F Plots showing nascent axon position on the soma of control cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 19 cells from six experiments ) . Numbers show total count of axons in each quadrant . G Plots showing nascent axon position on the soma of nocodazole treated cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 14 cells from three experiments ) . Numbers show total count of axons in each quadrant . H Plots showing the position of non - axonal protrusions on the soma of control cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 40 protrusions from 10 cells from three experiments ) . Numbers show total count of protrusions in each quadrant . I Plots showing the position of non - axonal protrusions on the soma of nocodazole treated cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 41 protrusions from 16 cells from three experiments ) . Numbers show total count of protrusions originating in each quadrant .

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

F Plots showing nascent axon position on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 19 cells from six experiments). Numbers show total count of axons in each quadrant. G Plots showing nascent axon position on the soma of nocodazole treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 14 cells from three experiments). Numbers show total count of axons in each quadrant. H Plots showing the position of non-axonal protrusions on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 40 protrusions from 10 cells from three experiments). Numbers show total count of protrusions in each quadrant. I Plots showing the position of non-axonal protrusions on the soma of nocodazole treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 41 protrusions from 16 cells from three experiments). Numbers show total count of protrusions originating in each quadrant.


J Graph showing the ratio of kif5c560 - YFP fluorescence intensity in the soma compared to the axon with or without nocodazole . Bars show mean and standard deviation . Control : n = 6 cells from four experiments ; mean = 1 . 81 , s . d . = 1 . 59 . Nocodazole : n = 7 cells from four experiments ; mean = 0 . 75 , s . d . = 0 . 19 . Data information : * * * P < 0 . 001 . * * * * P < 0 . 0001 .

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J Graph showing the ratio of kif5c560-YFP fluorescence intensity in the soma compared to the axon with or without nocodazole. Bars show mean and standard deviation. Control: n = 6 cells from four experiments; mean = 1.81, s.d. = 1.59. Nocodazole: n = 7 cells from four experiments; mean = 0.75, s.d. = 0.19. Data information: *** P < 0.001. **** P < 0.0001.


K Image sequence from confocal time lapse of a neuron that extends a nascent axon during nocodazole treatment labelled with a membrane marker and kif5c560 - YFP before ( - 10m ) and during ( 0h to 144m ) nocodazole treatment . A nascent axon - like protrusion is extended during nocodazole treatment ( 92m to 144m ) but kif5c560 - YFP does not accumulate there . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

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

K Image sequence from confocal time lapse of a neuron that extends a nascent axon during nocodazole treatment labelled with a membrane marker and kif5c560-YFP before (-10m) and during (0h to 144m) nocodazole treatment. A nascent axon-like protrusion is extended during nocodazole treatment (92m to 144m) but kif5c560-YFP does not accumulate there. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


A Transverse section from a confocal z - stack showing the whole neural tube of a utr - mCherry embryo , showing localisation to the basal surface . B Transverse sections from a confocal z - stack of a non - apical progenitor labelled with a membrane marker in a utr - mCherry embryo to identify the basal surface of the spinal cord . Image sequence from confocal time - lapse shows the non - apical progenitor ( - 44m ) undergoing mitosis to produce two neurons ( - 22m ) , of which one is not in contact with the basal surface . Both neurons extend nascent axons ( 0m ) . Data information : All scale bars = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Transverse section from a confocal z-stack showing the whole neural tube of a utr-mCherry embryo, showing localisation to the basal surface. B Transverse sections from a confocal z-stack of a non-apical progenitor labelled with a membrane marker in a utr-mCherry embryo to identify the basal surface of the spinal cord. Image sequence from confocal time-lapse shows the non-apical progenitor (-44m) undergoing mitosis to produce two neurons (-22m), of which one is not in contact with the basal surface. Both neurons extend nascent axons (0m). Data information: All scale bars = 5 µm.


C Transverse sections from a confocal z - stack of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr - mCherry embryo to identify the basal surface of the spinal cord . Three different sections show the middle of the soma , the axon initiation site , and the axon tip . Green and magenta peaks in graphs show relative positions of cell membrane and basal surface , respectively . D Transverse sections from confocal z - stacks of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr - mCherry embryo injected with lamMO . Three different sections show the middle of the soma , the axon initiation site , and the axon tip . Green and magenta peaks in graphs show relative positions of cell membrane and basal surface , respectively . E Graphs showing distance ( µm ) between the basal edge of the soma , the axon initiation site or the axon tip and the basal surface of the spinal cord . Measurements were performed by measuring between basal - most green and magenta peaks in graphs of relative grey values ( C , D ) . Bars show mean and standard deviation . WT : n = 5 cells from one experiment ; lamMO : n = 11 cells from two experiments . Soma : WT mean = 2 . 007 µm , s . d . = 1 . 055 ; lamMO mean = 1 . 871 µm , s . d . = 1 . 830 . Student ' s two - tailed test , P = 0 . 880 . Axon initiation site : WT mean = 1 . 366 µm , s . d . = 3 . 13 ; lamMO mean = 2 . 353 µm , s . d . = 2 . 125 . Student ' s two - tailed test , P = 0 . 468 . Axon tip : WT mean = 0 . 637 µm , s . d . = 1 . 078 ; lamMO mean = 2 . 903 µm , s . d . = 2 . 0433 . Student ' s two - tailed test , P = 0 . 037 . Data information : All scale bars = 5 µm . * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 Transverse sections from a confocal z-stack of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr-mCherry embryo to identify the basal surface of the spinal cord. Three different sections show the middle of the soma, the axon initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. D Transverse sections from confocal z-stacks of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr-mCherry embryo injected with lamMO. Three different sections show the middle of the soma, the axon initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. E Graphs showing distance (µm) between the basal edge of the soma, the axon initiation site or the axon tip and the basal surface of the spinal cord. Measurements were performed by measuring between basal-most green and magenta peaks in graphs of relative grey values (C, D). Bars show mean and standard deviation. WT: n = 5 cells from one experiment; lamMO: n = 11 cells from two experiments. Soma: WT mean = 2.007 µm, s.d. = 1.055; lamMO mean = 1.871 µm, s.d. = 1.830. Student's two-tailed test, P = 0.880. Axon initiation site: WT mean = 1.366 µm, s.d. = 3.13; lamMO mean = 2.353 µm, s.d. = 2.125. Student's two-tailed test, P = 0.468. Axon tip: WT mean = 0.637 µm, s.d. = 1.078; lamMO mean = 2.903 µm, s.d. = 2.0433. Student's two-tailed test, P = 0.037. Data information: All scale bars = 5 µm. * P < 0.05.


F Image sequence from confocal time lapse shows a neuron in a Sly - / - embryo labelled with membrane and centrosome markers before ( - 40m ) , during ( 0 ) and after axon initiation ( 20m ) . Images are transverse reconstructions from confocal z - stacks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Image sequence from confocal time lapse shows a neuron in a Sly-/- embryo labelled with membrane and centrosome markers before (-40m), during (0) and after axon initiation (20m). Images are transverse reconstructions from confocal z-stacks.


G Plots showing axon position on the soma relative to the cell centroid at 0 , 0 for dorsal and transverse view in Sly - / - embryos ( n = 18 cells from three experiments ) . Axon position is not random ( dorsal view P < 0 . 001 , mean = 98 . 8o ; transverse view P < 0 . 001 , mean = 161 . 1o ; Moore ' s modification of Rayleigh ' s test ) . H Plots showing merge of WT and Sly - / - axon positions on the cell body relative to cell centroid at 0 , 0 for dorsal and transverse views . Axon positions in WT and Sly - / - are not significantly different in dorsal view ( 0 . 2 < P < 0 . 5 ) but are in transverse view ( P < 0 . 001 ; Batschelet ' s alternative to Hotelling test ) . WT : n = 86 cells from eight experiments ; Sly - / - : n = 18 cells from three experiments .

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G Plots showing axon position on the soma relative to the cell centroid at 0,0 for dorsal and transverse view in Sly-/- embryos (n = 18 cells from three experiments). Axon position is not random (dorsal view P < 0.001, mean = 98.8o; transverse view P < 0.001, mean = 161.1o; Moore's modification of Rayleigh's test). H Plots showing merge of WT and Sly-/- axon positions on the cell body relative to cell centroid at 0,0 for dorsal and transverse views. Axon positions in WT and Sly-/- are not significantly different in dorsal view (0.2 < P < 0.5) but are in transverse view (P < 0.001; Batschelet's alternative to Hotelling test). WT: n = 86 cells from eight experiments; Sly-/-: n = 18 cells from three experiments.


I Graph showing distance between centrosome and base of axon at time of axon initiation in WT and Sly - / - embryos . Bars show mean and standard deviation . WT : n = 26 cells from three experiments , mean = 10 . 13 µm , s . d . = 3 . 35 . Sly - / - : n = 15 cells from two experiments , mean = 12 . 41 µm , s . d . = 3 . 281 . One - way ANOVA , P = 0 . 123 .

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 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

I Graph showing distance between centrosome and base of axon at time of axon initiation in WT and Sly-/- embryos. Bars show mean and standard deviation. WT: n = 26 cells from three experiments, mean = 10.13 µm, s.d. = 3.35. Sly-/-: n = 15 cells from two experiments, mean = 12.41 µm, s.d. = 3.281. One-way ANOVA, P = 0.123.


J Plots showing centrosome position relative to the cell centroid at 0 , 0 for dorsal and transverse view in Sly - / - embryos ( n = 15 cells from two experiments ) . Centrosome position is not random ( dorsal view P < 0 . 001 , mean = - 129 . 0o ; transverse view 0 . 001 < P < 0 . 005 , mean = - 57 . 0o ; Moore ' s modification of Rayleigh ' s test ) . K Plots showing merge of WT and Sly - / - centrosome positions on the cell body relative to cell centroid at 0 , 0 for dorsal and transverse views . Centrosome positions are not significantly different between WT and Sly - / - ( dorsal view 0 . 2 < P < 0 . 2 , transverse view 0 . 1 < P < 0 . 2 ; Batschelet ' s alternative to Hotelling test ) . WT : n = 26 cells from three experiments ; Sly - / - : n = 15 cells from two experiments .

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

J Plots showing centrosome position relative to the cell centroid at 0,0 for dorsal and transverse view in Sly-/- embryos (n = 15 cells from two experiments). Centrosome position is not random (dorsal view P < 0.001, mean = -129.0o; transverse view 0.001 < P < 0.005, mean = -57.0o; Moore's modification of Rayleigh's test). K Plots showing merge of WT and Sly-/- centrosome positions on the cell body relative to cell centroid at 0,0 for dorsal and transverse views. Centrosome positions are not significantly different between WT and Sly-/- (dorsal view 0.2 < P < 0.2, transverse view 0.1 < P < 0.2; Batschelet's alternative to Hotelling test). WT: n = 26 cells from three experiments; Sly-/-: n = 15 cells from two experiments.


L Plots showing the positions of the centrosome and base of the axon in Sly - / - embryos at the time of axon initiation relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 15 cells from two experiments ) . Left - hand plots : centrosome position is not random ( dorsal view P < 0 . 001 , mean = - 129 . 0o ; transverse view P < 0 . 001 , mean = - 57 . 0 ) and axon position is not random ( dorsal view P < 0 . 001 , mean = 95 . 1o ; transverse view P < 0 . 001 , mean = 168 . 8o , Moore ' s modification of Rayleigh ' s test ) . Centrosome and axon positions are significantly different ( dorsal view 0 . 001 > P ; transverse view 0 . 001 > P ; Moore ' s test for paired data ) . Right - hand plots : vectors connecting centrosome and nascent axon from the same cell are not random ( dorsal view P < 0 . 001 , mean = 66 . 1o ; transverse view P < 0 . 001 , mean = 150 . 9o ) .

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

L Plots showing the positions of the centrosome and base of the axon in Sly-/- embryos at the time of axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 15 cells from two experiments). Left-hand plots: centrosome position is not random (dorsal view P < 0.001, mean = -129.0o; transverse view P < 0.001, mean = -57.0) and axon position is not random (dorsal view P < 0.001, mean = 95.1o; transverse view P < 0.001, mean = 168.8o, Moore's modification of Rayleigh's test). Centrosome and axon positions are significantly different (dorsal view 0.001 > P; transverse view 0.001 > P; Moore's test for paired data). Right-hand plots: vectors connecting centrosome and nascent axon from the same cell are not random (dorsal view P < 0.001, mean = 66.1o; transverse view P < 0.001, mean = 150.9o).


D . Global measurement of the severing of filaments exposed to 20 nM mCherry - cofilin - 1 from time t = 0 onwards . N = 20 ( from 1 representative experiment ) and 50 ( 1 experiment ) for non - oxidized and oxidized actin filaments , respectively . P - value = 8 . 10 - 8 ( log - rank test ) . Data information : Thick solid and dashed lines are survival fractions calculated from the experimental data . Thin grey lines are single exponential fits . 95 % confidence intervals are shown as shaded surfaces .

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D. Global measurement of the severing of filaments exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 20 (from 1 representative experiment) and 50 (1 experiment) for non-oxidized and oxidized actin filaments, respectively. P-value = 8.10-8 (log-rank test). Data information: Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.


B . Global quantification of the severing events on oxidized and non - oxidized filaments exposed to unlabelled S3D - cofilin1 from time t = 0 onwards . From top to bottom , N = 50 ( from 1 experiment ) , 50 ( 1 experiment ) , 100 ( 2 experiments ) filaments . Data information All lines are survival fractions calculated from the experimental data 95 % confidence intervals are shown as shaded surfaces .

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B. Global quantification of the severing events on oxidized and non-oxidized filaments exposed to unlabelled S3D-cofilin1 from time t=0 onwards. From top to bottom, N = 50 (from 1 experiment), 50 (1 experiment), 100 (2 experiments) filaments. Data information All lines are survival fractions calculated from the experimental data 95% confidence intervals are shown as shaded surfaces.


A . Time - lapse images of Tpm - saturated actin filament depolymerizing from their barbed end ( BE ) , with or without MICAL1 . Depolymerization is initiated at time t = 0 from a non - oxidized actin filament . The increase in the depolymerization rate reflects the oxidation of actin with time . Tpm is labeled with neonGreen ( nGr ) , and actin is unlabeled .

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A. Time-lapse images of Tpm-saturated actin filament depolymerizing from their barbed end (BE), with or without MICAL1. Depolymerization is initiated at time t=0 from a non-oxidized actin filament. The increase in the depolymerization rate reflects the oxidation of actin with time. Tpm is labeled with neonGreen (nGr), and actin is unlabeled.


C . Time - lapse images showing the disassembly of Tpm domains . Filaments were saturated with 100 nM nGr - Tpm1 . 8 before the experiment and Tpm was removed from solution at time t = 0 ( the solution contains 0 . 6 µM unlabeled G - actin to prevent filament depolymerization ) . PE and BE indicate F - actin pointed and barbed end , respectively .

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C. Time-lapse images showing the disassembly of Tpm domains. Filaments were saturated with 100 nM nGr-Tpm1.8 before the experiment and Tpm was removed from solution at time t=0 (the solution contains 0.6 µM unlabeled G-actin to prevent filament depolymerization). PE and BE indicate F-actin pointed and barbed end, respectively.


A . Time - lapse images of filaments , oxidized or not , bare or Tpm - saturated , exposed to 1 µM mCherry - cofilin - 1 , from time t = 0 onwards .

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A. Time-lapse images of filaments, oxidized or not, bare or Tpm-saturated, exposed to 1 µM mCherry-cofilin-1, from time t=0 onwards.


A . Time - lapse of actin filaments constantly exposed to nGr - Tpm1 . 8 ( yellow ) , WT mCh - cofilin - 1 ( blue ) and S3D - cofilin - 1 , and to MICAL1 from time t = 100s onwards . Here we mixed WT with an excess of S3D - cofilin - 1 to mimic cellular conditions in which most of cofilin - 1 is inhibited by phosphorylation . While Tpm prevents cofilin from binding to non - oxidized filaments , MICAL1 rapidly oxidizes actin , allowing cofilin to bind and sever filaments .

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A. Time-lapse of actin filaments constantly exposed to nGr-Tpm1.8 (yellow), WT mCh-cofilin-1 (blue) and S3D-cofilin-1, and to MICAL1 from time t=100s onwards. Here we mixed WT with an excess of S3D-cofilin-1 to mimic cellular conditions in which most of cofilin-1 is inhibited by phosphorylation. While Tpm prevents cofilin from binding to non-oxidized filaments, MICAL1 rapidly oxidizes actin, allowing cofilin to bind and sever filaments.


E and F . TLC analysis of cGAMP , which is indicative of cGAS activation , upon incubation with E ) tRNA or F ) total RNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . The FL - hcGAS concentration was 0 . 265 mg / mL . The tRNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 85 mg / mL . The extracted total RNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 5 mg / mL . NC indicates the negative control that has only the FL - hcGAS . dsDNA ISD at a concentration of 0 . 05 mg / mL was used as a positive control . All the samples were prepared in 20 mM HEPES at pH 7 . 5 and 150 mM NaCl .

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E and F. TLC analysis of cGAMP, which is indicative of cGAS activation, upon incubation with E) tRNA or F) total RNA (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES at pH 7.5 and 150 mM NaCl.


F . Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments .

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F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.


G . The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells ( n = 13 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . H . The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells ( n = 9 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . I . Pearson ' s correlation coefficient ( CC ) analysis of the fluorescence signals of cGAS vs . RNA ( n = 13 , biological replicates ) , and mCherry vs . RNA ( n = 9 , biological replicates ) . The median values were calculated and used for evaluation of significance . * * * P < 0 . 001 ( two - tailed Mann - Whitney test ) .

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

G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).


D . Fluorescence microscopy images showing the replacement of FTSC - tRNA by Cy5 - ISD at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . E . Fluorescence microscopy images showing the replacement of FTSC - tRNA by TAMRA - 380 bp - dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . F . Quantification of the replacement in " D " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of Cy5 - ISD over FTSC - tRNA in the four parts were calculated , averaged and plotted . G . Quantification of the replacement in " E " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of TAMRA - 380 bp - dsDNA over FTSC - tRNA in the four parts were calculated , averaged and plotted .

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

D. Fluorescence microscopy images showing the replacement of FTSC-tRNA by Cy5-ISD at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy images showing the replacement of FTSC-tRNA by TAMRA-380 bp-dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in "D". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD over FTSC-tRNA in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in "E". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA over FTSC-tRNA in the four parts were calculated, averaged and plotted.