a ) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at the time of tissue collection . Clones connected to hair follicles were more likely to be larger in anagen compared to telogen ( Chi square test , * * * p = 0 . 0009 at 3 weeks , * * * p < 0 . 0001 at 5 weeks , * p = 0 . 0193 at 12 weeks , * * * p = 0 . 0001 at 24 weeks ) . However no difference between the two phases was observed in clones that were not attached to HFs . Clone numbers are indicated on the graph . All data are represented as mean ± s . e . m .

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

a) Anagen and telogen skin identified by skin pigmentation at the time of collection were obtained from each Skinbowmouse at different time points. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at the time of tissue collection. Clones connected to hair follicles were more likely to be larger in anagen compared to telogen (Chi square test, *** p=0.0009 at 3 weeks, *** p<0.0001 at 5 weeks, * p=0.0193 at 12 weeks, *** p=0.0001 at 24 weeks). However no difference between the two phases was observed in clones that were not attached to HFs. Clone numbers are indicated on the graph. All data are represented as mean ± s.e.m.


b ) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post - induction . Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling . Average clone size of attached ( circles ) and non - attached ( squares ) clones were plotted over the number of club hair . T - test , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Telogen skin was obtained from Skinbowmice induced at P42 at different time points from 1 to 24 weeks post-induction. Each skin sample was evaluated for the average number of club hair found in hair follicles reflecting its history of past hair cycling. Average clone size of attached (circles) and non-attached (squares) clones were plotted over the number of club hair. T-test, **** p<0.0001.


c ) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days . Histogram charts represent the relative frequency of clone size from clones attached ( top row ) or not ( bottom row ) to HF in anagen and telogen phase at induction . Even at a rate of one cell division per 24h , 7 cycles of divisions results in 128 cells . Therefore the findings reported are not incompatible with our claims as suggested . Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe . Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones . Chi square test , * * * p = 0 . 001 ; * * 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 O O O O O O O O O O O O O O O O O O O O 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) Skinbowmice were induced at a time were the back skin contained both areas of anagen and telogen and samples collected for evaluation at 7 days. Histogram charts represent the relative frequency of clone size from clones attached (top row) or not (bottom row) to HF in anagen and telogen phase at induction. Even at a rate of one cell division per 24h, 7 cycles of divisions results in 128 cells. Therefore the findings reported are not incompatible with our claims as suggested. Observation of spontaneous recombinations if they exist are therefore a very rare event that we have never been able to observe. Such rare event is unlikely to influence our analysis based on sizes of hundreds of clones. Chi square test, *** p=0.001; ** p=0.01.


( a - b ) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway ( a ) and corresponding scatter plots quantification . ( Scale bar 100µm ) ( b ) . Analysis focused on the basal layer of the IFE . Each dot represents one image . Proliferation in the IFE is significantly increased during the anagen phase except in mid - anagen . Unpaired t test , * * * * p < 0 . 0001 , * * p = 0 . 0020 . EA n = 32 , EMA n = 13 , MA n = 15 , C n = 21 , T n = 19 from 6 mice . Black arrows : positive Ki67 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-b) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway (a) and corresponding scatter plots quantification. (Scale bar 100µm) (b). Analysis focused on the basal layer of the IFE. Each dot represents one image. Proliferation in the IFE is significantly increased during the anagen phase except in mid-anagen. Unpaired t test, ****p<0.0001, **p=0.0020. EA n = 32, EMA n = 13, MA n = 15, C n = 21, T n = 19 from 6 mice. Black arrows: positive Ki67 cells.


( c ) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti - Ki67 antibody or upon BrdU incorporation . Ki67 + cells are labelled with Alexa 647 ( red ) . The density of Ki67 + cells increased in the anagen phase ( iii and iv ) compared to telogen ( i and ii ) . White arrows : ki67 + cells . ( Scale bar = 20µm ) . Similarly , short term BrdU pulse ( 2h ) resulted in significant staining of anagen epidermal areas ( vi , red nuclear stain ) reinforcing hair follicle openings as compared to telogen areas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti-Ki67 antibody or upon BrdU incorporation. Ki67+ cells are labelled with Alexa 647 (red). The density of Ki67+ cells increased in the anagen phase (iii and iv) compared to telogen (i and ii). White arrows: ki67+ cells. (Scale bar = 20µm). Similarly, short term BrdU pulse (2h) resulted in significant staining of anagen epidermal areas (vi, red nuclear stain) reinforcing hair follicle openings as compared to telogen areas.


a - b ) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . a ) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti - BrdU antibody ( red ) . White dots represent hair follicles at different chase time points .

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

a-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points.


a - b ) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day . Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase . b ) At 5 weeks of chase , BrdU staining ( green ) in skinbowskin revealed single cells distant from hair follicles . White dashed circles : hair follicles . White line : small clones . White arrows : BrdU + cells . ( scale bar = 20 µm ) .

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

a-b) Skinbowmice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. b) At 5 weeks of chase, BrdU staining (green) in skinbowskin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm).


c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . c ) Photomicrographs show optical sections from a confocal image stack acquired on a 0 . 5 mm back skin section immunostained with anti - ki67 antibody . Ki67 + cells are labelled with Alexa 647 ( red ) . Back skin from treated and untreated areas was collected 48hrs after TPA topical application .

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 B-CONTROLLED_VAR O O O

c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application.


c - d ) In Skinbowmice induced at P42 - 46 , TPA was applied for one day , 3 weeks post - induction and animals were sacrificed at 48hours following a short pulse of BrdU ( 2h ) . d ) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti - BrdU ( green ) . Small clones not attached to HF ( 2 examples figured by dashed lines ) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation .

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 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

c-d) In Skinbowmice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation.


e ) ( i to iv ) Representative individual sections from Z stacks , show small clones ( lines ) identified in back skin treated ( i and ii ) or not with TPA ( iii and iv ) . ( scale bar = 100 µm ) . ( v to viii ) Maximal intensity projection from stack acquired on whole mounted skin . A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA ( v to vi ) compared to skin treated with vehicle ( vii and viii ) . ( scale bars = 100 µm ) . f ) Reduction in small clone frequency in the areas treated with TPA . Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse . Mann Whitney test , * * p = 0 . 0079 . Data pooled from 3 mice represented as median with interquartile range . The number of clones analyzed is indicated on the graph . * p = 0 . 0272 . Data are represented as mean ± s . e . m .

O O O O O O O O O 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 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in back skin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.


a ) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42 . Data are represented as mean ± s . e . m .

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

a) Bar charts showing the relative surface of clones attached and not attached between 3 and 24 weeks after induction at P21 or P42. Data are represented as mean ± s.e.m.


A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNF - α - stimulated VE - cadherin WT ( WT ) or VE - cadherin Y731F ( Y731F ) primary endothelial cells , each transfected with control or SHP2 - specific siRNA . The transmigration rate is presented relative to that of WT cells transfected with control siRNA , set as 100 % . On the right , Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α - tubulin .

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

A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNF-α-stimulated VE-cadherin WT (WT) or VE-cadherin Y731F (Y731F) primary endothelial cells, each transfected with control or SHP2-specific siRNA. The transmigration rate is presented relative to that of WT cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.


B Transmigration of human neutrophils towards the chemokine IL - 8 through TNFα - stimulated human endothelial cells ( HUVEC ) , transfected with control or SHP2 - specific siRNA and were either not transduced ( - ) or transduced ( + ) with adenoviruses expressing human SHP2 ( Adv - SHP2 ) . The transmigration rate is presented relative to that of cells transfected with control siRNA , set as 100 % . On the right , Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α - tubulin .

B Transmigration of human neutrophils towards the chemokine IL-8 through TNFα-stimulated human endothelial cells (HUVEC), transfected with control or SHP2-specific siRNA and were either not transduced (-) or transduced (+) with adenoviruses expressing human SHP2 (Adv-SHP2). The transmigration rate is presented relative to that of cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.


C Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST , GST ‐ SHP2 ‐ WT , or GST ‐ SHP2 ‐ DA . Pulldowns and total cell lysates were analyzed by Western blotting for VE - cadherin and GST . Graph below represents quantification of two independent experiments , presented as fold change of bound VE - cadherin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST, GST‐SHP2‐WT, or GST‐SHP2‐DA. Pulldowns and total cell lysates were analyzed by Western blotting for VE-cadherin and GST. Graph below represents quantification of two independent experiments, presented as fold change of bound VE-cadherin.


TNFα - activated bEnd . 5 cells were incubated with mouse T cells for 0 - 30 min . SHP2 ( above ) or VE - cadherin ( below ) were immunoprecipitated ( IP ) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens . Molecular weight markers are indicated in kDa . Graph below represents quantification of five independent experiments , presented as fold change of bound VE - cadherin .

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

TNFα-activated bEnd.5 cells were incubated with mouse T cells for 0-30 min. SHP2 (above) or VE-cadherin (below) were immunoprecipitated (IP) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. Graph below represents quantification of five independent experiments, presented as fold change of bound VE-cadherin.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( A ) bEnd . 5 cells were incubated with antigen - stimulated mouse T cells for 0 - 15 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (A) bEnd.5 cells were incubated with antigen-stimulated mouse T cells for 0-15 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( B ) luEnd cells devoid of PECAM - 1 ( KO ) or re - transfected with PECAM - 1 ( RT ) were incubated with T cells for 30 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (B) luEnd cells devoid of PECAM-1 (KO) or re-transfected with PECAM-1 (RT) were incubated with T cells for 30 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( C ) HUVEC transfected with control or PECAM - 1 - specific siRNA were incubated with differentiated HL60 cells for 20 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( D ) bEnd . 5 cells were pre - treated with anti - PECAM - 1 ( Mec13 . 3 ) or anti - endomucin antibodies for 30 min prior to incubation with T cells for 30 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (D) bEnd.5 cells were pre-treated with anti-PECAM-1 (Mec13.3) or anti-endomucin antibodies for 30 min prior to incubation with T cells for 30 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above ) and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( E ) HUVEC , siRNA depleted for endogenous PECAM - 1 , were transduced with wild - type PECAM - 1 ( PECAM - 1 WT ) or Y663 , 686F PECAM - 1 double mutant ( PECAM - 1 YF ) and incubated with HL60 - derived neutrophils for 20 min . On the right , immunoblot analysis of lysates from siRNA treated cells before PECAM - 1 re - expression .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above) and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (E) HUVEC, siRNA depleted for endogenous PECAM-1, were transduced with wild-type PECAM-1 (PECAM-1 WT) or Y663,686F PECAM-1 double mutant (PECAM-1 YF) and incubated with HL60-derived neutrophils for 20 min. On the right, immunoblot analysis of lysates from siRNA treated cells before PECAM-1 re-expression.


F Transmigration of human neutrophils towards IL - 8 through TNFα - stimulated HUVEC expressing PECAM - 1 WT or PECAM - 1 - Y663 , 686F . The transmigration rate is presented relative to that of PECAM - 1 WT expressing cells , set as 100 % .

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

F Transmigration of human neutrophils towards IL-8 through TNFα-stimulated HUVEC expressing PECAM-1 WT or PECAM-1-Y663,686F. The transmigration rate is presented relative to that of PECAM-1 WT expressing cells, set as 100%.


Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C and monitored by the internalization of preincubated mAb BV6 , which was visualized in fixed cells with a secondary fluorescent antibody ( white ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( white ) . ( A ) HUVEC had been transfected prior to the experiment with control or PECAM - 1 - specific siRNA . Cell lysates were analyzed for expression levels of PECAM - 1 and tubulin ( below ) . Relative VE - cadherin internalization efficiency is given on the right .

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

Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). Relative VE-cadherin internalization efficiency is given on the right.


Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C and monitored by the internalization of preincubated mAb BV6 , which was visualized in fixed cells with a secondary fluorescent antibody ( white ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( white ) . ( B ) PECAM - 1 siRNA treated HUVEC were transduced with WT PECAM - 1 - EGFP or PECAM - 1 - Y663 , 686F - EGFP . Expression levels of transduced PECAM - 1 forms and of EGFP and VE - cadherin are shown in immunoblots ( on the right ) . Relative VE - cadherin internalization efficiency is given on the right .

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

Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (B) PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.


A Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated bEnd . 5 cells transfected with control or SHP2 - specific siRNA and pre - treated with anti - endomucin ( 7C7 . 1 ) or anti - PECAM - 1 ( Mec13 . 3 ) antibodies for 30 min at 37 ° C before addition of neutrophils . The transmigration rate is presented relative to that of control cells , set as 100 % .

A Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated bEnd.5 cells transfected with control or SHP2-specific siRNA and pre-treated with anti-endomucin (7C7.1) or anti-PECAM-1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.


B Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated WT or Y731F primary mouse endothelial cells pre - treated with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to that of control cells , set as 100 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated WT or Y731F primary mouse endothelial cells pre-treated with isotype-control or anti-PECAM-1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.


Extravasated leukocytes ( C ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

Extravasated leukocytes (C) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


adherent leukocytes ( D ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

adherent leukocytes (D) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


( D ) and rolling flux fraction of leukocytes ( E ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

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(D) and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2 . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens .

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A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens.


B bEnd . 3 cells were lysed and VE - cadherin or immature VE - cadherin was immunoprecipitated respectively with C5 or VD47 antibodies . Precipitates were blotted against pY731 - VE - cadherin , β - catenin , plakoglobin , p120 , α - catenin and VE - cadherin .

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B bEnd.3 cells were lysed and VE-cadherin or immature VE-cadherin was immunoprecipitated respectively with C5 or VD47 antibodies. Precipitates were blotted against pY731-VE-cadherin, β -catenin, plakoglobin, p120, α -catenin and VE-cadherin.


C VE - cadherin - catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated ( no SDS ) or treated with 0 . 2 % SDS for 30 min at room temperature , followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30 ° C . Precipitates were analyzed in immunoblots for pY731 - VE - cadherin , plakoglobin , β - catenin and VE - cadherin . D Quantification of pY731 blot signals of three independent experiments ( from C ) , with the signal intensity for untreated VE - cadherin set as 1 . Molecular weight markers are indicated in kDa .

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C VE-cadherin-catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731-VE-cadherin, plakoglobin, β-catenin and VE-cadherin. D Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE-cadherin set as 1. Molecular weight markers are indicated in kDa.


A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNFα - stimulated WT or Y731F primary endothelial cells , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator ( MAPTAM ) for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to DMSO - treated endothelial cells expressing WT VE - cadherin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin.


B TNFα - activated bEnd . 5 cells were treated with DMSO or MAPTAM , prior to incubation with antigen - stimulated mouse T cells for 30 min . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . Quantification of pY731 blot signals of five independent experiments are shown below , with the signal intensity for the control sample ( no T cells , vehicle - control ) set as 100 % .

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B TNFα-activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%.


A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNFα - stimulated WT or Y731F primary endothelial cells , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to DMSO - treated endothelial cells expressing WT VE - cadherin . C Transmigration assays treatment with Blebbistatin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin. C Transmigration assays treatment with Blebbistatin.


B TNFα - activated bEnd . 5 cells were treated with DMSO prior to incubation with antigen - stimulated mouse T cells for 30 min . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . Quantification of pY731 blot signals of five independent experiments are shown below , with the signal intensity for the control sample ( no T cells , vehicle - control ) set as 100 % . treatment with Blebbistatin .

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B TNFα-activated bEnd.5 cells were treated with DMSO prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%. treatment with Blebbistatin.


E HUVEC transduced with lacZ or with SHP2 were either untreated ( - ) or treated ( + ) with thrombin for 30 min followed by immunoblotting either VE - cadherin immunoprecipitates or cell lysates for the indicated antigens on the right .

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E HUVEC transduced with lacZ or with SHP2 were either untreated (-) or treated (+) with thrombin for 30 min followed by immunoblotting either VE-cadherin immunoprecipitates or cell lysates for the indicated antigens on the right.


HUVEC were transfected with control or VE - cadherin - specific siRNA and transduced with VE - cad FL or VE - cad FL - TS . Cell lysates were immunoblotted for VE - cadherin and actin .

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HUVEC were transfected with control or VE-cadherin-specific siRNA and transduced with VE-cad FL or VE-cad FL-TS. Cell lysates were immunoblotted for VE-cadherin and actin.


C Immunofluorescence staining for VE - cadherin of HUVEC transfected with control or VE ‑ cadherin specific siRNA ( VE - cad kd ) and transduced with VE - cad FL or VE - cad FL - TS . D HUVEC were either transfected with control or VE - cadherin targeting siRNA and transduced with VE ‑ cad FL or VE - cad FL ‑ TS . The tension sensor was immunoprecipitated from cell lysates using an anti - GFP antibody . Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens . Molecular weight markers are indicated in kDa . E , Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS , after thrombin stimulation ( 1 U / ml ) or under control conditions .

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C Immunofluorescence staining for VE-cadherin of HUVEC transfected with control or VE‑cadherin specific siRNA (VE-cad kd) and transduced with VE-cad FL or VE-cad FL-TS. D HUVEC were either transfected with control or VE-cadherin targeting siRNA and transduced with VE‑cad FL or VE-cad FL‑TS. The tension sensor was immunoprecipitated from cell lysates using an anti-GFP antibody. Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. E, Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS, after thrombin stimulation (1 U/ml) or under control conditions.


A - C Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL ( A , B ) or VE - cad FL - TS ( C ) . HUVEC were stimulated 4h with TNFα prior to adding IL - 8 for 4 min , followed by flow with buffer alone ( A ) or together with PMNs ( B , C ) . The connected data points represent FRET efficiency of same junctions compared before and after addition of flow / PMNs .

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A-C Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL (A, B) or VE-cad FL-TS (C). HUVEC were stimulated 4h with TNFα prior to adding IL-8 for 4 min, followed by flow with buffer alone (A) or together with PMNs (B, C). The connected data points represent FRET efficiency of same junctions compared before and after addition of flow/PMNs.


D Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS . Cells were either exposed to flow and PMNs or to flow alone , fixed using 4 % PFA and washed with PBS . FLIM measurements were performed at sites of transmigration ( PMNs ) or at junctions without PMNs ( Flow ) .

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D Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS. Cells were either exposed to flow and PMNs or to flow alone, fixed using 4 % PFA and washed with PBS. FLIM measurements were performed at sites of transmigration (PMNs) or at junctions without PMNs (Flow).


E Representative images of HUVEC expressing VE - cad FL at a site of PMN transmigration ( left micrographs ) or without PMN ( right micrographs ) . Maximum intensity projection of a Z - stack of YPet fluorescence ( VE - cadherin in green ) and CellTracker DeepRed ( PMN in red ) is shown for the upper micrographs . The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue . The amplitude averaged lifetime of YPet per pixel ( FLIM measurement ) of the same cells is shown in the bottom micrographs .

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E Representative images of HUVEC expressing VE-cad FL at a site of PMN transmigration (left micrographs) or without PMN (right micrographs). Maximum intensity projection of a Z-stack of YPet fluorescence (VE-cadherin in green) and CellTracker DeepRed (PMN in red) is shown for the upper micrographs. The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue. The amplitude averaged lifetime of YPet per pixel (FLIM measurement) of the same cells is shown in the bottom micrographs.


F HUVEC were grown on collagen - coated polyacrylamide gels of varying physiologic stiffness of 0 . 2 kPa and 20 kPa , and stimulated with TNFα for 17 h prior to adding HL60 - derived neutrophils for 20 min . VE - cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens . Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE - cadherin are shown on the

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F HUVEC were grown on collagen-coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa and 20 kPa, and stimulated with TNFα for 17 h prior to adding HL60-derived neutrophils for 20 min. VE-cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens. Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE-cadherin are shown on the right


B ) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre - and postsynaptic markers , but not glia marker ( GFAP ) in the SN fraction . Cytosolic markers were depleted , while nuclear markers were barely detected in the SN ( n = 4 biological replicates , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ; one sample t - test ; error bars indicate SEM ) .

B) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre- and postsynaptic markers, but not glia marker (GFAP) in the SN fraction. Cytosolic markers were depleted, while nuclear markers were barely detected in the SN (n=4 biological replicates, *** p<0.001, ** p<0.01, * p<0.05; one sample t-test; error bars indicate SEM).


C ) Synaptoneurosomes were NMDAR - stimulated and incubated with radioactive 35S - methionine / cysteine mix for 15 - 120 min . After labeling , proteins were separated on SDS - PAGE and newly synthesized proteins in SN labeled with [ 35S ] were visualized by autoradiography ( left panel ) . To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol ( CAP , 50 μg / ml ) or with puromycin ( PURO , 3mM ) respectively ( right panel ) . After 1 hour after the stimulation , a significant increase in 35S - labelled proteins was observed ( n = 6 biological replicates , * * * p < 0 . 001 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) and the CAP treatment did not affect the overall levels of de novo synthetized proteins ( n = 6 biological replicates , ns ; p = 0 . 220 ) . In contrast , PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes ( n = 6 biological replicates , * * p = 0 . 0091 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Error bars indicate SEM .

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

C) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 15-120 min. After labeling, proteins were separated on SDS-PAGE and newly synthesized proteins in SN labeled with [35S] were visualized by autoradiography (left panel). To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol (CAP, 50 μg/ml) or with puromycin (PURO, 3mM) respectively (right panel). After 1 hour after the stimulation, a significant increase in 35S-labelled proteins was observed (n=6 biological replicates, *** p<0.001; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) and the CAP treatment did not affect the overall levels of de novo synthetized proteins (n=6 biological replicates, ns; p=0.220). In contrast, PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes (n=6 biological replicates, ** p=0.0091, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( D - F ) Results of mass spectrometry ( label free quantification - LFQ ) analysis identifying proteins with increased abundance in NMDAR - stimulated ( 20 min ) synaptoneurosomes . ( D ) Volcano plot ( Whitney - Mann test , n = 4 per group ) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated . Mitochondrial proteins are depicted in orange . The vertical line defines the p - value statistical significance cut - off . ( E ) Biological functions of identified proteins annotated using DAVID . Third top functional category identified as mitochondrial ones . ( F ) Pie - chart showing percentage share of mitochondrial proteins among all identified upregulated proteins .

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(D-F) Results of mass spectrometry (label free quantification - LFQ) analysis identifying proteins with increased abundance in NMDAR-stimulated (20 min) synaptoneurosomes. (D) Volcano plot (Whitney-Mann test, n=4 per group) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. (E) Biological functions of identified proteins annotated using DAVID. Third top functional category identified as mitochondrial ones. (F) Pie-chart showing percentage share of mitochondrial proteins among all identified upregulated proteins.


( A - D ) Results of high - resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes . Volcano plots ( Whitney - Mann test , Benjamini Hochberg correction , n = 5 per group ) showing abundance of identified proteins in NMDAR - stimulated ( 20 min ) synaptoneurosomes as compared to unstimulated using iTRAQ8 - plex ( A ) and TMT10 - plex ( B ) labelling methods . Mitochondrial proteins are depicted in orange . The vertical line defines the p - value statistical significance cut - off . For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified , the data is presented on Figure EV2 . ( C ) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation ( assembled from all three proteomic analyzes : LFQ , iTRAQ8 ,

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(A-D) Results of high-resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes. Volcano plots (Whitney-Mann test, Benjamini Hochberg correction, n=5 per group) showing abundance of identified proteins in NMDAR-stimulated (20 min) synaptoneurosomes as compared to unstimulated using iTRAQ8-plex (A) and TMT10-plex (B) labelling methods. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified, the data is presented on Figure EV2. (C) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation (assembled from all three proteomic analyzes: LFQ, iTRAQ8, TMT10


( E ) RNA absorbance profiles from polyribosomal fractionation of unstimulated and NMDAR - stimulated ( 20 min ) synaptoneurosomes . Upon NMDAR stimulation , a fraction of mRNAs is shifted towards heavy - polyribosomal fraction , which represents actively translating ribosomes . The fraction of polyribosomes used for RNA - seq experiment is depicted .

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(E) RNA absorbance profiles from polyribosomal fractionation of unstimulated and NMDAR-stimulated (20 min) synaptoneurosomes. Upon NMDAR stimulation, a fraction of mRNAs is shifted towards heavy-polyribosomal fraction, which represents actively translating ribosomes. The fraction of polyribosomes used for RNA-seq experiment is depicted.


( F ) Comparison of proteomic data with RNA - seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples . Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA - seq values of polyribosome associated mRNA with total mRNA ( y axis ) . Green dots represent mRNAs encoding proteins upregulated upon stimulation ( identified in MS experiments ) while orange dots represent mRNAs encoding mitochondrial proteins . Density curves for transcripts encoding mitochondrial proteins ( orange ) and upregulated proteins ( green ) detected by the mass spectrometry are present on the right side of the plot . Please note that both classes represent high abundant mRNAs with good ribosome association values . Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red .

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(F) Comparison of proteomic data with RNA-seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples. Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA-seq values of polyribosome associated mRNA with total mRNA (y axis). Green dots represent mRNAs encoding proteins upregulated upon stimulation (identified in MS experiments) while orange dots represent mRNAs encoding mitochondrial proteins. Density curves for transcripts encoding mitochondrial proteins (orange) and upregulated proteins (green) detected by the mass spectrometry are present on the right side of the plot. Please note that both classes represent high abundant mRNAs with good ribosome association values. Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red.


( A ) Respiration activity of permeabilized synaptoneurosomes ( upper panel ) as compared to permeabilized HEK293T ( lower panel ) . Oxygen concentration ( blue line ) and oxygen consumption ( red line ) measured using O2k oxygraph are shown .

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(A) Respiration activity of permeabilized synaptoneurosomes (upper panel) as compared to permeabilized HEK293T (lower panel). Oxygen concentration (blue line) and oxygen consumption (red line) measured using O2k oxygraph are shown.


( B ) Blue native gel electrophoresis ( BN - PAGE ) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria .

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(B) Blue native gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria.


( C ) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN - PAGE followed by Western blot using antibodies specific for : complex I ( Ndufb8 ) , complex II ( Sdha ) , complex III ( Uqcrc2 ) , complex IV ( Cox6a ) and complex V ( Atp5a ) . Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria .

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(C) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN-PAGE followed by Western blot using antibodies specific for: complex I (Ndufb8), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox6a) and complex V (Atp5a). Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria.


( D ) Selected bands ( 1 - 8 ) visible in blue - native gel were excised and analyzed using mass spectrometry ( LC - MS ) . Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands .

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(D) Selected bands (1-8) visible in blue-native gel were excised and analyzed using mass spectrometry (LC-MS). Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands.


( A ) Synaptoneurosomes were NMDAR - stimulated and incubated with radioactive 35S - methionine / cysteine mix for 1h . Samples were separated using blue - native electrophoresis ( BN - PAGE ) . Protein complexes were visualized by Coomassie staining ( left ) and autoradiography ( right ) . Autoradiography of the BN - PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes .

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(A) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 1h. Samples were separated using blue-native electrophoresis (BN-PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN-PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.


To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m - chlorophenyl hydrazone ( 10 μM CCCP ) ( B ) Protein complexes were separated on BN - PAGE and visualized by Coomassie staining and autoradiography . 1 hour after the stimulation , significant increase in 35S - labelled proteins was observed ( B , n = 4 biological replicates , * , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in the presence of CCCP ( B ; n = 4 biological replicates , * p = 0 . 02 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test )

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

To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * , ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test)


To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture ( containing 1 μM valinomycin , 20 μM oligomycin , 8 μM antimycin ) ( C ) . Additionaly , in order to block protein synthesis , SN were pretreated with puromycin ( PURO , 3mM ) ( C ) . Protein complexes were separated on BN - PAGE and visualized by Coomassie staining and autoradiography . 1 hour after the stimulation , significant increase in 35S - labelled proteins was observed C , n = 5 biological replicates , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in the presence of VOA ( C ; n = 5 biological replicates , * * p = 0 . 0044 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Also , significant inhibition of 35S - methionine / cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin ( C , n = 5 biological replicates , * p = 0 . 0236 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Error bars indicate SEM .

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

To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) (C). Additionaly, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3mM) (C). Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed C, n=5 biological replicates, ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of VOA (C; n=5 biological replicates, ** p=0.0044, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S-methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n=5 biological replicates, * p=0.0236, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( B ) Equal amounts of synaptoneurosomal samples isolated from mouse brains ( Fmr1 KO and wild - type littermates ) were permeabilized by saponin and assayed on MitoPlates for 2 hours . Results are presented as the average rate / min / µg of protein , + / - SEM ( n = 3 per genotype ; two - way ANOVA , post - hoc Sidak ' s multiple comparisons test ; * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Equal amounts of synaptoneurosomal samples isolated from mouse brains (Fmr1 KO and wild-type littermates) were permeabilized by saponin and assayed on MitoPlates for 2 hours. Results are presented as the average rate/min/µg of protein, +/- SEM (n=3 per genotype; two-way ANOVA, post-hoc Sidak's multiple comparisons test; *** p< 0.0001).


( C ) Mitochondrial O2 consumption rates in SN were measured by high - resolution respirometry in the presence of following respiratory substrates : malate , pyruvate , and glutamate for Complexes I - IV , succinate for Complexes II - IV , glycerol 3 - phosphate for mGpDH - IV and ascorbate and TMPD for complex IV , and normalized to protein content . Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD ( * p = 0 . 0483 and * p = 0 . 0360 respectively , n = 9 per genotype , paired two - tailed t - test ) . Data are presented as a box - and - whiskers graph ( the box extends from 25th to 75th percentiles , central horizontal line is plotted at the median , whiskers show 5th - 95th percentile ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondrial O2 consumption rates in SN were measured by high-resolution respirometry in the presence of following respiratory substrates: malate, pyruvate, and glutamate for Complexes I-IV, succinate for Complexes II-IV, glycerol 3-phosphate for mGpDH-IV and ascorbate and TMPD for complex IV, and normalized to protein content. Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD (*p=0.0483 and *p=0.0360 respectively, n=9 per genotype, paired two-tailed t-test). Data are presented as a box-and-whiskers graph (the box extends from 25th to 75th percentiles, central horizontal line is plotted at the median, whiskers show 5th - 95th percentile).


( D ) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild - types . Mitochondria are indicated by arrows ; presynaptic part in blue , postsynaptic part in green . 3 mice per genotype were analyzed . Scale bars , 0 . 5 µm .

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

(D) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild-types. Mitochondria are indicated by arrows; presynaptic part in blue, postsynaptic part in green. 3 mice per genotype were analyzed. Scale bars, 0.5 µm.


( A ) The bar graph represents top 10 biological pathways upregulated in gene ontology ( GO ) based Reactome pathway analysis using a set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald Test ) , 3 biological replicates ) in RNA - seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where the gene expression matrix was transformed into z - score . The heat map was generated from the common genes present in the three GO terms indicated by the three black lines . The numbers on the bars indicate the p - value of that particular GO term .

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

(A) The bar graph represents top 10 biological pathways upregulated in gene ontology (GO) based Reactome pathway analysis using a set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 biological replicates) in RNA-seq analysis in IRGM shRNA knockdown HT29 cells compared to control shRNA cells. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( D ) The qRT - PCR validation of RNA - seq data in control and IRGM KD HT29 cells .

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

(D) The qRT-PCR validation of RNA- seq data in control and IRGM KD HT29 cells.


( E ) The bar graph represents the top pathways upregulated in GO - based Reactome pathway analysis using set of genes induced ( 1 . 5 fold , p < 0 . 05 ( Wald Test ) , 3 mice each group ) in the brain of Irgm1 - / - mice compared to Irgm1 + / + wild type mice . Heatmaps were generated for sentinel interferon - regulated genes ( three biological replicates ) using ' ComplexHeatmap ' library using ' R ' Bioconductor package where the gene expression matrix was transformed into z - score . The heat map was generated from the common genes present in the three GO terms indicated by the three black lines . The numbers on the bars indicate the p - value of that particular GO term .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 bar graph represents the top pathways upregulated in GO-based Reactome pathway analysis using set of genes induced (1.5 fold, p<0.05 (Wald Test), 3 mice each group) in the brain of Irgm1-/- mice compared to Irgm1+/+ wild type mice. Heatmaps were generated for sentinel interferon-regulated genes (three biological replicates) using 'ComplexHeatmap' library using 'R' Bioconductor package where the gene expression matrix was transformed into z-score. The heat map was generated from the common genes present in the three GO terms indicated by the three black lines. The numbers on the bars indicate the p-value of that particular GO term.


( H ) The qRT - PCR validation of RNA - seq data in Irgm1 + / + and Irgm1 - / - mouse brain .

(H) The qRT-PCR validation of RNA- seq data in Irgm1+/+ and Irgm1-/- mouse brain.


( A ) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1 - / - mouse brain and BMDMs .

(A) Heat map of nucleic acid sensor proteins upregulated in IRGM KD HT29 cells and Irgm1-/- mouse brain and BMDMs.


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( B ) HT29 control ( henceforth IRGM + / + ) and single allele CRISPR knockout IRGM cells ( henceforth IRGM + / - ) ,

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

Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (B) HT29 control (henceforth IRGM+/+) and single allele CRISPR knockout IRGM cells (henceforth IRGM+/-),


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( C ) HT29 cells stably expressing control shRNA or IRGM shRNA ,

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

Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (C) HT29 cells stably expressing control shRNA or IRGM shRNA,


Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of ( D ) Irgm1 + / + and Irgm1 - / - mouse brain ( n = 3 mice ) .

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

Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (D) Irgm1+/+ and Irgm1-/- mouse brain (n=3 mice).


( E ) Western blot analysis to determine levels of adaptor proteins in control and IRGM + / - HT29 cells .

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

(E) Western blot analysis to determine levels of adaptor proteins in control and IRGM+/- HT29 cells.


( F ) SDD - AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells . Western blot analysis with cytoplasmic fraction was also performed .

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

(F) SDD-AGE followed by western blot analysis with a mitochondrial fraction from control and IRGM siRNA knockdown THP1 cells. Western blot analysis with cytoplasmic fraction was also performed.


Western blot analysis performed with lysates of ( G ) control and stable IRGM shRNA knockdown HT29 cells ( H ) IRGM + / + and IRGM + / - HT29

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

Western blot analysis performed with lysates of (G) control and stable IRGM shRNA knockdown HT29 cells (H) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( I ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of TBK1 protein .

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

Western blot analysis performed with lysates of (I) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of TBK1 protein.


Western blot analysis performed with lysates of ( J ) IRGM + / + and IRGM + / - HT29

Western blot analysis performed with lysates of (J) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( K - L ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of IRF proteins .

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

Western blot analysis performed with lysates of (K-L) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of IRF proteins.


Western blot analysis performed with lysates of ( M ) IRGM + / + and IRGM + / - HT29

Western blot analysis performed with lysates of (M) IRGM+/+ and IRGM+/- HT29 cells


Western blot analysis performed with lysates of ( N ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of STAT proteins ( n = 3 mice ) .

Western blot analysis performed with lysates of (N) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of STAT proteins (n=3 mice).


Western blot analysis performed with lysates of ( O ) IRGM + / + and IRGM + / - HT29 cells . 2 biological replicates are shown .

Western blot analysis performed with lysates of (O) IRGM+/+ and IRGM+/- HT29 cells. 2 biological replicates are shown.


Western blot analysis performed with lysates of ( P ) Irgm1 + / + and Irgm1 - / - mouse brain to determine levels of ISG proteins ( n = 2 mice ) .

Western blot analysis performed with lysates of (P) Irgm1+/+ and Irgm1-/- mouse brain to determine levels of ISG proteins (n=2 mice).


( Q ) Representative confocal image of immunofluorescence assay performed with IRGM + / + and IRGM + / - HT29 cells stained with MX1 antibodies . Scale bar , 10 µm .

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

(Q) Representative confocal image of immunofluorescence assay performed with IRGM+/+ and IRGM+/- HT29 cells stained with MX1 antibodies. Scale bar, 10 µm.


( A ) Immunoprecipitation ( IP ) analysis of the interaction between endogenous IRGM and endogenous RIG - I , cGAS , MDA5 , TLR3 , TLR4 , TLR7 , NLRC4 , and AIM2 in THP - 1 cell lysates . IgG ( H ) , IgG heavy chain .

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

(A) Immunoprecipitation (IP) analysis of the interaction between endogenous IRGM and endogenous RIG-I, cGAS, MDA5, TLR3, TLR4, TLR7, NLRC4, and AIM2 in THP-1 cell lysates. IgG (H), IgG heavy chain.


( B - D ) Co - immunoprecipitation ( Co - IP ) analysis of the interaction between ( B ) GFP - IRGM and Flag - RIG - I , ( C ) GFP - IRGM , and Flag - cGAS , ( D ) GFP - IRGM and Flag - TLR3 in HEK293T cell lysates . L . E , Long exposure of input blot . IgG ( L ) , IgG Light chain ; IgG ( H ) , IgG heavy chain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Co-immunoprecipitation (Co-IP) analysis of the interaction between (B) GFP-IRGM and Flag-RIG-I, (C) GFP-IRGM, and Flag-cGAS, (D) GFP-IRGM and Flag-TLR3 in HEK293T cell lysates. L.E, Long exposure of input blot. IgG (L), IgG Light chain; IgG (H), IgG heavy chain.


( E ) GST pull - down assay of in - vitro translated and radiolabelled myc - tagged cGAS , RIG - I , and TLR3 with GST or GST - tagged IRGM .

(E) GST pull-down assay of in-vitro translated and radiolabelled myc-tagged cGAS, RIG-I, and TLR3 with GST or GST-tagged IRGM.


( F ) Western blot analysis with the lysates of HT - 29 cells stably transfected with control or Flag - IRGM plasmid and probed with the indicated antibodies .

(F) Western blot analysis with the lysates of HT-29 cells stably transfected with control or Flag-IRGM plasmid and probed with the indicated antibodies.


( G - I ) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP - IRGM and ( G ) Flag - RIG - I or ( H ) Flag - cGAS or ( I ) or Flag - TLR3 .

O O O O O O O O O O O O O O O O O O O O O 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-I) Western blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP-IRGM and (G) Flag-RIG-I or (H) Flag-cGAS or (I) or Flag-TLR3.


( J - K ) The qRT - PCR analysis with RNA isolated from ( J ) control and Flag - IRGM stable HT - 29 cells ( K ) THP - 1 cells transiently transfected with control or Flag - IRGM plasmid .

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

(J-K) The qRT-PCR analysis with RNA isolated from (J) control and Flag-IRGM stable HT-29 cells (K) THP-1 cells transiently transfected with control or Flag-IRGM plasmid.


Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated .

Luciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated.


( O ) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 ( 300 nM ; 4 h and 8 h ) treated Flag - IRGM expressing HT29 stable cell lines .

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

(O) Western blotting analysis with lysates of control and untreated or Bafilomycin A1 (300 nM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.


( P ) Western blotting analysis with lysates of control and untreated or chloroquine ( 50 µM ; 4 h and 8 h ) treated Flag - IRGM expressing HT29 stable cell lines .

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

(P) Western blotting analysis with lysates of control and untreated or chloroquine (50 µM; 4 h and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.


( Q ) The qRT - PCR analysis with RNA isolated from untreated or Bafilomycin A1 ( 300 nM ; 4 h , 8 h , and 12 h ) treated control or Flag - IRGM expressing HT - 29 stable cells as indicated .

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

(Q) The qRT-PCR analysis with RNA isolated from untreated or Bafilomycin A1 (300 nM; 4 h, 8 h, and 12 h) treated control or Flag-IRGM expressing HT-29 stable cells as indicated.


( A ) The cell lysates of control or BECLIN1 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM were subjected to immunoblotting with indicated antibodies .

(A) The cell lysates of control or BECLIN1 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.


( B ) The cell lysates of control or ATG5 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM were subjected to immunoblotting with indicated antibodies .

(B) The cell lysates of control or ATG5 siRNA transfected THP-1 cells expressing GFP or GFP-IRGM were subjected to immunoblotting with indicated antibodies.


( C - D ) qRT - PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

(C-D) qRT-PCR analysis with RNA isolated from control or BECLIN1 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


qRT - PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

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

qRT-PCR analysis with RNA isolated from control or ATG5 siRNA transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


( G - H ) qRT - PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

(G-H) qRT-PCR analysis with RNA isolated from control or p62 siRNA or BECLIN1 siRNA transfected HT29 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


Co - IP analysis of the interaction between GFP IRGM with ( I ) Flag - p62 or Flag - NDP52 or Flag - NBR1 in HEK293T cell lysates .

Co-IP analysis of the interaction between GFP IRGM with (I) Flag-p62 or Flag-NDP52 or Flag-NBR1 in HEK293T cell lysates.


Co - IP analysis of the interaction between GFP IRGM with ( J ) Flag - Optineurin or Flag - TAX1BP1 in HEK293T cell lysates .

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

Co-IP analysis of the interaction between GFP IRGM with (J) Flag-Optineurin or Flag-TAX1BP1 in HEK293T cell lysates.


( K - M ) Co - IP analysis of the interaction between GFP IRGM with ( K ) Flag - cGAS , ( L ) Flag - RIG - I , ( M ) Flag - TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates .

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

(K-M) Co-IP analysis of the interaction between GFP IRGM with (K) Flag-cGAS, (L) Flag-RIG-I, (M) Flag-TLR3 in control siRNA or p62 siRNA transfected HEK293T cell lysates.


( N ) The cell lysates of control or p62 siRNA transfected THP - 1 cells expressing GFP or GFP - IRGM ( as indicate ) were subjected to immunoblotting with indicated antibodies .

(N) The cell lysates of control or p62 siRNA transfected THP-1 cells expressing GFP or GFP- IRGM (as indicate) were subjected to immunoblotting with indicated antibodies.


( O ) The qRT - PCR analysis with RNA isolated from control or p62 transfected THP - 1 cells expressing 3X - Flag epitope or Flag - IRGM as indicated .

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

(O) The qRT-PCR analysis with RNA isolated from control or p62 transfected THP-1 cells expressing 3X-Flag epitope or Flag-IRGM as indicated.


( P - R ) Co - IP analysis of the interaction between HA - p62 and ( P ) Flag - RIG - I , ( Q ) Flag - TLR3 , ( R ) Flag - cGAS in the absence and presence of GFP or GFP - IRGM in HEK293T cell lysates .

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

(P-R) Co-IP analysis of the interaction between HA-p62 and (P) Flag-RIG-I, (Q) Flag-TLR3, (R) Flag-cGAS in the absence and presence of GFP or GFP-IRGM in HEK293T cell lysates.


( A ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 antibody . Scale bar , 5 µm . Zoom panels are digital magnifications .

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

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 antibody. Scale bar, 5 µm. Zoom panels are digital magnifications.


( B ) The graph depicts the percentage of cells with swollen / rounded mitochondria in Irgm1 + / + and Irgm1 - / - mouse BMDMs .

(B) The graph depicts the percentage of cells with swollen/rounded mitochondria in Irgm1+/+ and Irgm1-/- mouse BMDMs.


( C ) Representative confocal ( STED ) images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies . Line profile : Co - localization analysis using line intensity profiles .

O O O O O O O O O O O O O O O O O O O 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) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies. Line profile: Co-localization analysis using line intensity profiles.


( D ) Representative confocal ( STED ) images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies . Line profile : Co - localization analysis using line intensity profiles .

(D) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies. Line profile: Co-localization analysis using line intensity profiles.


( E ) Representative confocal images of control siRNA and IRGM siRNA transfected THP - 1 cells processed for IF analysis with Ubiquitin ( green ) and TOM20 ( red ) antibodies . Scale bar , 10 µm .

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

(E) Representative confocal images of control siRNA and IRGM siRNA transfected THP-1 cells processed for IF analysis with Ubiquitin (green) and TOM20 (red) antibodies. Scale bar, 10 µm.


( F ) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP - 1 cells , probed with indicated antibodies .

(F) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells, probed with indicated antibodies.


( G ) Western blot analysis of mitochondrial fraction of Irgm1 + / + and Irgm1 - / - mouse BMDM cells , probed with indicated antibodies ( n = 2 mice ) .

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(G) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells, probed with indicated antibodies (n=2 mice).


( H ) Western blot analysis of mitochondrial fraction of Irgm1 + / + and Irgm1 - / - mouse BMDM cells untreated or treated with Bafilomycin A1 ( 300 nM , 3 h ) , probed with indicated antibodies .

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(H) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.


( I ) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP - 1 cells untreated or treated with Bafilomycin A1 ( 300 nM , 3 h ) , probed with indicated antibodies .

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(I) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.


( J ) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1 + / + and Irgm1 - / - mouse BMDM cells . The experiments were performed using the XF24 extracellular flux analyzer , and the flow chart showed the measurement of oxygen consumption rate ( OCR )

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(J) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1+/+ and Irgm1-/- mouse BMDM cells. The experiments were performed using the XF24 extracellular flux analyzer, and the flow chart showed the measurement of oxygen consumption rate (OCR)


( K ) Representative confocal images of control and IRGM siRNA transfected THP - 1 cells stained with CMXRos red dye .

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(K) Representative confocal images of control and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye.


( L ) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP - 1 cells stained with CMXRos red dye ( 10 nM , 30 mins ) . ( M ) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP - 1 cells stained with CMXRos .

(L) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye (10 nM, 30 mins). (M) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP-1 cells stained with CMXRos.


( N ) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP - 1 cells stained with JC - 1 dye ( 2 µM , 30 mins ) . At low mitochondrial membrane potential , JC - 1 is predominantly a monomer that yields green fluorescence , whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission .

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(N) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP-1 cells stained with JC-1 dye (2 µM, 30 mins). At low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence, whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission.


( P ) Representative flow cytometry analysis of control and IRGM siRNA transfected THP - 1 cells stained with MitoSox red dye ( 1µM , 20 min ) . The percentage of control and IRGM knockdown cells with increased red fluorescence ( mitochondrial ROS generation ) is depicted .

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(P) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with MitoSox red dye (1µM, 20 min). The percentage of control and IRGM knockdown cells with increased red fluorescence (mitochondrial ROS generation) is depicted.


( Q ) Representative flow cytometry analysis of control and IRGM siRNA transfected THP - 1 cells stained with CellRox - green and CellRox - orange dye ( 1µM , 30 min ) .

(Q) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with CellRox-green and CellRox-orange dye (1µM, 30 min).


( A ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with Tom20 ( red ) and dsDNA ( green ) antibody .

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(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 (red) and dsDNA (green) antibody.


( B ) Representative STED microscopy images of control THP - 1 cells processed for IF analysis with cGAS ( green ) , dsDNA ( red ) , and Lamin B1 ( cyan ) antibodies . ( C ) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP - 1 cells .

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(B) Representative STED microscopy images of control THP-1 cells processed for IF analysis with cGAS (green), dsDNA (red), and Lamin B1 (cyan) antibodies. (C) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP-1 cells.


( D - E ) Representative STED microscopy images of Irgm1 - / - mouse BMDMs ( D ) transfected with mCherry - cGAS and immunostained with dsDNA ( green ) antibody . The white line depicts the periphery of the cells . Scale bar , 3 µm . ( E ) The graph depicts the percentage of cells with micronuclei in Irgm1 + / + and Irgm1 - / - mouse BMDMs .

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(D-E) Representative STED microscopy images of Irgm1-/- mouse BMDMs (D) transfected with mCherry-cGAS and immunostained with dsDNA (green) antibody. The white line depicts the periphery of the cells. Scale bar, 3 µm. (E) The graph depicts the percentage of cells with micronuclei in Irgm1+/+ and Irgm1-/- mouse BMDMs.


( F ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs .

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(F) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs.


( G ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from control and IRGM siRNA transfected HT - 29 cells .

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(G) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control and IRGM siRNA transfected HT-29 cells.


( H ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT - 29 ( Rho0 ) cells .

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(H) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.


( I ) The qRT - PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT - 29 ( Rho0 ) cells .

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(I) The qRT-PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells.


( J ) The qRT - PCR analysis with cytosolic DNA ( minus mitochondria ) isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs transfected with DNase I ( 15 μg , 6hr ) .

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(J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr).


( K ) The qRT - PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP - 1 cells electroporated with DNase I ( 5 μg , 1hr ) as indicated .

(K) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with DNase I (5 μg, 1hr) as indicated.


( L - M ) Representative confocal images of Irgm1 + / + and Irgm1 - / - mouse BMDMs processed for IF analysis with ( L ) dsRNA ( green ) and Tom20 ( red ) antibodies . ( M ) The graph depicts the percentage of Irgm1 + / + and Irgm1 - / - mouse BMDMs with dsRNA aggregates .

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(L-M) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with (L) dsRNA (green) and Tom20 (red) antibodies. (M) The graph depicts the percentage of Irgm1+/+ and Irgm1-/- mouse BMDMs with dsRNA aggregates.


( N ) Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with RIG - I ( red ) and dsRNA ( green ) antibodies . Line profile : Co - localization analysis using line intensity profiles .

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(N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles.


Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with ( O ) Mda5 ( red ) and dsRNA ( green ) antibodies Co - localization analysis using line intensity profiles .

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Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies Co-localization analysis using line intensity profiles.


Representative confocal images of Irgm1 - / - mouse BMDMs processed for IF analysis with ( P ) Mda5 ( red ) , dsRNA ( green ) and Tia1 ( blue ) antibodies ( Scale bar , 3 µm ) . Line profile : Co - localization analysis using line intensity profiles .

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Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles.


( A ) qRT - PCR analysis with total RNA isolated from Irgm1 + / + and Irgm1 - / - mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG - I siRNA or cGAS siRNA and RIG - I siRNA as indicated .

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(A) qRT-PCR analysis with total RNA isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with control siRNA or cGAS siRNA or RIG-I siRNA or cGAS siRNA and RIG-I siRNA as indicated.


( B ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG - I siRNA or transfected with three siRNAs as indicated .

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(B) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and cGAS siRNA or IRGM siRNA and RIG-I siRNA or transfected with three siRNAs as indicated.


( C ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated .

(C) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STING siRNA or IRGM siRNA and MAVS siRNA or transfected with all the three siRNAs as indicated.


( D ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated .

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(D) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and MDA5 siRNA as indicated.


( E ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated .

(E) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and IRF3 siRNA as indicated.


( F ) qRT - PCR analysis with total RNA isolated from HT - 29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated .

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(F) qRT-PCR analysis with total RNA isolated from HT-29 cells transfected with control siRNA or IRGM siRNA or doubly transfected with IRGM siRNA and STAT1 siRNA or IRGM siRNA and STAT2 siRNA as indicated.