Figure 1B ) Egg chambers flatten along the coverslip , allowing for follicle cell morphologies to be compared in two dimensions . In ( B ) , three live w1118 ( control ) egg chambers expressing the membrane marker Basigin : : YFP are shown in multiple planes of focus , with a z - reconstruction at the bottom . Because the egg chambers flatten at the coverslip , the basal follicle cell morphologies can be revealed in a single focal plane . To account for differences in follicle cell height between the three egg chambers , which increase in maturity from left to right , separate focal planes are shown for the apical morphologies . A diagram illustrating the position of the three focal planes with respect to the flattened egg chamber is shown in ( B " ) . Scale bars = C - E ) Follicle cell cross - sectional area decreases ( C ) and cell shape becomes more regular ( D ) and hexagonal ( E ) as early - stage egg chambers mature . These measurements were derived from at least three egg chambers from at least three flies per AR range . Bars in ( C ) and ( D ) represent mean and standard deviation . Statistical significance for cell side distributions was calculated using a chi - squared test . Number of cells used for morphological analysis : 1 . 0 - 1 . 2 apical n = 23 , basal n = 27 ; 1 . 2 - 1 . 4 apical n = 114 , basal n = 61 ; 1 . 4 - 1 . 6 , apical n = 160 , basal n = 149 . Vertex counts : 1 . 0 - 1 . 2 apical n = 38 , basal n = 33 ; 1 . 2 - 1 . 4 apical n = 73 , basal n = 70 ; 1 . 4 - 1 . 6 , apical n = 284 , basal n = 259 .

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

Figure 1B) Egg chambers flatten along the coverslip, allowing for follicle cell morphologies to be compared in two dimensions. In (B), three live w1118 (control) egg chambers expressing the membrane marker Basigin::YFP are shown in multiple planes of focus, with a z-reconstruction at the bottom. Because the egg chambers flatten at the coverslip, the basal follicle cell morphologies can be revealed in a single focal plane. To account for differences in follicle cell height between the three egg chambers, which increase in maturity from left to right, separate focal planes are shown for the apical morphologies. A diagram illustrating the position of the three focal planes with respect to the flattened egg chamber is shown in (B"). Scale bars =C-E) Follicle cell cross-sectional area decreases (C) and cell shape becomes more regular (D) and hexagonal (E) as early-stage egg chambers mature. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars in (C) and (D) represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for morphological analysis: 1.0-1.2 apical n=23, basal n=27; 1.2-1.4 apical n=114, basal n=61; 1.4-1.6, apical n=160, basal n=149. Vertex counts: 1.0-1.2 apical n =38, basal n=33; 1.2-1.4 apical n=73, basal n=70; 1.4-1.6, apical n=284, basal n=259.


Figure 2Egg chamber elongation is unaffected by reduced cell proliferation . String - shRNA expressed for 24 hours alters epithelial shape and cell number ( A Chambers shown in ( A ) have approximately equal widths , with the cross - sectional area corresponding to developmental stage 6 . Scale bars = 20 μm . Number of chambers analyzed : w1118 1 . 0 - 1 . 2 n = 24 , 1 . 2 - 1 . 4 n = 14 , 1 . 4 - 1 . 6 n = 18 String - shRNA 1 . 0 - 1 . 2 n = 11 , 1 . 2 - 1 . 4 n = 10 , 1 . 4 - 1 . 6 n = 15 . Bars represent mean and standard deviationEgg chamber elongation is unaffected by reduced cell proliferation . String - shRNA expressed for 24 hours alters epithelial shape and cell numbe but not the relationship between egg chamber aspect ratio and cross - sectional area , which is quantified in ( B ) Number of chambers analyzed : w1118 1 . 0 - 1 . 2 n = 24 , 1 . 2 - 1 . 4 n = 14 , 1 . 4 - 1 . 6 n = 18 String - shRNA 1 . 0 - 1 . 2 n = 11 , 1 . 2 - 1 . 4 n = 10 , 1 . 4 - 1 . 6 n = 15 . Bars represent mean and standard deviationC , D ) Cell proliferation is necessary for normal tissue morphology Follicle cell shape regularity ( C ) and polygonality ( D ) measurements in control and String - shRNA egg chambers of different aspect ratios . These measurements were derived from at least three egg chambers from at least three flies per AR range . Bars represent mean and standard deviation . Statistical significance for cell side distributions was calculated using a chi - squared test . Number of cells used for analysis : w1118 1 . 0 - 1 . 2 n = 23 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 ; String - shRNA 1 . 0 - 1 . 2 , n = 6 , 1 . 2 - 1 . 4 n = 19 , 1 . 4 - 1 . 6 n =

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

Figure 2Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell number (A Chambers shown in (A) have approximately equal widths, with the cross-sectional area corresponding to developmental stage 6. Scale bars = 20 μm. Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviationEgg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell numbe but not the relationship between egg chamber aspect ratio and cross-sectional area, which is quantified in (B) Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviationC, D) Cell proliferation is necessary for normal tissue morphology Follicle cell shape regularity (C) and polygonality (D) measurements in control and String-shRNA egg chambers of different aspect ratios. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for analysis: w1118 1.0-1.2 n= 23, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=6, 1.2-1.4 n=19, 1.4-1.6 n=17


Figure 3Epithelial cell size and shape is affected by decreased proliferation . Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1 . 4 ( A ) . Epithelial cell size and shape is affected by decreased proliferation . Cell outlines are marked with Basigin : : YFP . Scale bars = 5 μm . Cells become significantly larger ( B ) Cell area shown on a log4 scale in ( B ) . Number of cells analyzed : w1118 1 . 0 - 1 . 2 n = 14 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 Statistical significance calculated using a Mann - Whitney test . Data information : Bars represent mean and standard deviation . Epithelial cell size and shape is affected by decreased proliferation . Cell outlines are marked with Basigin : : YFP . Scale bars = 5 μm . Cells become more aligned with the tissue elongation axis when proliferation is stopped using String - shRNA ( C ) . Number of cells analyzed : w1118 1 . 0 - 1 . 2 n = 14 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 ; String - shRNA 1 . 0 - 1 . 2 , n = 10 , 1 . 2 - 1 . 4 n = 19 , 1 . 4 - 1 . 6 n = 17 . Statistical significance calculated using a Mann - Whitney test . Data information : Bars represent mean and standard deviation . D , E ) The follicular epithelium ' thins out ' over the egg chamber equator when proliferation is blocked . Cartoon demonstrates the tissue cell shape observed in the follicular epithelium in control ( w1118 ) and String - shRNA treated egg chambers . Quantification of the phenomenon shown in ( D ) reveals that cells are shorter over the equator than at the anterior pole in elongated egg chambers when proliferation is blocked . Statistical significance calculated using unpaired t - test with Welch ' s correction . Number of cells analyzed : w1118 1 . 0 - 1 . 2 n = 21 , 1 . 2 - 1 . 4 n = 11 , 1 . 4 - 1 . 6 n = 10 ; String - shRNA 1 . 0 - 1 . 2 , n = 9 , 1 . 2 - 1 . 4 n = 11 , 1 . 4 - 1 . 6 n = 13 . Data information : Bars represent mean and standard deviation . F ) Recoil is observed when cuts are made in apical cortex with a 2 - photon laser with egg chambers in saggital view . Images show cortex before and after cutting . Scale bars = 10 μm . Yellow arrows indicate edges of ablated region . G ) Recoil is higher when cuts are made at the apical cortex at the equator of egg chambers , rather than at the poles . Quantification of experiment shown in ( F ) . Statistical significance tested by Mann - Whitney test . Number of laser cuts : equator n = 30 , pole n = 20 . Data information Laser ablation experiments performed on tissue from sqhAX3 , sqh : : GFP flies . H ) Recoil is observed when larger cuts are made in apical cortex with a UV laser . Images show cortex before and after cutting . Red lines indicate the lines over which recoil was calculated , parallel to the axes of the egg chamber . Scale bars = 5 μm . Yellow arrows indicate edges of ablated region . I ) Recoil tends to be higher towards the AP ( elongation ) axis compared to the the UD ( rotation ) axis . Graph shows the ratio of recoil speed plotted on a log2 scale in the AP versus the UD axis . Red line at x = 1 indicates equal recoil in both axes . Values above 1 indicate higher recoil speeds in the AP axis lower than 1 indicate higher recoil in the UD axis . AP / UD Ratio Median = 1 . 242 ; Mean = 2 . 1 . Wilcoxon Signed Rank Test to test for significance of the recoil ratio AP / UD shows that the median is significantly different from 1 to p = 0 . 0172 . Number of cuts = 20 . Bar represents median . Laser ablation experiments performed on tissue from sqhAX3 , sqh : : GFP flies .

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

Figure 3Epithelial cell size and shape is affected by decreased proliferation. Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1.4 (A).Epithelial cell size and shape is affected by decreased proliferation. Cell outlines are marked with Basigin::YFP. Scale bars = 5 μm. Cells become significantly larger (B) Cell area shown on a log4 scale in (B). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160 Statistical significance calculated using a Mann-Whitney test. Data information: Bars represent mean and standard deviation.Epithelial cell size and shape is affected by decreased proliferation. Cell outlines are marked with Basigin::YFP. Scale bars = 5 μm. Cells become more aligned with the tissue elongation axis when proliferation is stopped using String-shRNA (C). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=10, 1.2-1.4 n=19, 1.4-1.6 n=17. Statistical significance calculated using a Mann-Whitney test. Data information: Bars represent mean and standard deviation.D, E) The follicular epithelium 'thins out' over the egg chamber equator when proliferation is blocked. Cartoon demonstrates the tissue cell shape observed in the follicular epithelium in control (w1118) and String-shRNA treated egg chambers. Quantification of the phenomenon shown in (D) reveals that cells are shorter over the equator than at the anterior pole in elongated egg chambers when proliferation is blocked. Statistical significance calculated using unpaired t-test with Welch's correction. Number of cells analyzed: w1118 1.0-1.2 n= 21, 1.2-1.4 n=11, 1.4-1.6 n=10; String-shRNA 1.0-1.2, n=9, 1.2-1.4 n=11, 1.4-1.6 n=13. Data information: Bars represent mean and standard deviation.F) Recoil is observed when cuts are made in apical cortex with a 2-photon laser with egg chambers in saggital view. Images show cortex before and after cutting. Scale bars = 10 μm. Yellow arrows indicate edges of ablated region. G) Recoil is higher when cuts are made at the apical cortex at the equator of egg chambers, rather than at the poles. Quantification of experiment shown in (F). Statistical significance tested by Mann-Whitney test. Number of laser cuts: equator n=30, pole n=20. Data information Laser ablation experiments performed on tissue from sqhAX3, sqh::GFP flies.H) Recoil is observed when larger cuts are made in apical cortex with a UV laser. Images show cortex before and after cutting. Red lines indicate the lines over which recoil was calculated, parallel to the axes of the egg chamber. Scale bars = 5 μm. Yellow arrows indicate edges of ablated region. I) Recoil tends to be higher towards the AP (elongation) axis compared to the the UD (rotation) axis. Graph shows the ratio of recoil speed plotted on a log2 scale in the AP versus the UD axis. Red line at x=1 indicates equal recoil in both axes. Values above 1 indicate higher recoil speeds in the AP axis lower than 1 indicate higher recoil in the UD axis. AP/UD Ratio Median = 1.242 ; Mean = 2.1. Wilcoxon Signed Rank Test to test for significance of the recoil ratio AP/UD shows that the median is significantly different from 1 to p = 0.0172. Number of cuts = 20. Bar represents median.Laser ablation experiments performed on tissue from sqhAX3, sqh::GFP flies.


Figure 4B ) i ) Planar division orientation is initially random in the follicular epithelium , with a significant bias towards the egg chamber elongation axis evident at stage 6 ( asterisk , p = 0 . 0031 , Wilcoxon Signed Rank Test , Theoretical Mean of 45 ; p = 0 . 0218 when comparison to a randomly generated list of the same size ) . Number of cells analyzed : stage 2 / 3 n = 16 , stage 4 n = 27 stage 5 n = 30 , stage 6 n = 40 . ii ) Quantification of the angle of tissue expansion as calculated by vector addition of the minor and major axis of egg chambers . Number of egg chambers analyzed : stage 2 / 3 n = 10 , stage 4 n = 15 , stage 5 n = 15 , stage 6 n = 10 . iii ) Quantification of cell elongation angles ( as shown in E ) with respect to the chamber elongation axis by stage from live egg chambers . Number of cells analyzed : stage 2 / 3 n = 18 , stage 4 n = 49 stage 5 n = 64 , stage 6 n = 97 . Bars represent mean and standard deviationC ) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2 - shRNA and Pak - shRNA egg chambers . Mann Whitney test w1118 versus Pak shRNA p = 0 . 0094 . Neither the Fat2 - shRNA nor Pak - shRNA distributions are stastically different from random ( Wilcoxon Signed Rank Test , Theoretical Mean of 45 ) . Number of cells analyzed : w1118 n = 97 , Fat2 - shRNA n = 44 , Pak - shRNA n = 39 . Egg chambers analyzed : w1118 n = 40 , Fat2 - shRNA n = 21 , Pak - shRNA n = 21D ) Neighboring cells can form new attachments underneath ( basal to ) a mitotic cell that has moved apically . Scale bars = 5 μm . Thin yellow lines in the XZ reconstructions indicate the z - position of the two XY focal planesE ) Workflow for determining follicle shape shapes . Segmentation of an egg chamber with an aspect ratio of 1 . 5 reveals that follicle cell elongation is usually perpendicular to the elongation axis of the egg chamber . Cell outlines were marked with Basigin : : YFP . Scale bars = 5 μmF ) Live confocal imaging shows that the long axis of a follicle cell at interphase does not predict the orientation of division . Long axis orientations at the timepoints shown were determined as in ( E ) . Scale bars = 5 μm . G ) The interphase long axis does not correlate with the division angle in both the follicular epithelium ( FE ) and embyronic neuroectoderm ( ENE ) . n = 10 cells ( FE ) , 18 cells ( ENE )

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

Figure 4B) i) Planar division orientation is initially random in the follicular epithelium, with a significant bias towards the egg chamber elongation axis evident at stage 6 (asterisk, p = 0.0031, Wilcoxon Signed Rank Test, Theoretical Mean of 45; p = 0.0218 when comparison to a randomly generated list of the same size). Number of cells analyzed: stage 2/3 n=16, stage 4 n=27 stage 5 n=30, stage 6 n=40. ii) Quantification of the angle of tissue expansion as calculated by vector addition of the minor and major axis of egg chambers. Number of egg chambers analyzed: stage 2/3 n=10, stage 4 n=15, stage 5 n=15, stage 6 n=10. iii) Quantification of cell elongation angles (as shown in E) with respect to the chamber elongation axis by stage from live egg chambers. Number of cells analyzed: stage 2/3 n=18, stage 4 n=49 stage 5 n=64, stage 6 n=97. Bars represent mean and standard deviationC) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2-shRNA and Pak-shRNA egg chambers. Mann Whitney test w1118 versus Pak shRNA p = 0.0094. Neither the Fat2-shRNA nor Pak-shRNA distributions are stastically different from random (Wilcoxon Signed Rank Test, Theoretical Mean of 45). Number of cells analyzed: w1118 n =97, Fat2-shRNA n=44, Pak-shRNA n=39. Egg chambers analyzed: w1118 n=40, Fat2-shRNA n=21, Pak-shRNA n=21D) Neighboring cells can form new attachments underneath (basal to) a mitotic cell that has moved apically. Scale bars = 5 μm. Thin yellow lines in the XZ reconstructions indicate the z-position of the two XY focal planesE) Workflow for determining follicle shape shapes. Segmentation of an egg chamber with an aspect ratio of 1.5 reveals that follicle cell elongation is usually perpendicular to the elongation axis of the egg chamber. Cell outlines were marked with Basigin::YFP. Scale bars = 5 μmF) Live confocal imaging shows that the long axis of a follicle cell at interphase does not predict the orientation of division. Long axis orientations at the timepoints shown were determined as in (E). Scale bars = 5 μm.G) The interphase long axis does not correlate with the division angle in both the follicular epithelium (FE) and embyronic neuroectoderm (ENE). n=10 cells (FE), 18 cells (ENE)


Figure 5B , C ) Pins and Mud are symmetric around the entire cell cortex in ( B ) mitotic follicle cells and ( C ) mitotic cells in the ENE . ( B " and C " ) Confocal imaging of immunostained fixed tissue . Scale bE ) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0 . Timepoints shown in minutes relative anaphase . Scale bars = 5 μm .

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

Figure 5B, C) Pins and Mud are symmetric around the entire cell cortex in (B) mitotic follicle cells and (C) mitotic cells in the ENE. (B" and C") Confocal imaging of immunostained fixed tissue. Scale bE) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0. Timepoints shown in minutes relative anaphase. Scale bars = 5 μm.


Figure 6Live confocal imaging of Myosin - II shown by Zipper : : YFP ( A at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue - level organization during stage 6Live confocal imaging of Myosin - II shown b Spaghetti squash - mCherry ( B ) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue - level organization during stage 6 . ( B ) The localization Sqh - mCherry is shown relative to adherens junction marked by Shg : :

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

Figure 6Live confocal imaging of Myosin-II shown by Zipper::YFP (A at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6Live confocal imaging of Myosin-II shown b Spaghetti squash-mCherry (B) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6. (B) The localization Sqh-mCherry is shown relative to adherens junction marked by Shg::GFP


Figure 7A ) Apical Myo - II localization as marked by Sqh - GFP is disrupted in round ( stage 2 / 3 ) and elongated ( stage 6 ) egg chambers . Red arrows point to the seperation of Myo - II at cell junctions , indicating that actomyosin is no longer correctly linked to adherens junctions . Live confocal imaging . Scale bars = 5 μm . Tissue topology is affected by disruption of Canoe , but not Mud ( D ) Statistical significance for cell side distributions was calculated using a chi - squared test . Number of cells analyzed : w1118 1 . 4 - 1 . 6 n = 284 ; Canoe - shRNA 1 . 4 - 1 . 6 n = 310 ; mud3 / mud4 1 . 4 - 1 . 6 n = 251Tissue topology is affected by disruption of Canoe , but not Mud Representative image of a Canoe - shRNA follicle epithelium . Cell outlines are marked with Basigin : : YFP . This egg chamber has no mitotic cells and AR of 1 . 58 . Scale bars = 5

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

Figure 7A) Apical Myo-II localization as marked by Sqh-GFP is disrupted in round (stage 2/3) and elongated (stage 6) egg chambers. Red arrows point to the seperation of Myo-II at cell junctions, indicating that actomyosin is no longer correctly linked to adherens junctions. Live confocal imaging. Scale bars = 5 μm.Tissue topology is affected by disruption of Canoe, but not Mud (D) Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells analyzed: w1118 1.4-1.6 n=284; Canoe-shRNA 1.4-1.6 n=310; mud3/mud4 1.4-1.6 n=251Tissue topology is affected by disruption of Canoe, but not Mud Representative image of a Canoe-shRNA follicle epithelium. Cell outlines are marked with Basigin::YFP. This egg chamber has no mitotic cells and AR of 1.58. Scale bars = 5 μm


Figure 1B Representative confocal microscopy of human PBMCs stained with DAPI ( white arrows ) co - cultured with previously labeled UC - MSC with MitoTracker - Green ( top image ) and specific antibody for human CD90 ( bottom image ) . C Percentage of mitochondrial transfer ( MitoT ) on total human CD45 + cells after 24 hrs co - culture with MitoTracker - Green labeled bone marrow ( BM ) , menstrual ( Mens ) or umbilical cord ( UC ) - MSCs ( MSC : PBMC ratio 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of BM , Mens and UC - MSCs ) . D FACS analysis of MitoT on T CD3 + ( n = 4 ) , B CD19 + ( n = 4 ) and natural killer ( NK ) CD56 + ( n = 3 ) cells after 24 hrs co - culture with MitoGreen labeled UC - MSCs ( ratio 1 : 25 ) ( Biological replicates , with different donors of UC - MSCs ) . E FACS analysis of MitoT on CD3 + CD4 + or CD3 + CD8 + T cell populations after 24 hrs co - culture with labeled UC - MSCs ( ratio 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of UC - MSCs ) . F Representative FACS plots of MitoT on CD45 + CD3 + T cells at increasing amounts of UC - MSC . G Percentage of MitoT on CD45 + CD3 + human cells after 24 hrs co - culture with increasing ratios of UC - MSC : PBMCs ( n = 5 biological replicates , with 5 different UC - MSCs and 5 different PBMCs ) . H FACS analysis of MitoT on CD45 + CD3 + T cells after short times of co - culture with UC - MSCs at two different temperatures , at a ratio 1 : 25 ( n = 3 biological replicates , with 3 different UC - MSCs and 3 different PBMCs ) . I Mean fluorescence intensity ( MFI ) of MitoTracker - Green + UC - MSCs at baseline or after 72 hours PBMC co - culture . Representative FACS plot ( left panel ) and average of MFI ( n = 4 biological replicates , with 4 different UC - MSCs ) ( right panel ) . J Confocal microscopy of human PBMCs after co - culture with MitoTracker - Green stained UC - MSCs . Images showed MSC - mitochondria ( green ) inside the CD3 + T cells ( red ) . K qPCR analysis of human β2 - microglobulin ( B2M ) , human specific mitochondrial ( Mito ) and mouse specific ( β - actin ) gene expression levels in UC - MSCs , mouse derived spleen cells or FACS sorted CD45 + mouse cells after 24 hrs co - culture with UC - MSCs ( n = 3 biological replicates ) . n / d = not detected . M FACS analysis of in vivo MitoT from UC - MSCs to Balb / c mouse mesenteric lymph node and spleen cells , 24 hours after intraperitoneal transplantation of 5 * 106 MitoTracker - labeled MSCs or without cells ( control ) ( n = 1 ) .

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

Figure 1B Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co-cultured with previously labeled UC-MSC with MitoTracker-Green (top image) and specific antibody for human CD90 (bottom image).C Percentage of mitochondrial transfer (MitoT) on total human CD45+ cells after 24 hrs co-culture with MitoTracker-Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)-MSCs (MSC:PBMC ratio 1:25) (n=3 biological replicates, with 3 different donors of BM, Mens and UC-MSCs).D FACS analysis of MitoT on T CD3+ (n=4), B CD19+ (n=4) and natural killer (NK) CD56+ (n=3) cells after 24 hrs co-culture with MitoGreen labeled UC-MSCs (ratio 1:25) (Biological replicates, with different donors of UC-MSCs).E FACS analysis of MitoT on CD3+CD4+ or CD3+CD8+ T cell populations after 24 hrs co-culture with labeled UC-MSCs (ratio 1:25) (n=3 biological replicates, with 3 different donors of UC-MSCs).F Representative FACS plots of MitoT on CD45+CD3+ T cells at increasing amounts of UC-MSC.G Percentage of MitoT on CD45+CD3+ human cells after 24 hrs co-culture with increasing ratios of UC-MSC:PBMCs (n=5 biological replicates, with 5 different UC-MSCs and 5 different PBMCs).H FACS analysis of MitoT on CD45+CD3+ T cells after short times of co-culture with UC-MSCs at two different temperatures, at a ratio 1:25 (n=3 biological replicates, with 3 different UC-MSCs and 3 different PBMCs).I Mean fluorescence intensity (MFI) of MitoTracker-Green+ UC-MSCs at baseline or after 72 hours PBMC co-culture. Representative FACS plot (left panel) and average of MFI (n=4 biological replicates, with 4 different UC-MSCs) (right panel).J Confocal microscopy of human PBMCs after co-culture with MitoTracker-Green stained UC-MSCs. Images showed MSC-mitochondria (green) inside the CD3+ T cells (red).K qPCR analysis of human β2-microglobulin (B2M), human specific mitochondrial (Mito) and mouse specific (β-actin) gene expression levels in UC-MSCs, mouse derived spleen cells or FACS sorted CD45+ mouse cells after 24 hrs co-culture with UC-MSCs (n=3 biological replicates). n/d = not detected.M FACS analysis of in vivo MitoT from UC-MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 hours after intraperitoneal transplantation of 5*106 MitoTracker-labeled MSCs or without cells (control) (n=1).


Figure 2B Confocal microscopy images of human CD3 + T cells with endogenous MT - labeled with MitoTracker Red CMXRos ( white arrows , left panel ) or after mitoception with MitoTracker Green labeled - MT from UC - MSCs ( white asterisk , right panel ) . C FACS analysis of MitoT on CD3 + human T cells after mitoception with increasing amounts of UC - MSC MT corresponding to the same cell number ratios used in co - culture experiments ( n = 4 biological replicates , with 4 different donors of UC - MSCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test . D Representative transmission electron microscopy images of FACS - sorted human CD3 + MitoTneg and CD3 + MitoTpos cells following mitoception with UC - MSC MT . Red arrows show human mitochondria . Quantification of human MT in sorted CD3 + MitoTneg and MitoTpos cells after mitoception with UC - MSCs , by transmission electron microscopy ( n = 25 independent cells analyzed per group ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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Figure 2B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel).C FACS analysis of MitoT on CD3+ human T cells after mitoception with increasing amounts of UC-MSC MT corresponding to the same cell number ratios used in co-culture experiments (n=4 biological replicates, with 4 different donors of UC-MSCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.D Representative transmission electron microscopy images of FACS-sorted human CD3+MitoTneg and CD3+MitoTpos cells following mitoception with UC-MSC MT. Red arrows show human mitochondria.Quantification of human MT in sorted CD3+ MitoTneg and MitoTpos cells after mitoception with UC-MSCs, by transmission electron microscopy (n=25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t-test.


Figure 3B Volcano plot analysis derived from FPKM values showing DE genes ( p < 0 . 05 , Student ' s t test ) in sorted MitoTneg and MitoTpos CD3 + cells ( from 4 different donors of PBMCs ) . C Heatmap depiction of RNA - seq analysis of significantly ( p < 0 . 01 , Student ' s t test ) DE coding genes from sorted MitoTpos and MitoTneg CD3 + cells ( from 4 different donors of PBMCs ) . E Differentially expressed genes from the significantly enriched pathways listed in figure 2D . Highlighted in red are key markers of Treg phenotype . F qRT - PCR analysis of genes related to T cell activation after mitoception with UC - MSC MT ( n = 4 biological replicates , with 4 different donors of PBMCs ) . The graph depicts fold expression in CD3 + MitoTpos cells relative to CD3 + MitoTneg cells , which are set at 1 . Graph shows mean ± SEM and statistical analysis by Student ' s t - test ( * p < 0 . 05 ) .

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Figure 3B Volcano plot analysis derived from FPKM values showing DE genes (p<0.05, Student's t test) in sorted MitoTneg and MitoTpos CD3+ cells (from 4 different donors of PBMCs).C Heatmap depiction of RNA-seq analysis of significantly (p<0.01, Student's t test) DE coding genes from sorted MitoTpos and MitoTneg CD3+ cells (from 4 different donors of PBMCs).E Differentially expressed genes from the significantly enriched pathways listed in figure 2D. Highlighted in red are key markers of Treg phenotype.F qRT-PCR analysis of genes related to T cell activation after mitoception with UC-MSC MT (n=4 biological replicates, with 4 different donors of PBMCs). The graph depicts fold expression in CD3+MitoTpos cells relative to CD3+MitoTneg cells, which are set at 1. Graph shows mean ± SEM and statistical analysis by Student's t-test (*p<0.05).


Figure 4A Representative bright - field microscopy images of FACS - sorted CD4 + naïve MitoTneg ( upper panel ) and MitoTpos ( lower panel ) cells after 5 days in culture with plain media ( no cytokines added ) . Red arrows are indicative of activation / proliferation clusters . B Representative FACS plots of Treg ( CD127lowCD25 + FoxP3 + ) populations after T cell differentiation on media with or without cytokines and antibody supplements ( anti - CD3 , IL - 2 and TGF - b1 ) . C FACS analysis of Treg induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells in plain ( n = 5 ) or differentiation media ( n = 6 ) ( Biological replicates , with different donors of PBMCs ) . D ICOS expression on FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) and bar graph ( right panel ) of ICOS high population ( n = 4 biological replicates ) . E CCR7 expression on sorted CD4 + naïve MitoTneg and MitoTpos cells after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) and bar graph ( right panel ) of CCR7 + population ( n = 4 biological replicates ) . F FACS analysis of Th1 ( CD4 + IFNγ + ) induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells in plain media ( n = 3 biological replicates ) . n / s = not significant .

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Figure 4A Representative bright-field microscopy images of FACS-sorted CD4+ naïve MitoTneg (upper panel) and MitoTpos (lower panel) cells after 5 days in culture with plain media (no cytokines added). Red arrows are indicative of activation/proliferation clusters.B Representative FACS plots of Treg (CD127lowCD25+FoxP3+) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti-CD3, IL-2 and TGF-b1).C FACS analysis of Treg induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain (n=5) or differentiation media (n=6) (Biological replicates, with different donors of PBMCs).D ICOS expression on FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of ICOS high population (n=4 biological replicates).E CCR7 expression on sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of CCR7+ population (n=4 biological replicates).F FACS analysis of Th1 (CD4+IFNγ+) induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain media (n=3 biological replicates). n/s= not significant.


Figure 5Representative proliferation FACS histogram of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells ( ratio 1 : 1 ) after 5 - 7 days of CD4 + naïve cell activation with Treg induction / differentiation media ( anti - CD3 , IL - 2 and TGF - b1 ) . PBMC alone represents positive control of proliferation and PBMC co - culture with MitoTneg correspond to the inhibition control with induced Treg cells . C Percentage of immunosuppression of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells after 5 - 7 days of CD4 + naïve cell activation with differentiation media ( ratio MitoT cells : PBMC ) ( n = 3 biological replicates , with different donors of PBMCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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Figure 5Representative proliferation FACS histogram of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells (ratio 1:1) after 5-7 days of CD4+ naïve cell activation with Treg induction/differentiation media (anti-CD3, IL-2 and TGF-b1). PBMC alone represents positive control of proliferation and PBMC co-culture with MitoTneg correspond to the inhibition control with induced Treg cells.C Percentage of immunosuppression of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells after 5-7 days of CD4+ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n=3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


Figure 6B Representative FACS plots of mitoception ( % of MitoTracker + ) in CD4 + or CD8 + T cells from mitocepted PBMC compared to control PBMC , before intravenous injection . C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) . Control group in grey , were untreated mice ( n = 6 mice ) . * p < 0 . 05 by Mann Whitney t - test . D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount ( 12x106 cells ) of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) ( p = 0 . 0218 by log - rank test ) . E FACS analysis of human Treg ( CD4 + CD25 + FoxP3 + ) cell engraftment in mouse spleen following transplantation of PBMC - MitoT or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) . F FACS analysis of human pro - inflammatory T helper1 ( CD4 + IFNγ + , left panel ) and T cytotoxic ( CD8 + IFNγ + , right panel ) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) . G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC - MitoT , PBMC and non - transplanted control samples , stained with Masson ' s trichrome staining . Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts . Arrowheads represents a reduced number of Paneth cells . H - I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) . J Quantification of Paneth cells per crypt , from at least 50 crypts per PBMC - MitoT ( n = 8 mice ) , PBMC transplanted ( n = 5 mice ) or non - transplanted control ( n = 3 ) mouse . Representative histopathologic images of GVHD in lung sections obtained from PBMC - MitoT , PBMC and non - transplanted control , stained with Masson ' s trichrome staining . L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) . M FACS analysis of total human CD4 + cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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

Figure 6B Representative FACS plots of mitoception (% of MitoTracker+) in CD4+ or CD8+T cells from mitocepted PBMC compared to control PBMC, before intravenous injection.C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group). Control group in grey, were untreated mice (n=6 mice). * p<0.05 by Mann Whitney t-test.D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount (12x106 cells) of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group) (p=0.0218 by log-rank test).E FACS analysis of human Treg (CD4+CD25+FoxP3+) cell engraftment in mouse spleen following transplantation of PBMC-MitoT or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).F FACS analysis of human pro-inflammatory T helper1 (CD4+IFNγ+, left panel) and T cytotoxic (CD8+IFNγ+, right panel) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC-MitoT, PBMC and non-transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells.H-I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).J Quantification of Paneth cells per crypt, from at least 50 crypts per PBMC-MitoT (n=8 mice), PBMC transplanted (n=5 mice) or non-transplanted control (n=3) mouse.Representative histopathologic images of GVHD in lung sections obtained from PBMC-MitoT, PBMC and non-transplanted control, stained with Masson's trichrome staining.L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).M FACS analysis of total human CD4+ cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


Figure 1 ( C ) Top panel : Representative images show PS labeling in the WT dLGN at P6 and P13 . RGC inputs were labeled with CTB488 . Bottom panel : Enlarged images of the boxed region in top panels . Circles indicate PS + RGC inputs . Arrows point to the enlarged PS + RGC inputs in the insets . ( D ) Quantification of the percentage of PS + RGC inputs in total RGC inputs at P6 and P13 . ( E ) Co - labeling of vGlut2 and PSVue in the dLGN at P6 . Circles indicate colocalized vGlut2 and PSVue . ( F ) Co - labeling of Homer1 and PSVue in the dLGN at P6 . Circles indicate colocalized Homer1 and PSVue . ( G ) Orthogonal sections showed a triple positive PS + / CTB488 + / CD68 + RGC inputs inside of microglia . ( H ) Orthogonal sections showed a triple positive PS + / vGlut2 + / CD68 + synapse inside of microglia . ( I ) A representative image of microglia ( upper - left ) is surface rendered ( bottom - left ) . The white arrow points to cells which might be apoptotic cells labeled by PSVue . RGC inputs and PSVue outside or inside of microglia are shown in the two right panels . Scare bar , 20 µm . ( J ) Quantification of the percentage of PS + and PS - RGC inputs outside of microglia in total inputs . N = 4 . ( K ) Quantification of the percentage of engulfed PS + and PS - RGC inputs in total engulfed inputs . N = 4 .

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Figure 1(C) Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. (D) Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13.(E) Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue.(F) Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue.(G) Orthogonal sections showed a triple positive PS+/CTB488+/CD68+ RGC inputs inside of microglia.(H) Orthogonal sections showed a triple positive PS+/vGlut2+/CD68+ synapse inside of microglia.(I) A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 µm.(J) Quantification of the percentage of PS+ and PS- RGC inputs outside of microglia in total inputs. N = 4. (K) Quantification of the percentage of engulfed PS+ and PS- RGC inputs in total engulfed inputs. N=4.


Figure 2 ( D ) The binding experiment using flow cytometry show that only the GAIN domain binds to PS , similar as Annexin V binding . ( E ) In the competition experiment , AF647 channel shows that GAIN domain binds 60 . 1 % of the PS + Ba / F3 cells ( left ) . Correspondingly , FITC channel reveals 39 . 7 % Annexin V binding to PS + Ba / F3 cells ( right ) . ( G ) Direct binding of hFc - tagged GPR56 GAIN proteins to PS and other phospholipids . HFc was used as a negative control , and GAS6 as a positive control .

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

Figure 2(D) The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding.(E) In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right).(G) Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control.


Figure 3 ( B ) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice . ( D ) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16 . 5 neocortex . Arrows indicate cortical ectopias that are shown in insets . ( E ) Quantification of ectopia size per section . ( F ) The distribution of ectopia from rostral to caudal cortex . ( G ) . Myelin basic protein ( MBP ) staining of corpus callosum in P28 controls , Gpr56 null and Gpr56 S4 mice . ( H ) Quantification of MBP intensity in corpus callosum . ( I ) Gpr56 transcript levels in WT microglia at P5 and P14 . ( J ) An overview of vGlut2 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the dotted boxes show where synapses are quantified . K ) Representative images of vGlut2 / Homer1 staining in the dLGN of control , Gpr56 null , and Gpr56 S4 brains at P10 . Arrows pointing to the enlarged synapse in the insets . L ) Relative vGlut2 / Homer1 synapse density in dLGN . N = 10 ( Ctrl ) , N = 6 ( Gpr56 null ) , N = 3 ( Gpr56 S4 ) . ( N ) Representative images and surface rendered microglia ( green ) in which CTB + ( red ) RGC inputs were engulfed . ( O ) Quantification of the percentage of engulfed RGC inputs in controls , Gpr56 null and Gpr56 S4 microglia .

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

Figure 3(B) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice.(D) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16.5 neocortex. Arrows indicate cortical ectopias that are shown in insets. (E) Quantification of ectopia size per section. (F) The distribution of ectopia from rostral to caudal cortex.(G). Myelin basic protein (MBP) staining of corpus callosum in P28 controls, Gpr56 null and Gpr56 S4 mice.(H) Quantification of MBP intensity in corpus callosum.(I) Gpr56 transcript levels in WT microglia at P5 and P14.(J) An overview of vGlut2 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.K) Representative images of vGlut2/Homer1 staining in the dLGN of control, Gpr56 null, and Gpr56 S4 brains at P10. Arrows pointing to the enlarged synapse in the insets. L) Relative vGlut2/Homer1 synapse density in dLGN. N=10 (Ctrl), N=6 (Gpr56 null), N=3 (Gpr56 S4).(N) Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. (O) Quantification of the percentage of engulfed RGC inputs in controls, Gpr56 null and Gpr56 S4 microglia.


Figure 4 ( A ) Gpr56 is deleted in CKO microglia . RNAscope shows Gpr56 transcripts co - localize with microglia ( arrow ) in controls , but not in CKO , and is absent in all cell types in the global Gpr56 KO ( Gpr56 null ) . Arrowhead indicates non - microglia cells expressing Gpr56 . RNAscope was performed in the prefrontal cortex of P30 mice . Scale bar , 20 µm . ( B ) Quantification of Gpr56 fluorescence signals in ( A ) . N = 20 - 50 cells for each . ( C ) Confocal images of vGlut2 ( green ) labeling retinal ganglion cell ( RGC ) presynaptic terminals and Homer1 ( red ) for post - synapses in the dLGN of CKO and controls at P10 . Overlapped vGlut2 and Homer1 are quantified as synapses , indicated by white circles . Arrows pointing to the synapse enlarged in the upper right - hand inset . ( D ) A time course of dLGN synapse density ( vGlut2 + / Homer1 + ) between WT and controls . ( E ) Representative super - resolution images and 3D - reconstructed images of vGlut2 and Homer1 staining of P8 dLGN . Green objects represent surface rendering of vGlut2 + presynaptic terminals . Red spots are rendered Homer1 + post - synapses . Magenta spots represent Homer1 + post - synapses within a distance of 300nm from the nearest vGlut2 + surface . ( F ) Quantification of Homer1 + spots adjacent to vGlut2 surface within 100nm , 200nm , and 300nm , respectively . N = 3 . ( G ) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice . ( H , I ) Quantification of vGlut2 and Homer1 expression in CKOs and controls . N = 3 , * p = 0 . 027 . ( J ) Quantification of synapse density ( vGlut2 + / Homer1 + ) in the dLGN of both male and female iCKO mice at P10 . N = 3 for male , N = 4 for female . ( K ) An overview of vGlut1 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the dotted boxes show where synapses are quantified . ( L ) Representative images of vGlut1 + ( green ) presynaptic terminals and Homer1 + ( red ) postsynapses in the dLGN of CKO and controls at P10 . Arrows pointing to the synapse enlarged in inset . ( M ) Quantification of synapse density ( vGlut1 / homer1 ) at P10 in the dLGN of CKO mice and controls . N = 3 ( WT ) , N = 5 ( CKO ) , p = 0 . 053 .

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

Figure 4(A) Gpr56 is deleted in CKO microglia. RNAscope shows Gpr56 transcripts co-localize with microglia (arrow) in controls, but not in CKO, and is absent in all cell types in the global Gpr56 KO (Gpr56 null). Arrowhead indicates non-microglia cells expressing Gpr56. RNAscope was performed in the prefrontal cortex of P30 mice. Scale bar, 20 µm. (B) Quantification of Gpr56 fluorescence signals in (A). N = 20-50 cells for each. (C) Confocal images of vGlut2 (green) labeling retinal ganglion cell (RGC) presynaptic terminals and Homer1 (red) for post-synapses in the dLGN of CKO and controls at P10. Overlapped vGlut2 and Homer1 are quantified as synapses, indicated by white circles. Arrows pointing to the synapse enlarged in the upper right-hand inset.(D) A time course of dLGN synapse density (vGlut2+/Homer1+) between WT and controls.(E) Representative super-resolution images and 3D-reconstructed images of vGlut2 and Homer1 staining of P8 dLGN. Green objects represent surface rendering of vGlut2+ presynaptic terminals. Red spots are rendered Homer1+ post-synapses. Magenta spots represent Homer1+ post-synapses within a distance of 300nm from the nearest vGlut2+ surface.(F) Quantification of Homer1+ spots adjacent to vGlut2 surface within 100nm, 200nm, and 300nm, respectively. N = 3.(G) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice. (H, I) Quantification of vGlut2 and Homer1 expression in CKOs and controls. N = 3, *p = 0.027. (J) Quantification of synapse density (vGlut2+/Homer1+) in the dLGN of both male and female iCKO mice at P10. N = 3 for male, N = 4 for female. (K) An overview of vGlut1 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.(L) Representative images of vGlut1+ (green) presynaptic terminals and Homer1+ (red) postsynapses in the dLGN of CKO and controls at P10. Arrows pointing to the synapse enlarged in inset. (M) Quantification of synapse density (vGlut1/homer1) at P10 in the dLGN of CKO mice and controls. N=3 (WT), N=5 (CKO), p = 0.053.


Figure 5 ( A ) Representative images show hippocampal vGlut2 and Homer1 immunostaining . White box outlines the region of interest and yellow box shows the regions where confocal images were taken . ( B ) Confocal images of synaptic immunostaining in CA1 striatum lacunosum - moleculare ( str . lac / mol ) at P10 . Arrows pointing to the enlarged synapse in the insets . ( C , D ) Quantification of synapse density in CA1 str . lac / mol in iCKO versus control at P10 ( C ) , and CKO versus control at P21 ( D ) . At P10 , N ( Ctrl ) = 4 , N ( iCKO ) = 3 , p = 0 . 02 . At P21 , N = 8 , p = 0 . 005 . ( E ) Representative images show hippocampal vGlut1 and Homer1 immunostaining . ( F ) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10 . ( G , H ) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 ( G ) , and CKO versus control at P21 ( H ) . At P10 , N = 5 , p = 0 . 83 . At P21 , N = 5 , p = 0 . 89 .

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

Figure 5(A) Representative images show hippocampal vGlut2 and Homer1 immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken.(B) Confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare (str. lac/mol) at P10. Arrows pointing to the enlarged synapse in the insets. (C, D) Quantification of synapse density in CA1 str. lac/mol in iCKO versus control at P10 (C), and CKO versus control at P21 (D). At P10, N(Ctrl) = 4, N(iCKO) = 3, p = 0.02. At P21, N = 8, p = 0.005.(E) Representative images show hippocampal vGlut1 and Homer1 immunostaining.(F) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10. (G, H) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H). At P10, N = 5, p = 0.83. At P21, N = 5, p = 0.89.


Figure 6 ( B ) CTB - labeled dLGN shows reduced eye - segregation at P30 in CKO mice . The left column shows contralateral dLGN labeled with CTB488 ( green ) , and the middle one is ipsilateral dLGN with CTB647 ( magenta ) . The right column represents the dLGN pseudocolored according to the R - value for each pixel ( R = log ( Fipsi / Fcontra ) ) . ( C ) A histogram distribution chart of R - value for all pixels within dLGN represents the degree of eye - specific segregation . When R is 0 , it means equal ipsilateral and contralateral fluorescence intensity at a pixel . A greater R - value means a bigger difference of ipsi - to - contraleteral fluorescence intensity . The narrower distribution of CKO in the inset indicates reduced segregation . ( D ) The variance of R distributions in P30 control and CKO mice . N = 4 ( Ctrl ) , N = 6 ( CKO ) , p = 0 . 004 , mean ± SD . ( F ) Maximal NMDAR mediated currents measured in the dLGN of P28 ~ P34 mice , p = 0 . 03 , N = 14 ( Ctrl ) , 23 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM . ( G ) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice . ( H ) Quantification of NMDAR1 expression in CKO mice and control . ( I ) Maximal AMPAR mediated currents measured in the dLGN of P28 ~ P34 mice . P = 0 . 302 , N = 13 ( Ctrl ) , 17 ( CKO ) cells from 5 , 7 mice , respectively . Mean ± SEM . ( J ) Western blot of GluR1 with microdissected P30 dLGN tissue . ( K ) Quantification of GluR1 expression in CKO mice and controls .

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Figure 6(B) CTB-labeled dLGN shows reduced eye-segregation at P30 in CKO mice. The left column shows contralateral dLGN labeled with CTB488 (green), and the middle one is ipsilateral dLGN with CTB647 (magenta). The right column represents the dLGN pseudocolored according to the R-value for each pixel (R = log(Fipsi/Fcontra)). (C) A histogram distribution chart of R-value for all pixels within dLGN represents the degree of eye-specific segregation. When R is 0, it means equal ipsilateral and contralateral fluorescence intensity at a pixel. A greater R-value means a bigger difference of ipsi-to-contraleteral fluorescence intensity. The narrower distribution of CKO in the inset indicates reduced segregation. (D) The variance of R distributions in P30 control and CKO mice. N = 4 (Ctrl), N = 6 (CKO), p = 0.004, mean ± SD. (F) Maximal NMDAR mediated currents measured in the dLGN of P28~P34 mice, p = 0.03, N = 14 (Ctrl), 23 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.(G) Western blot of NMDAR1 using microdissected dLGN tissue from P30 mice. (H) Quantification of NMDAR1 expression in CKO mice and control. (I) Maximal AMPAR mediated currents measured in the dLGN of P28~P34 mice. P=0.302, N = 13 (Ctrl), 17 (CKO) cells from 5, 7 mice, respectively. Mean ± SEM.(J) Western blot of GluR1 with microdissected P30 dLGN tissue. (K) Quantification of GluR1 expression in CKO mice and controls.


Figure 7 ( A ) Colocalized RGC inputs and PSVue signals in P6 control and CKO . White circles indicate PS + RGC inputs . Arrows pointing to the RGC inputs enlarged in the insets . ( B ) The percentage of PS + RGC inputs in total inputs in CKO and controls . ( C ) Representative images of engulfed PS + and PS - RGC inputs by microglia in control and CKO . ( D ) Quantification of engulfed PS + RGC inputs by microglia , which is calculated as : Volume of engulfed PS + RGC inputs / Volume of microglia cell . ( E ) Quantification of engulfed PS - RGC inputs in control and CKO .

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

Figure 7(A) Colocalized RGC inputs and PSVue signals in P6 control and CKO. White circles indicate PS+ RGC inputs. Arrows pointing to the RGC inputs enlarged in the insets. (B) The percentage of PS+ RGC inputs in total inputs in CKO and controls.(C) Representative images of engulfed PS+ and PS- RGC inputs by microglia in control and CKO. (D) Quantification of engulfed PS+ RGC inputs by microglia, which is calculated as: Volume of engulfed PS+ RGC inputs / Volume of microglia cell. (E) Quantification of engulfed PS- RGC inputs in control and CKO.


f1 ( a - d ) Formation of protein inclusions in SY5Y cells expressing FL ( a ) or the indicated truncated forms of Sph1 ( b - d ) was induced by 12 h lactacystin treatment . After extensive washes , cells were maintained in serum - supplemented ( serum + ) or serum - free ( serum − ) media , and in the absence ( Control ) or presence of 10 mM 3 - MA , an inhibitor of macroautophagy . Graphs show the relative fold difference in the number of cells containing either Sph1 Agm ( left ) or Agg ( right ) observed under different recovery conditions . Values are mean + s . e . ( n = 4 ) . * Significant versus serum + condition . See also Supplementary Figs S1 - S5 .

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f1(a-d) Formation of protein inclusions in SY5Y cells expressing FL (a) or the indicated truncated forms of Sph1 (b-d) was induced by 12 h lactacystin treatment. After extensive washes, cells were maintained in serum-supplemented (serum+) or serum-free (serum−) media, and in the absence (Control) or presence of 10 mM 3-MA, an inhibitor of macroautophagy. Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (left) or Agg (right) observed under different recovery conditions. Values are mean+s.e. (n=4). *Significant versus serum+ condition. See also Supplementary Figs S1-S5.


f2 ( a - c ) Top : fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL ( a ) , or the indicated truncated Sph1 proteins ( b - c ) treated with lactacystin ( 12 h ) and recovered in basal conditions ( serum + ) . Graphs show the relative fold difference in the number of cells containing either Sph1 Agm ( middle ) or Agg ( bottom ) . Values are mean + s . e . ( n = 3 ) . * Significant versus control . ( d - f ) Relative fold difference in the number of FL ( d ) or truncated ( e - f ) Sph1 Agm ( left ) and Agg ( right ) in WT and Atg5 KO mice MEFs incubated in serum + or serum − conditions . Values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition .

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f2(a-c) Top: fluorescence images of Agm and Agg observed in MEFs from WT and Atg5 KO mice expressing FL (a), or the indicated truncated Sph1 proteins (b-c) treated with lactacystin (12 h) and recovered in basal conditions (serum+). Graphs show the relative fold difference in the number of cells containing either Sph1 Agm (middle) or Agg (bottom). Values are mean+s.e. (n=3). *Significant versus control.(d-f) Relative fold difference in the number of FL (d) or truncated (e-f) Sph1 Agm (left) and Agg (right) in WT and Atg5 KO mice MEFs incubated in serum+ or serum− conditions. Values are mean+s.e. (n=3). *Significant versus serum+ condition.


f3 ( a - d ) Left : fluorescence images of SY5Y cells co - expressing HA - p38 ( red ) and GFP ( green ) forms of FL ( a ) or the indicated truncated Sph1 ( b - d ) . Cells were treated with lactacystin ( 12 h ) and recovered in the presence or absence of serum . Graphs : percentage of p38 existing alone in Agm or co - aggregating with Sph1 in the forms of Agm or Agg ( left ) , and susceptibility of p38 existing on its own in Agm or co - aggregating with Sph1 proteins in Agm toward serum - stimulated autophagic clearance ( right ) . Values are expressed as folds Agm in the presence of serum and are mean + s . e . ( n = 3 ) . * Significant versus serum + condition calculated using Student ' s t - test . ( e ) Autophagic susceptibility of Agm formed by p38 and ANK1 - p38 fusion proteins . Values are mean + s . e . ( n = 3 ) . * Significant versus control group calculated using Bonferroni test . See also Supplementary Fig . S6 .

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

f3(a-d) Left: fluorescence images of SY5Y cells co-expressing HA-p38 (red) and GFP (green) forms of FL (a) or the indicated truncated Sph1 (b-d). Cells were treated with lactacystin (12 h) and recovered in the presence or absence of serum. Graphs: percentage of p38 existing alone in Agm or co-aggregating with Sph1 in the forms of Agm or Agg (left), and susceptibility of p38 existing on its own in Agm or co-aggregating with Sph1 proteins in Agm toward serum-stimulated autophagic clearance (right). Values are expressed as folds Agm in the presence of serum and are mean+s.e. (n=3). *Significant versus serum+ condition calculated using Student's t-test.(e) Autophagic susceptibility of Agm formed by p38 and ANK1-p38 fusion proteins. Values are mean+s.e. (n=3). *Significant versus control group calculated using Bonferroni test. See also Supplementary Fig. S6.


f4 ( a ) Top : colocalization of LAMP1 ( red ) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti - GFP ( green ) in SY5Y cells . Arrows indicate colocalization . Bottom : percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum + or serum − media , treated or not with lysosomal inhibitors ( N / L ) . ( b ) Percentage LAMP1 colocalization with Agm in cells co - expressing HA - p38 and the indicated Sph1 proteins maintained in the absence of serum . ( c ) Left : colocalization of LAMP1 ( red ) with GFP - p38 and - ANK1 - p38 Agm in SY5Y cells maintained in the absence of serum . Arrows indicate colocalization . Right : percentage LAMP1 colocalization with p38 and ANK1 - p38 Agm in cells maintained in the presence or absence of serum . ( d , e ) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP - tagged Sph1 truncations ( d ) , or p38 and ANK1 - p38 ( e ) . Top : phase - contrast fluorescence microscopy ; middle : electron micrographs of the boxed region ; bottom : higher magnification examples of autophagic vacuoles ( yellow arrows ) . # Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs . ( f ) Top : colocalization of LC3 ( red ) with GFP - p38 and - ANK1 - p38 Agm in SY5Y cells maintained in the absence of serum . Arrows indicate colocalization . Bottom : percentage colocalization of LC3 with the fluorescent proteins . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , c , f ) or p38 Agm ( b ) calculated using Bonferroni test . See also Supplementary Fig . S7 .

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f4(a) Top: colocalization of LAMP1 (red) with Agm and Agg of ACA and CACT Sph1 proteins highlighted by anti-GFP (green) in SY5Y cells. Arrows indicate colocalization. Bottom: percentage LAMP1 colocalization with Agm and Agg in cells maintained in serum+ or serum− media, treated or not with lysosomal inhibitors (N/L).(b) Percentage LAMP1 colocalization with Agm in cells co-expressing HA-p38 and the indicated Sph1 proteins maintained in the absence of serum.(c) Left: colocalization of LAMP1 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Right: percentage LAMP1 colocalization with p38 and ANK1-p38 Agm in cells maintained in the presence or absence of serum.(d,e) Correlative light and electron microscopy of SY5Y cells expressing the indicated GFP-tagged Sph1 truncations (d), or p38 and ANK1-p38 (e). Top: phase-contrast fluorescence microscopy; middle: electron micrographs of the boxed region; bottom: higher magnification examples of autophagic vacuoles (yellow arrows). #Location of the Agm or Agg structures in both the fluorescence images and the corresponding micrographs.(f) Top: colocalization of LC3 (red) with GFP-p38 and -ANK1-p38 Agm in SY5Y cells maintained in the absence of serum. Arrows indicate colocalization. Bottom: percentage colocalization of LC3 with the fluorescent proteins. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,c,f) or p38 Agm (b) calculated using Bonferroni test. See also Supplementary Fig. S7.


f5 ( a ) Fluorescence images showing colocalization patterns of the indicated GFP - p38 variants and Sph1 ( green ) with endogenous p62 ( red ) in SY5Y cells . Arrows indicate colocalization . ( b ) Quantitative FRAP analysis of mCherry - p62 expressed alone ( control ) or co - expressed with the indicated GFP - Sph1 and - p38 variants . Top : time course of changes in relative fluorescence intensity ( RFI ) ; bottom : percentage of p62 in the diffusing fraction and diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten cells analysed over three different experiments . Representative images are shown in Supplementary Fig . S8 . ( c - d ) Top : IBs for p62 and actin in control and p62 knockdown ( p62 KD # 325 or # 788 ) SY5Y cells ( c ) ; and in control and p62 KO MEFs ( d ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in the same cells in response to serum removal . ( e ) Fluorescence images of GFP - p38 , - ANK1 - p38 and - NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1 . Arrows indicate colocalization . ( f , g ) Top : IBs for NBR1 , p62 and actin in control and NBR1 knockdown ( NBR1 KD # 161 ) SY5Y cells ( f ) , and in control and p62 / NBR1 double knockdown ( Double KD # A or # B ) SY5Y cells ( g ) . Bottom : autophagic clearance of GFP - ANK1 - p38 Agm in the same cells . For all densitometric analyses , values are mean + s . e . ( n = 3 ) . * Significant versus serum + condition . See also Supplementary Fig . S8 - S11 .

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f5(a) Fluorescence images showing colocalization patterns of the indicated GFP-p38 variants and Sph1 (green) with endogenous p62 (red) in SY5Y cells. Arrows indicate colocalization.(b) Quantitative FRAP analysis of mCherry-p62 expressed alone (control) or co-expressed with the indicated GFP-Sph1 and -p38 variants. Top: time course of changes in relative fluorescence intensity (RFI); bottom: percentage of p62 in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Representative images are shown in Supplementary Fig. S8.(c-d) Top: IBs for p62 and actin in control and p62 knockdown (p62 KD #325 or #788) SY5Y cells (c); and in control and p62 KO MEFs (d). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells in response to serum removal.(e) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous NBR1. Arrows indicate colocalization.(f,g) Top: IBs for NBR1, p62 and actin in control and NBR1 knockdown (NBR1 KD #161) SY5Y cells (f), and in control and p62/NBR1 double knockdown (Double KD #A or #B) SY5Y cells (g). Bottom: autophagic clearance of GFP-ANK1-p38 Agm in the same cells. For all densitometric analyses, values are mean+s.e. (n=3). *Significant versus serum+ condition. See also Supplementary Fig. S8-S11.


f6 ( a - d ) Fluorescence images of GFP - p38 , - ANK1 - p38 and - NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin - 1 ( a ) , Vps34 ( b ) , Atg14L ( c ) and DFCP - 1 ( d ) . Arrows indicate colocalization .

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f6(a-d) Fluorescence images of GFP-p38, -ANK1-p38 and -NA Sph1 Agm in SY5Y cells immunostained for endogenous beclin-1 (a), Vps34 (b), Atg14L (c) and DFCP-1 (d). Arrows indicate colocalization.


f7 ( a ) Quantitative FRAP analysis of the indicated GFP - p38 variants . Top : representative images . Middle : time course of changes in relative fluorescence intensity ( RFI ) . Bottom : percentage of proteins in the diffusing fraction and diffusion time ( t1 / 2 ) . Values are mean + s . e . of ten cells analysed over three different experiments . Scale bars , 10 μm ( b ) Top : schematic showing all lysine ( K ) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site ( s ) . Bottom : relative fold difference in the number of Agm formed by WT and the different ' K to R ' mutants of ANK1 - p38 fusion protein in SY5Y cells maintained in the presence or absence of serum . ( c ) IBs for HA ( top ) and GFP ( bottom ) of total lysates ( Input ) and GFP immunoprecipitates ( IP ) from the cell lysates of SY5Y cells co - transfected with the indicated HA - ubiquitin constructs and GFP - p38 or - ANK1 - p38 WT or mutant K385R in the presence of lactacystin . ( d ) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination . Values were corrected by levels of GFP immunoprecipitated . ( e ) Top : IF for GFP and K63 - linked ubiquitin ( red ) of SY5Y cells expressing GFP - p38 or - ANK1 - p38 WT and mutant K385R ( green ) . Arrows indicate colocalization . Bottom : percentage of Agm positive for K63 - linked ubiquitin . ( f ) Quantitative FRAP analysis of GFP - ANK1 - p38 K385R and GFP - ANK1 - p38 as in ( a ) . Values are mean + s . e . of ten cells analysed over three different experiments . ( g ) Top : representative images of GFP - ANK1 - p38 K385R Agm in SY5Y cells immunostained for Atg14L , Vps34 and DFCP - 1 . Bottom : percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L , Vps34 and DFCP - 1 under serum − conditions . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition or p38 Agm . # Significant versus ANK1 - p38 Agm . Significance calculated using Bonferroni test . See also Supplementary Fig . S8 .

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f7(a) Quantitative FRAP analysis of the indicated GFP-p38 variants. Top: representative images. Middle: time course of changes in relative fluorescence intensity (RFI). Bottom: percentage of proteins in the diffusing fraction and diffusion time (t1/2). Values are mean+s.e. of ten cells analysed over three different experiments. Scale bars, 10 μm(b) Top: schematic showing all lysine (K) residues in ANK1 domain of Sph1 that may serve as potential ubiquitination site(s). Bottom: relative fold difference in the number of Agm formed by WT and the different 'K to R' mutants of ANK1-p38 fusion protein in SY5Y cells maintained in the presence or absence of serum.(c) IBs for HA (top) and GFP (bottom) of total lysates (Input) and GFP immunoprecipitates (IP) from the cell lysates of SY5Y cells co-transfected with the indicated HA-ubiquitin constructs and GFP-p38 or -ANK1-p38 WT or mutant K385R in the presence of lactacystin. (d) Quantification of the levels of each p38 protein ubiquitinated via K63 compared with K48 ubiquitination. Values were corrected by levels of GFP immunoprecipitated.(e) Top: IF for GFP and K63-linked ubiquitin (red) of SY5Y cells expressing GFP-p38 or -ANK1-p38 WT and mutant K385R (green). Arrows indicate colocalization. Bottom: percentage of Agm positive for K63-linked ubiquitin.(f) Quantitative FRAP analysis of GFP-ANK1-p38 K385R and GFP-ANK1-p38 as in (a). Values are mean+s.e. of ten cells analysed over three different experiments.(g) Top: representative images of GFP-ANK1-p38 K385R Agm in SY5Y cells immunostained for Atg14L, Vps34 and DFCP-1. Bottom: percentage of p38 variant and NA Sph1 Agm that colocalized with Atg14L, Vps34 and DFCP-1 under serum− conditions. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition or p38 Agm. #Significant versus ANK1-p38 Agm. Significance calculated using Bonferroni test. See also Supplementary Fig. S8.


f8 ( a , b ) Autophagic clearance of Agm ( a ) or Agg ( b ) formed by FL WT and mutant K385R or K394R GFP - Sph1 in SY5Y cells , in response to serum removal ( a ) or upon blockage of basal autophagy with 3 - MA ( b ) . ( c , d ) Left : Agm ( c ) or Agg ( d ) of FL GFP - Sph1 ( green ) immunostained for endogenous K63 - linked ubiquitin ( red ) in SY5Y cells . Arrows in ( c ) indicate colocalization . Right : percentage of Agm ( c ) or Agg ( d ) positive for K63 - linked ubiquitin . ( e ) Formation of WT and mutant K385R FL Sph1 ( left ) and ANK1 - p38 ( right ) Agm in response to proteasome inhibition . Values in all graphs represent mean + s . e . ( n = 3 ) . * Significant versus serum + condition ( a , b ) or WT FL Sph1 or ANK1 - p38 ( c - e ) .

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f8(a,b) Autophagic clearance of Agm (a) or Agg (b) formed by FL WT and mutant K385R or K394R GFP-Sph1 in SY5Y cells, in response to serum removal (a) or upon blockage of basal autophagy with 3-MA (b).(c,d) Left: Agm (c) or Agg (d) of FL GFP-Sph1 (green) immunostained for endogenous K63-linked ubiquitin (red) in SY5Y cells. Arrows in (c) indicate colocalization. Right: percentage of Agm (c) or Agg (d) positive for K63-linked ubiquitin.(e) Formation of WT and mutant K385R FL Sph1 (left) and ANK1-p38 (right) Agm in response to proteasome inhibition. Values in all graphs represent mean+s.e. (n=3). *Significant versus serum+ condition (a,b) or WT FL Sph1 or ANK1-p38 (c-e).


Figure 1E ) Caco - 2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h . Western blotting of cell lysates is shown .

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Figure 1E) Caco-2 cells were transfected with the indicated siRNAs for 48 h followed by treatment with IFNβ for 24 h. Western blotting of cell lysates is shown.


Figure 2D ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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Figure 2D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


Figure 3HEK293T - ACE2 - GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h . D ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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Figure 3HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h. D) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


Figure 4HEK293T - ACE2 - GFP stable cells were transiently transfected with mouse ( m ) IFITM plasmids or vector control for 24 h . C ) Graph depicts normalized infection percentage measurements from 2 - 3 independent experiments , each performed in triplicate , from cells infected Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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Figure 4HEK293T-ACE2-GFP stable cells were transiently transfected with mouse (m) IFITM plasmids or vector control for 24 h. C) Graph depicts normalized infection percentage measurements from 2-3 independent experiments, each performed in triplicate, from cells infected Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


Figure 5D ) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in ( C ) and one experiment with MOI 0 . 1 . Bars represent averages with individual data points shown as circles . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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Figure 5D) Graph depicts normalized infection percentage measurements from two experiments with cells infected as in (C) and one experiment with MOI 0.1. Bars represent averages with individual data points shown as circles. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


Figure 6U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu - 3 cells transfected with mCherry and SARS - CoV - 2 Spike ( S ) or vector control . Cells were co - cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye . A ) Representative example fluorescent imaging of the indicated co - cultures showing the presence of EGFP / mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs . Scale bar indicates 50 μm . B ) Quantification of nuclei per EGFP / mCherry double positive cell syncytia in co - cultures involving the indicated IFITM3 constructs as compared to vector control . Bars depict averages of three independent experiments with circles representing the individual data points . For each condition , nuclei within 50 - 75 individual syncytia were counted . Error bars represent SD . # p < 0 . 05 compared to vector control by ANOVA followed by Tukey ' s multiple comparisons test .

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Figure 6U2OS cells transfected with EGFP and the indicated IFITM3 constructs or vector control were mixed Calu-3 cells transfected with mCherry and SARS-CoV-2 Spike (S) or vector control. Cells were co-cultured for 24 h and imaged by fluorescent microscopy to visualize EGFP and mCherry as well as nuclei via Hoechst dye. A) Representative example fluorescent imaging of the indicated co-cultures showing the presence of EGFP/mCherry double positive cell syncytia dependent upon S expression as well as varying effects of the IFITM3 constructs. Scale bar indicates 50 μm. B) Quantification of nuclei per EGFP/mCherry double positive cell syncytia in co-cultures involving the indicated IFITM3 constructs as compared to vector control. Bars depict averages of three independent experiments with circles representing the individual data points. For each condition, nuclei within 50-75 individual syncytia were counted. Error bars represent SD. #p<0.05 compared to vector control by ANOVA followed by Tukey's multiple comparisons test.


Figure 7HEK293T - ACE2 - GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h . B ) Graph depicts normalized infection percentage measurements from 3 independent experiments , each performed in triplicate , from cells infected as in ( B ) . Bars represent averages with individual data points shown as circles . Error bars represent SD . The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection . # p < 0 . 05 by ANOVA followed by Tukey ' s multiple comparisons test . NS , not significant . Not indicated , additional comparisons of interest with p < 0 . 05 include IFITM3 ( - ) vs . IFITM3 - Y20 ( - ) , IFITM3 ∆ 59 - 68 ( - ) , and IFITM3 - S61A , N64A , T65A ( - ) .

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Figure 7HEK293T-ACE2-GFP stable cells were transiently transfected with human IFITM plasmids or vector control plus TMPRSS2 or vector control for 24 h. B) Graph depicts normalized infection percentage measurements from 3 independent experiments, each performed in triplicate, from cells infected as in (B). Bars represent averages with individual data points shown as circles. Error bars represent SD. The blue dotted line is added to the graph to assist with comparisons to vector control levels of infection. #p<0.05 by ANOVA followed by Tukey's multiple comparisons test. NS, not significant. Not indicated, additional comparisons of interest with p<0.05 include IFITM3 (-) vs. IFITM3-Y20 (-), IFITM3∆59-68 (-), and IFITM3-S61A,N64A,T65A (-).