / ventricular zone .

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Figure 7(A) Locomoting neurons in the upper IZ of the cerebral cortices at E17, electroporated with the indicated plasmids plus pCAG-EGFP at E14. (B) The ratio of locomoting neurons with more than three primary neurites. Each score represents the mean of ratios with the individual points. Control: n = 4 brains, Rab21-sh115: n = 5 brains, Rab21-sh115+Caveolin-1: n = 6 brains. (C) The box-and-whisker plot shows average leading process length of the locomoting neurons in the IZ. Control: n = 115 cells, Rab21-sh115: n = 188 cells, Rab21-sh115+Caveolin-1: n = 185 cells. ((D-E) Cerebral cortices at P0, electroporated with the indicated plasmids plus pCAG-EGFP at E14. The lower graphs in (D) and the graph in (E) show the estimation of cell migration, as measured by EGFP fluorescence intensities in distinct regions of the cerebral cortices using Leica SP5 software. Each bar in the graph in (E) represents the mean percentage of relative intensities. Rab21-sh115: n = 4 brains, Rab21-sh115+CAG-wt-caveolin-1: n = 6 brains. II-IV: layers II-IV of the cortical plate, V-VI: layers V-VI of the cortical plate, IZ: intermediate zone, WM: white matter, SVZ/VZ: subventricular zone/ventricular zone.


Figure 1 ( F ) Examples of expression levels of Treg signature genes Foxp3 , Il10ra , Nrp1 , Ikzf4 and Ccr6 ; and examples of expression levels of genes upregulated in Tet2 / 3 DKO Treg cells Eomes , Ccl4 , Chek1 and Fn1 . The Y - axis shows FPKM ( Fragment Per Kilobase per Million reads ) , the X - axis shows the different cell types . Error bars show mean ± S . D . from two ( CD4 + naïve T cells ) or four ( WT and Tet2 / 3 DKO Treg ) biological replicates .

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Figure 1(F) Examples of expression levels of Treg signature genes Foxp3, Il10ra, Nrp1, Ikzf4 and Ccr6; and examples of expression levels of genes upregulated in Tet2/3 DKO Treg cells Eomes, Ccl4, Chek1 and Fn1. The Y-axis shows FPKM (Fragment Per Kilobase per Million reads), the X-axis shows the different cell types. Error bars show mean ± S.D. from two (CD4+ naïve T cells) or four (WT and Tet2/3 DKO Treg) biological replicates.


Figure 2 ( D ) Genome browser view of CMS - IP data showing CMS ( 5hmC ) distribution at Il2ra and Foxp3 locus for CD4 + naïve T cells and WT Treg cells . Hydroxymethylated regions ( hmRs ) and DhmRs are shown on the top ; conservation ( cons ) is shown at the bottom .

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Figure 2(D) Genome browser view of CMS-IP data showing CMS (5hmC) distribution at Il2ra and Foxp3 locus for CD4+ naïve T cells and WT Treg cells. Hydroxymethylated regions (hmRs) and DhmRs are shown on the top; conservation (cons) is shown at the bottom.


Figure 3 ( E ) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4 + naïve T cells , WT Treg cells and Tet2 / 3 DKO Treg cells . Differentially hydroxymethylated regions ( DhmRs ) and differentially methylated CpGs ( DM - CpGs ) are shown on top of the tracks . Conservation track ( Cons ) and denotation of all CpGs are shown at the bottom . WGBS data are from two biological replicate samples .

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Figure 3(E) Genome browser view of DNA methylation patterns of Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. Differentially hydroxymethylated regions (DhmRs) and differentially methylated CpGs (DM-CpGs) are shown on top of the tracks. Conservation track (Cons) and denotation of all CpGs are shown at the bottom. WGBS data are from two biological replicate samples.


Figure 4 ( E ) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4 + naïve T cells , WT Treg cells and Tet2 / 3 DKO Treg cells . DhmRs , DM - CpGs and DARs ( shaded ) are shown on top of the tracks . Conservation track ( Cons ) is shown at the bottom .

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Figure 4(E) Genome browser view of chromatin accessibilities at Hopx and Il10 loci for CD4+ naïve T cells, WT Treg cells and Tet2/3 DKO Treg cells. DhmRs, DM-CpGs and DARs (shaded) are shown on top of the tracks. Conservation track (Cons) is shown at the bottom.


Figure 5 ( H - J ) Box - and - whisker plots of DNA methylation levels for DM - CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs ( H ) , DM - CpGs less methylated in WT Treg cells vs CD4 + naïve T cells ( I ) and DM - CpGs less methylated in WT Treg cells vs Tet2 / 3 DKO Treg cells ( J ) . DM - CpGs denotes the CpGs in the respective cell types used for the comparison . Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1 . 5 times the interquartile range . P - values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey ' s post - hoc test . Data are from two biological replicates .

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Figure 5(H-J) Box-and-whisker plots of DNA methylation levels for DM-CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated in WT Treg cells vs CD4+ naïve T cells (I) and DM-CpGs less methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.


Figure 7 ( D ) Graphs depicting the percentage of viable cells for TGFβ iTregs ( left ) and TGFβ + RA + VitC iTregs ( right ) differentiated with 0 U / ml , 1 U / ml , 5 U / ml and 10 U / ml IL - 2 . Error bars show mean ± S . D . from three biological replicates .

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Figure 7(D) Graphs depicting the percentage of viable cells for TGFβ iTregs (left) and TGFβ+RA+VitC iTregs (right) differentiated with 0 U/ml, 1 U/ml, 5 U/ml and 10 U/ml IL-2. Error bars show mean ± S.D. from three biological replicates.


Figure 8 ( C ) ChIP - qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U / ml IL - 2 restimulation . GMPR is a negative control region . Data are from two biological replicates .

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Figure 8(C) ChIP-qPCR showing STAT5 occupancy at Foxp3 CNS2 and IL2Rα IN1b regions in TGFβ and TGFβ + RA + VitC iTregs without or with 3 U/ml IL-2 restimulation. GMPR is a negative control region. Data are from two biological replicates.


Figure 1A . Expression of Dot1L in different B - cell populations ( Shi et al . , 2015 ) . B1 : B1 cells , MZB : marginal zone B , FOB : follicular B , GCB : germinal center B , SPLPB : spleen plasma blast , SPLPC : spleen plasma cells , and BMPC : Bone marrow plasma cells . Expression is shown as transcript per million ( TPM ) . Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates ( WT ; n = 2 ) . . Bars indicate mean values .

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Figure 1A. Expression of Dot1L in different B-cell populations (Shi et al., 2015). B1: B1 cells, MZB: marginal zone B, FOB: follicular B, GCB: germinal center B, SPLPB: spleen plasma blast, SPLPC: spleen plasma cells, and BMPC: Bone marrow plasma cells. Expression is shown as transcript per million (TPM). Results represent the data from two biological replicates except SPLPB and BMPC which lack replicates (WT; n=2)..Bars indicate mean values.


Figure 2A - G . Statistical analysis of absolute number of total nucleated splenocytes , splenic B - and T - cells , and indicated mature B cell subsets in WT and KO mice . Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group ( A : ( WT ) n = 16 , ( KO ) n = 17 ; B ( B - cells ) : ( WT ) n = 16 , ( KO ) n = 17 ; B ( T - cells ) : ( WT ) n = 8 , ( KO ) n = 9 ; C - E : ( WT ) n = 9 , ( KO ) n = 9 ; F : ( WT ) n = 6 , ( KO ) n = 6 ; G : ( WT ) n = 9 , ( KO ) n = 9 ) .

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Figure 2A-G. Statistical analysis of absolute number of total nucleated splenocytes, splenic B- and T-cells, and indicated mature B cell subsets in WT and KO mice. Results represent the data pooled from at least two independent experiments and numbers represent biological replicates for each group (A: (WT) n = 16, (KO) n = 17; B (B-cells): (WT) n = 16, (KO) n = 17; B (T-cells): (WT) n = 8, (KO) n = 9; C-E: (WT) n = 9, (KO) n = 9; F: (WT) n = 6, (KO) n = 6; G: (WT) n = 9, (KO) n = 9).


Figure 3D - E . Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone ( IgG3 switching ) ( D ) or with LPS + IL - 4 ( IgG1 switching ) ( E ) . Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group ( D : ( WT ) n = 8 , ( KO ) n = 9 ; E : ( WT ) n = 11 , ( KO ) n = 13 ) . F . Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti - CD40 + IL - 4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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Figure 3D-E. Statistical analysis of switching of WT and KO naïve B cells activated for four days with LPS alone (IgG3 switching) (D) or with LPS + IL-4 (IgG1 switching) (E). Results represent the data pooled from at least four independent experiments and numbers represent biological replicates for each group (D: (WT) n = 8, (KO) n = 9; E: (WT) n = 11, (KO) n = 13). F. Statistical analysis of IgG1 switching of naïve B cells after four days of activation with anti-CD40 + IL-4 in the presence of the DOT1L inhibitor Pinometostat or DMSO as a control. Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


Figure 4C . Statistical analysis of serum titers of LCMV - specific IgM and IgG of WT and KO mice before ( D0 ) and 14 days after ( D14 ) LCMV Armstrong infection . Results represent the data from two experiments and numbers represent biological replicates for each group ( WT ; n = 7 , KO ; n = 8 ) . D . Statistical analysis of serum titers of NP - specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge . Adjusted p - value were calculated using two - way ANOVA . Error bars indicate mean ± standard error of mean ( SEM ) . Results represent the data from one experiment and numbers represent biological replicates for each group ( WT ; n = 6 , KO ; n = 6 ) . * * * denotes p - value < 0 . 0001 and * * denotes p - value < 0 . 001 .

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Figure 4C. Statistical analysis of serum titers of LCMV-specific IgM and IgG of WT and KO mice before (D0) and 14 days after (D14) LCMV Armstrong infection. Results represent the data from two experiments and numbers represent biological replicates for each group (WT; n = 7, KO; n = 8).D. Statistical analysis of serum titers of NP-specific IgG1 and IgM quantified by ELISA from WT and KO mice at the indicated days following immune challenge. Adjusted p-value were calculated using two-way ANOVA. Error bars indicate mean ± standard error of mean (SEM). Results represent the data from one experiment and numbers represent biological replicates for each group (WT; n = 6, KO; n = 6).*** denotes p- value < 0.0001 and ** denotes p- value < 0.001.


Figure 5A . Representative flow cytometry plots showing gating strategy to identify and compare plasma cells ( Sca - I + CD138 + ) after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analyses of their relative numbers ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) . B . Representative flow cytometry plots showing gating strategy to identify and compare activated B cells ( CD138 + Blimp1 + ) , Pre - PB cells ( CD138 - Blimp1 + ) and plasma blast ( PB ) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analyses of their relative numbers ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) . C . Representative flow cytometry plots showing the relative surface density for B220 ( CD45R ) after four days of stimulation with LPS either in the presence of DOT1L inhibitor , Pinometostat or DMSO as a control ( left panel ) and statistical analysis of MFI of B220 for each treatment ( right panel ) . Results represent the data from one experiment and numbers represent biological replicates for each treatment ( n = 4 ) .

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Figure 5A. Representative flow cytometry plots showing gating strategy to identify and compare plasma cells (Sca-I+CD138+) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).B. Representative flow cytometry plots showing gating strategy to identify and compare activated B cells (CD138+ Blimp1+), Pre-PB cells (CD138- Blimp1+) and plasma blast (PB) cells after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analyses of their relative numbers (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).C. Representative flow cytometry plots showing the relative surface density for B220 (CD45R) after four days of stimulation with LPS either in the presence of DOT1L inhibitor, Pinometostat or DMSO as a control (left panel) and statistical analysis of MFI of B220 for each treatment (right panel). Results represent the data from one experiment and numbers represent biological replicates for each treatment (n = 4).


Figure 6B . Differential ( FDR < 0 . 05 ) expression of Bach2 , Prdm1 ( encoding BLIMP - 1 ) , Cd138 ( a plasma - cell marker ) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO . Data was generated from three independent biological replicates for each genotype . Bars and error bars indicate mean ± SD . Statistical significance is indicated by FDR after the Benjamini - Hochberg multiple testing correction performed by edgeR package using R language .

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Figure 6B. Differential (FDR < 0.05) expression of Bach2, Prdm1 (encoding BLIMP-1), Cd138 (a plasma-cell marker) and Myc transcripts as indicated by counts per million after TMM normalization from WT and KO. Data was generated from three independent biological replicates for each genotype. Bars and error bars indicate mean ± SD. Statistical significance is indicated by FDR after the Benjamini-Hochberg multiple testing correction performed by edgeR package using R language.


Figure 7D . H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells , as determined by reads per genomic content ( RPGC ) . Data represent three independent biological replicates .

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Figure 7D. H3K79me2 methylation at the Ezh2 locus from WT activated and naïve B cells, as determined by reads per genomic content (RPGC). Data represent three independent biological replicates.


f1 ( a ) The Kaplan - Meier survival curves for KRas ; Atg5 + / + ( n = 12 ) , KRas ; Atg5fl / + ( n = 19 ) and KRas ; Atg5fl / fl ( n = 18 ) littermate mice injected intranasally with AdCre ( 2 . 5 × 107 p . f . u . ) . P0 . 01 ( log - rank test ) between the KRas ; Atg5fl / fl cohort and controls ( b ) Western blot analysis for Atg5 , LC3I and LC3II and p62 in purified primary lung tumour cells . Representative data are shown for tumour cells isolated from KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 18 weeks after AdCre inhalation . β - actin is shown as a loading control . ( c ) Immunohistochemistry to detect LC3I , p62 and Atg5 in tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 18 weeks after AdCre inhalation . Note highly elevated levels of LC3I and p62 , both indicative of defective autophagy , in KRas ; Atg5fl / fl tumours . Scale bars , 50 μm . ( d , e ) Impaired formation of autophagosomes ( arrows ) in KRas ; Atg5fl / fl tumour cells . ( d , e ) Impaired formation of autophagosomes ( arrows ) in KRas ; Atg5fl / fl tumour cells . ( d ) Representative electron microscopy images are shown for KRas ; Atg5fl / + and KRas ; Atg5fl / fl tumours18 weeks after AdCre inhalation . Scale bars , 5 μm for upper and 2 μm for lower panels . M , mitochondria ; * , lamellar bodies ( Corpuscula lamellariae ) , rare cell organelles containing surfactant lipoproteins characteristic for type II pneumocytes . ( e ) Mean percentages ( ± s . e . m . ) of tumour cells with detectable autophagosomes or autolysosomes , counted on electron microscopy images . n ≥ 8 mice per genotype and at least 20 intact cells for each electron microscopy section were counted . * * * P0 . 001 ( χ2 - test of a generalized linear model with logit link assessing the genotype effect ) .

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f1(a) The Kaplan-Meier survival curves for KRas;Atg5+/+ (n=12), KRas;Atg5fl/+ (n=19) and KRas;Atg5fl/fl (n=18) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). P0.01 (log-rank test) between the KRas;Atg5fl/fl cohort and controls(b) Western blot analysis for Atg5, LC3I and LC3II and p62 in purified primary lung tumour cells. Representative data are shown for tumour cells isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. β-actin is shown as a loading control.(c) Immunohistochemistry to detect LC3I, p62 and Atg5 in tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 18 weeks after AdCre inhalation. Note highly elevated levels of LC3I and p62, both indicative of defective autophagy, in KRas;Atg5fl/fl tumours. Scale bars, 50 μm. (d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells.(d,e) Impaired formation of autophagosomes (arrows) in KRas;Atg5fl/fl tumour cells. (d) Representative electron microscopy images are shown for KRas;Atg5fl/+ and KRas;Atg5fl/fl tumours18 weeks after AdCre inhalation. Scale bars, 5 μm for upper and 2 μm for lower panels. M, mitochondria; *, lamellar bodies (Corpuscula lamellariae), rare cell organelles containing surfactant lipoproteins characteristic for type II pneumocytes. (e) Mean percentages (±s.e.m.) of tumour cells with detectable autophagosomes or autolysosomes, counted on electron microscopy images. n≥8 mice per genotype and at least 20 intact cells for each electron microscopy section were counted. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).


f2 ( a ) Representative electron microscopy images of autolysosomes ( left panels ) , autophagosomes ( middle panels ) and mitophagy ( right panels ) , all indicated by arrows , in lungtumour cells from control KRas ; Atg5fl / + mice18 weeks after AdCre inhalation . Note the near - to - complete absence of such structures in the lungtumour cells from KRas ; Atg5fl / fl littermates . Very rarely , we found cells with one autolysosome ( left panels ) or one single autophagosome ( middle panels ; arrows ) in KRas ; Atg5fl / fl tumours , whereas such structures can frequently be seen within single KRas ; Atg5fl / + tumour cells . Of note , the presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig . 1e . Also note aberrant mitochondrial morphologies and increased mitochondrial numbers in KRas ; Atg5fl / fl tumour cells . * indicates lamellar bodies . Scale bars , 2 μm . ( b ) Mean numbers ( ± s . e . m . ) of mitochondria per lungtumour cell from KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice18 weeks after tumour induction . Numbers of mitochondria were determined by electron microscopy . n ≥ 8 mice per genotype and a minimum of 20 intact cells were counted per sample . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( c ) Mitochondria ( arrows ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl lung tumour cells 18 weeks after AdCre inhalation . Note markedly altered mitochondrial morphology , that is , disorganized matrix structures , rounding and enlargement in the autophagy - defective KRas ; Atg5fl / fl cells . Au , autophagosomes ; Au - L , autolysosome ; * , lamellar bodies . Scale bars , 2 μm . ( d - g ) Bioenergetics profiling of purified tumour cells from KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice ( e ) 6 weeks and ( g ) 18 weeks after tumour induction . Representative experimental data are shown . Oxygen consumption rates ( OCR ) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser . OCR ± s . e . m . were recorded to construct bioenergetics profiles for basal respiration ( BR ) , ATP turnover ( AT ) , respiratory capacity ( RC ) , spare respiratory capacity ( SRC ) and proton leakage ( PL ) . A minimum of seven different samples were analysed for each group . * P0 . 05 ; * * P0 . 01 ( unpaired , two - sided t - test ) .

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

f2(a) Representative electron microscopy images of autolysosomes (left panels), autophagosomes (middle panels) and mitophagy (right panels), all indicated by arrows, in lungtumour cells from control KRas;Atg5fl/+ mice18 weeks after AdCre inhalation. Note the near-to-complete absence of such structures in the lungtumour cells from KRas;Atg5fl/fl littermates. Very rarely, we found cells with one autolysosome (left panels) or one single autophagosome (middle panels; arrows) in KRas;Atg5fl/fl tumours, whereas such structures can frequently be seen within single KRas;Atg5fl/+ tumour cells. Of note, the presence of a single autophagosome or a single autolysosome was scored as a positive event in Fig. 1e. Also note aberrant mitochondrial morphologies and increased mitochondrial numbers in KRas;Atg5fl/fl tumour cells. * indicates lamellar bodies. Scale bars, 2 μm. (b) Mean numbers (±s.e.m.) of mitochondria per lungtumour cell from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice18 weeks after tumour induction. Numbers of mitochondria were determined by electron microscopy. n≥8 mice per genotype and a minimum of 20 intact cells were counted per sample. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link).(c) Mitochondria (arrows) in KRas;Atg5fl/+ and KRas;Atg5fl/fl lung tumour cells 18 weeks after AdCre inhalation. Note markedly altered mitochondrial morphology, that is, disorganized matrix structures, rounding and enlargement in the autophagy-defective KRas;Atg5fl/fl cells. Au, autophagosomes; Au-L, autolysosome; *, lamellar bodies. Scale bars, 2 μm.(d-g) Bioenergetics profiling of purified tumour cells from KRas;Atg5fl/+ and KRas;Atg5fl/fl mice (e) 6 weeks and (g) 18 weeks after tumour induction. Representative experimental data are shown. Oxygen consumption rates (OCR) were used to detect the bioenergetics profiles of purified tumour cells using a Seahorse Bioscience 24XF extracellular flux analyser. OCR±s.e.m. were recorded to construct bioenergetics profiles for basal respiration (BR), ATP turnover (AT), respiratory capacity (RC), spare respiratory capacity (SRC) and proton leakage (PL). A minimum of seven different samples were analysed for each group. *P0.05; **P0.01(unpaired, two-sided t-test).


f3 ( a ) Increased tumour initiation in KRas ; Atg5fl / fl mice as compared with KRas ; Atg5fl / + littermates . Representative histological sections at 2 weeks after AdCre inhalation are shown . Insets show typical lung lesions . Haematoxylin and eosin staining . Scale bars , 2 mm for whole lung section ; 50 μm for insets . ( b ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation . n = 6 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( c ) Tumour growth curves based on mean tumour - to - lung area ratios ( ± s . e . m . ) determined in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates at the indicated time points . At least three different planes from each lung were analysed . n ≥ 6 per cohort for each time point . * P0 . 05 , * * P0 . 01 ( unpaired , two - sided Wicoxon test ) . ( d ) MicroCT analysis of lung tumours of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice 18 weeks after AdCre inhalation . Representative data from individual mice are shown . Scale bars , 5

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

f3(a) Increased tumour initiation in KRas;Atg5fl/fl mice as compared with KRas;Atg5fl/+ littermates. Representative histological sections at 2 weeks after AdCre inhalation are shown. Insets show typical lung lesions. Haematoxylin and eosin staining. Scale bars, 2 mm for whole lung section; 50 μm for insets.(b) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation. n=6 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link).(c) Tumour growth curves based on mean tumour-to-lung area ratios (±s.e.m.) determined in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates at the indicated time points. At least three different planes from each lung were analysed. n≥6 per cohort for each time point. *P0.05, **P0.01 (unpaired, two-sided Wicoxon test).(d) MicroCT analysis of lung tumours of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice 18 weeks after AdCre inhalation. Representative data from individual mice are shown. Scale bars, 5 mm


f4 ( a ) Distribution and ( b ) absolute numbers of hyperplastic lesions , adenomas and adenocarcinomas in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice6 , 12 and 18 weeks post AdCre infection . Data are shown as means ± s . e . m . ; n = 6 per group . Combining the tumour progression data from 6 , 12 and 18 weeks , a cumulative linked mixed model ( clmm ) analysis showed also a significant dependency of tumour progression on the genotype ; P0 . 05 ( χ2 - test of clmms assessing the genotype effect ) . Applying generalized linear - mixed effects ( glmer ) models with logit link to individual time points , shows a significant dependency of the transition from hyperplastic lesions to adenomas ( 12 weeks , P0 . 05 ) and from adenomas to adenocarcinomas ( 12 weeks , P0 . 01 and 18 weeks , P0 . 01 ) on the mouse genotype ( the last three P - values are from χ2 - tests of a generalized linear models with logit link assessing the genotype effect ) . ( c , d ) Histological analyses ( haematoxylin and eosin staining ) of lung tumours in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . ( c ) Nuclear and cellular polymorphy , more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 weeks after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours ( asterisk ) . ( c , d ) Histological analyses ( haematoxylin and eosin staining ) of lungtumours in KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . ( c ) Nuclear and cellular polymorphy , more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas ; Atg5fl / + mice as compared with KRas ; Atg5fl / fl tumours . Sections were analysed 18 weeks after AdCre inhalation . Scale bars , 50 μm . ( d ) KRas ; Atg5fl / fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours ( asterisk ) .

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f4(a) Distribution and (b) absolute numbers of hyperplastic lesions, adenomas and adenocarcinomas in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice6, 12 and 18 weeks post AdCre infection. Data are shown as means±s.e.m.; n=6 per group. Combining the tumour progression data from 6, 12 and 18 weeks, a cumulative linked mixed model (clmm) analysis showed also a significant dependency of tumour progression on the genotype; P0.05 (χ2-test of clmms assessing the genotype effect). Applying generalized linear-mixed effects (glmer) models with logit link to individual time points, shows a significant dependency of the transition from hyperplastic lesions to adenomas (12 weeks, P0.05) and from adenomas to adenocarcinomas (12 weeks, P0.01 and 18 weeks, P0.01) on the mouse genotype (the last three P-values are from χ2-tests of a generalized linear models with logit link assessing the genotype effect).(c,d) Histological analyses (haematoxylin and eosin staining) of lung tumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).(c,d) Histological analyses (haematoxylin and eosin staining) of lungtumours in KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. (c) Nuclear and cellular polymorphy, more dense and sometimes irregular chromatin as well as more nucleoli in tumour cells of KRas;Atg5fl/+ mice as compared with KRas;Atg5fl/fl tumours. Sections were analysed 18 weeks after AdCre inhalation. Scale bars, 50 μm. (d) KRas;Atg5fl/fl tumours exhibited markedly increased apoptotic figures and necrotic areas in the centre of larger tumours (asterisk).


f5 ( a ) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot . β - actin is shown as a loading control . ( b ) Atg5 mRNA expression was determined by quantitative PCR . Data are shown as mean relative mRNA expression ± s . e . m . in KRas ; Atg5fl / fl tumours as compared with tumours from KRas ; Atg5fl / + mice ( set to 1 ) . Values were normalized to β - actin mRNA expression . n = 3 per group .

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f5(a) Primary pneumocytes were infected with AdCre for 96 h and Atg5 protein expression determined by western blot. β-actin is shown as a loading control.(b) Atg5 mRNA expression was determined by quantitative PCR. Data are shown as mean relative mRNA expression±s.e.m. in KRas;Atg5fl/fl tumours as compared with tumours from KRas;Atg5fl/+ mice (set to 1). Values were normalized to β-actin mRNA expression. n=3 per group.


f6 ( b ) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho - Chk2 in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 18 weeks after AdCre inhalation . For γH2AX and phospho - Chk2 staining , sections were scored independently in a double - blinded manner . Data are shown as mean percentages ( ± s . e . m . ) of positive cells per total cell numbers . n = 6 mice per genotype . Scale bars , 50 μm . * * * P0 . 001 ( χ2 - test of a generalized linear model with logit link assessing the genotype effect ) . ( c ) Representative histological lung sections of KRas ; Atg5fl / + ; p53fl / fl and KRas ; Atg5fl / fl ; p53fl / fl littermate mice16 weeks after AdCre inhalation . Scale bars , 2 mm . ( d ) Distribution of hyperplastic lesions , adenomas and adenocarcinomas ( ± s . e . m . ) in KRas ; Atg5fl / + ; p53fl / fl and KRas ; Atg5fl / fl ; p53fl / fl littermates 16 weeks post AdCre infection ( n = 4 per genotype ) . ( e ) The Kaplan - Meier survival curves for KRas ; Atg5fl / + ; p53fl / fl ( n = 8 ) and KRas ; Atg5fl / fl ; p53fl / fl ( n = 9 ) littermate mice injected intranasally with AdCre ( 2 . 5 × 107 p . f . u . ) . There was no statistical difference between the cohorts ( log - rank test ) .

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f6(b) Representative immunohistochemistry and quantitative assessment of γH2AX and phospho-Chk2 in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 18 weeks after AdCre inhalation. For γH2AX and phospho-Chk2 staining, sections were scored independently in a double-blinded manner. Data are shown as mean percentages (±s.e.m.) of positive cells per total cell numbers. n=6 mice per genotype. Scale bars, 50 μm. ***P0.001 (χ2-test of a generalized linear model with logit link assessing the genotype effect).(c) Representative histological lung sections of KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermate mice16 weeks after AdCre inhalation. Scale bars, 2 mm. (d) Distribution of hyperplastic lesions, adenomas and adenocarcinomas (±s.e.m.) in KRas;Atg5fl/+;p53fl/fl and KRas;Atg5fl/fl;p53fl/fl littermates 16 weeks post AdCre infection (n=4 per genotype).(e) The Kaplan-Meier survival curves for KRas;Atg5fl/+;p53fl/fl (n=8) and KRas;Atg5fl/fl;p53fl/fl (n=9) littermate mice injected intranasally with AdCre (2.5 × 107 p.f.u.). There was no statistical difference between the cohorts (log-rank test).


f7 ( a , b ) Representative immunohistochemistry and quantitative assessment of ( a ) tumour - infiltrating CD3ε - positive T cells and ( b ) FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 6 weeks after AdCre inhalation . Data are shown as mean percentages ( ± s . e . m . ) of the indicated cell types per total cell numbers in the tumour areas . Only cells within tumours were scored . At least six mice per genotype were analysed . Scale bars , 50 μm . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) . ( c ) Representative immunohistochemistry and quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 2 weeks after AdCre inhalation . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + cells among total cell numbers in the tumour areas . Only cells within tumours were scored . At least six mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) . ( d ) FACS blots to detect CD25 + FoxP3 + Treg cells in lungs 2 weeks after AdCre inhalation . Cells were gated on CD4 + populations . Numbers indicate percentages among total CD4 + cells in the lungs . ( e ) Mediastinal lymph node and lung cells were isolated from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods . The left panel shows the percentage of CD4 + T cells among all lung - infiltrating CD45 + haematopoietic cells ( ± s . e . m . ) . The right panel shows the percentage of FoxP3 + cells among CD4 + T cells in mediastinal lymph nodes and lungs ( ± s . e . m . ) . n = 4 per genotype ; * P0 . 05 ( unpaired , two - sided t - test ) .

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

f7(a,b) Representative immunohistochemistry and quantitative assessment of (a) tumour-infiltrating CD3ε-positive T cells and (b) FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 6 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of the indicated cell types per total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. ***P0.001(χ2-test assessing the genotype effect in a generalized linear model with logit link).(c) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least six mice per genotype were analysed. Scale bars, 50 μm. **P0.01(χ2-test assessing the genotype effect in a generalized linear model with logit link).(d) FACS blots to detect CD25+FoxP3+ Treg cells in lungs 2 weeks after AdCre inhalation. Cells were gated on CD4+ populations. Numbers indicate percentages among total CD4+ cells in the lungs.(e) Mediastinal lymph node and lung cells were isolated from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates 2 weeks after AdCre inhalation and stained for flow cytometric analysis as described in Methods. The left panel shows the percentage of CD4+ T cells among all lung-infiltrating CD45+ haematopoietic cells (±s.e.m.). The right panel shows the percentage of FoxP3+ cells among CD4+ T cells in mediastinal lymph nodes and lungs (±s.e.m.). n=4 per genotype; *P0.05 (unpaired, two-sided t-test).


f8 ( a ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti - CD25 Ab . n = 4 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( b ) Histological lung sections of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice treated with an isotype control Ab or anti - CD25 - immunodepleting Abs . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology . Representative lung images ( haematoxylin and eosin ( HE ) staining ) are shown for each genotype and treatment . Scale bars , 2 mm . ( c ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) in KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or an Ab specific to FR4 . n = 4 per group . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . ( d ) Histological lung sections of KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice treated with isotype control Abs or Abs specific to FR4 . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology . Representative lung images ( HE staining ) are shown for each genotype and treatment . Scale bars , 2 mm .

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

f8(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated either with an isotype control Ab or a depleting anti-CD25 Ab. n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). (b) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with an isotype control Ab or anti-CD25-immunodepleting Abs. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (haematoxylin and eosin (HE) staining) are shown for each genotype and treatment. Scale bars, 2 mm.(c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) in KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with an isotype control Ab or an Ab specific to FR4. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). (d) Histological lung sections of KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice treated with isotype control Abs or Abs specific to FR4. Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for histopathology. Representative lung images (HE staining) are shown for each genotype and treatment. Scale bars, 2 mm.


f9 ( b ) Protein expression of Hif1α , Atg5 and glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) in isolated KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes ( analysed at 4 days after AdCre infection ) and tumours purified from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermate mice at week 2 after AdCre inhalation . Quantifications of the hypoxia - inducible factor ( HIF ) 1α / GAPDH ratios are shown for each lane ; two independent blots are quantified . ( c ) CD39 , Atg5 and as a control β - actin protein expression in isolated Atg5fl / + , Atg5fl / fl , KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes . Protein lysated were analysed at 4 days after AdCre infections by western blot . Quantifications of the CD39 / β - actin ratios are shown for each lane , normalized to the Atg5fl / + samples . Two independent blots are quantified . ( d , e ) CD39 , Atg5 and as a control β - actin protein expression in isolated KRas ; Atg5fl / + and KRas ; Atg5fl / fl pneumocytes treated with the ( d ) HIF1α inhibitor 3 - ( 2 - ( 4 - adamantan - 1 - yl - phenoxy ) - acetylamino ) - 4 - hydroxybenzoic acid methyl ester or left untreated and ( e ) the Nrf2 inhibitor Brusatol ( 100 nM ) or the ROS scavenger N - acetyl - L - cysteine ( 5 mM ) . Protein lysates were analysed on day 4 by western blot . Quantifications of the CD39 / β - actin ratios are shown for each lane . The inhibitors were present during the entire 4 day culture period .

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

f9(b) Protein expression of Hif1α, Atg5 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes (analysed at 4 days after AdCre infection) and tumours purified from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermate mice at week 2 after AdCre inhalation. Quantifications of the hypoxia-inducible factor (HIF)1α/GAPDH ratios are shown for each lane; two independent blots are quantified.(c) CD39, Atg5 and as a control β-actin protein expression in isolated Atg5fl/+, Atg5fl/fl, KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes. Protein lysated were analysed at 4 days after AdCre infections by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane, normalized to the Atg5fl/+ samples. Two independent blots are quantified.(d,e) CD39, Atg5 and as a control β-actin protein expression in isolated KRas;Atg5fl/+ and KRas;Atg5fl/fl pneumocytes treated with the (d) HIF1α inhibitor 3-(2-(4-adamantan-1-yl-phenoxy)-acetylamino)-4-hydroxybenzoic acid methyl ester or left untreated and (e) the Nrf2 inhibitor Brusatol (100 nM) or the ROS scavenger N-acetyl-L-cysteine (5 mM). Protein lysates were analysed on day 4 by western blot. Quantifications of the CD39/β-actin ratios are shown for each lane. The inhibitors were present during the entire 4 day culture period.


f10 ( a ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) and ( b ) representative histological lung sections ( haematoxylin and eosin ( HE ) staining ) of KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 ( intraperitoneally ; i . p . 10 mg kg − 1 daily ) to inhibit CD39 activity . n = 4 per group . * * * P0 . 001 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm . ( c ) Quantification of numbers of hyperplastic lesions / per lung ( mean values ± s . e . m . ) and ( d ) representative histological lung sections ( HE staining ) of KRas ; Atg5fl / + and KRas ; Atg5fl / fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 ( i . p . 10 mg kg − 1 daily ) . n = 4 per group . * * P0 . 01 ( χ2 - test assessing the genotype effect in a generalized linear model with log link ) . Scale bars , 2 mm . ( e , f ) Representative immunohistochemistry and quantitative assessment of FoxP3 + Tregs in lung tumours from KRas ; Atg5fl / + and KRas ; Atg5fl / fl littermates treated with vehicle control or the CD39 blocker polyoxometalate - 1 ( POM1 ) and treated with vehicle or the adenosine receptor inhibitor PSB1115 ( 4 - ( 2 , 3 , 6 , 7 - tetrahydro - 2 , 6 - dioxo - 1 - propyl - 1H - purin - 8 - yl ) - benzenesulphonic acid potassium salt ) . Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology . Data are shown as mean percentages ( ± s . e . m . ) of FoxP3 + cells among total cell numbers in the tumour areas . Only cells within tumours were scored . At least four mice per genotype were analysed . Scale bars , 50 μm . * * P0 . 01 . NS , not significant ; ( χ2 - test assessing the genotype effect in a generalized linear model with logit link ) .

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

f10(a) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (b) representative histological lung sections (haematoxylin and eosin (HE) staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice2 weeks after AdCre inhalation treated in vivo either with either vehicle control or POM1 (intraperitoneally; i.p. 10 mg kg−1 daily) to inhibit CD39 activity. n=4 per group. ***P0.001 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.(c) Quantification of numbers of hyperplastic lesions/per lung (mean values±s.e.m.) and (d) representative histological lung sections (HE staining) of KRas;Atg5fl/+ and KRas;Atg5fl/fl mice 2 weeks after AdCre inhalation treated either with either vehicle control or the adenosine receptor blocker PSB1115 (i.p. 10 mg kg−1 daily). n=4 per group. **P0.01 (χ2-test assessing the genotype effect in a generalized linear model with log link). Scale bars, 2 mm.(e,f) Representative immunohistochemistry and quantitative assessment of FoxP3+ Tregs in lung tumours from KRas;Atg5fl/+ and KRas;Atg5fl/fl littermates treated with vehicle control or the CD39 blocker polyoxometalate-1 (POM1) and treated with vehicle or the adenosine receptor inhibitor PSB1115 (4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl)-benzenesulphonic acid potassium salt). Mice were killed 2 weeks after AdCre inhalation and their lungs were processed for immunohistopathology. Data are shown as mean percentages (±s.e.m.) of FoxP3+ cells among total cell numbers in the tumour areas. Only cells within tumours were scored. At least four mice per genotype were analysed. Scale bars, 50 μm. **P0.01. NS, not significant; (χ2-test assessing the genotype effect in a generalized linear model with logit link).


Figure 1A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes ( CellROX green , Molecular Probes , USA ) in HC ( n = 4 ) and DM ( n = 4 ) platelets . ( * * p = 0 . 032 vs . HC ) . B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC ( # 1 - 3 ) and DM ( # 1 - 8 ) patient platelets . Quantification analysis on HC ( n = 5 ) and DM ( n = 18 ) individuals . ( * * p = 0 . 0025 vs . HC ) . GAPDH served as the loading control . C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets . Graph indicates HC and DM platelets positive for phosphorylated p53 signal . The y - axis indicates percentage of phosphorylated p53 positive cell from total cells ( * * p = 0 . 0064 vs . HC ) . Representative figure from n = 5 . D Mitochondrial membrane potential ( ΔΨm ) was measured by MitoTracker staining ( 50nM for 15 min ) in HC and DM patient platelets . Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets . The y - axis indicates fold of signal intensity in HC and DM platelet ( * * p = 1 . 1608E - 05 vs HC ) . Signal intensity of each group was converted to fold change compared with HC values . Representative figure from HC ( n = 6 ) and DM ( n = 4 ) . E ΔΨm and platelet apoptosis were measured by flow cytometry analysis . ΔΨm was detected using TMRM and apoptosis was assessed with Annexin - V ( PS externalization ) . Representative figure from n = 3 . F Western blot analysis of apoptosis - related pp53 , p53 , cytochrome c , and active caspase 3 in HC and DM platelets . Tubulin was used as the loading control . Representative figure from n = 3 . G Western blot analysis showing release of cytochrome c ( Cyto c ) into the cytosol in DM . Representative figure from n = 3 . H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets ( DM ) compared to a typical healthy mitochondria ( HC ) . Arrows indicate disrupted outer membranes . I Quantification of mitochondria in HC ( n = 33 ) and DM ( n = 17 ) patient platelets . The y - axis indicates average number of mitochondria in HC and DM single platelet EM view ( * * p = 5 . 50811E - 05 vs . HC ) . J Quantification of healthy and damaged mitochondria in HC and DM platelets .

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

Figure 1A ROS levels were measured by flow cytometry after staining with specific oxidative stress detection dyes (CellROX green, Molecular Probes, USA) in HC (n=4) and DM (n=4) platelets. (**p=0.032 vs. HC).B Western blot analysis of ROS downstream signaling molecules p53 and phosphorylated p53 in HC (#1-3) and DM (#1-8) patient platelets. Quantification analysis on HC (n=5) and DM (n=18) individuals. (**p=0.0025 vs. HC). GAPDH served as the loading control.C Fluorescent immunostaining for phosphorylated p53 in HC and DM patient platelets. Graph indicates HC and DM platelets positive for phosphorylated p53 signal. The y-axis indicates percentage of phosphorylated p53 positive cell from total cells (**p=0.0064 vs. HC). Representative figure from n=5.D Mitochondrial membrane potential (ΔΨm) was measured by MitoTracker staining (50nM for 15 min) in HC and DM patient platelets. Graph indicates signal intensity of MitoTracker fluorescence in HC and DM platelets. The y-axis indicates fold of signal intensity in HC and DM platelet (**p=1.1608E-05 vs HC). Signal intensity of each group was converted to fold change compared with HC values. Representative figure from HC (n=6) and DM (n=4).E ΔΨm and platelet apoptosis were measured by flow cytometry analysis. ΔΨm was detected using TMRM and apoptosis was assessed with Annexin-V (PS externalization). Representative figure from n=3.F Western blot analysis of apoptosis-related pp53, p53, cytochrome c, and active caspase 3 in HC and DM platelets. Tubulin was used as the loading control. Representative figure from n=3.G Western blot analysis showing release of cytochrome c (Cyto c) into the cytosol in DM. Representative figure from n=3.H EM of platelets mitochondria from HC and DM patients demonstrating examples of mitochondria damage in DM platelets (DM) compared to a typical healthy mitochondria (HC). Arrows indicate disrupted outer membranes.I Quantification of mitochondria in HC (n=33) and DM (n=17) patient platelets. The y-axis indicates average number of mitochondria in HC and DM single platelet EM view (**p=5.50811E-05 vs. HC).J Quantification of healthy and damaged mitochondria in HC and DM platelets.