Hippocampal cultures were transfected with FLAG - β2AR α11 . 2 - HA at 6 days in vitro ( DIV ) , treated with vehicle ( water ) or 1 M isoproterenol ( ISO ) for 5 min at 18 DIV , fixed surface labeled for HA FLAG . ( A , B ) Representative immunofluorescence microscopy obtained by wide - field microscopy at lower higher resolutions ( scale bar in A is 5 m ) . ( C ) of between centers of HA FLAG ( * * p < 0 . 001 , Mann - Whitney Rank Sum ) .

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Hippocampal cultures were transfected with FLAG-β2AR and α11.2-HA at 6 days in vitro (DIV), treated with vehicle (water) or 1 M isoproterenol (ISO) for 5 min at 18 DIV, fixed and surface labeled for HA and FLAG.(A,B) Representative immunofluorescence microscopy images obtained by wide-field microscopy at lower and higher resolutions (scale bar in A is 5 m).(C) Quantification of distance between centers of HA and FLAG puncta (**p<0.001, Mann-Whitney Rank Sum Test).


Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) a second application of ISO for 5 min ( lane 6 ) , before solubilization ultracentrifugation . ( A ) β2AR was IPed before IB for α11 . 2 ( top part of IB ) , GluA1 ( middle part of IB ) , β2AR ( bottom part of IB ) , as indicated . ( B , C ) For , α11 . 2 GluA1 IB signals were normalized to β2AR ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of β2AR from α11 . 2 ( A , lanes 2 - 6 ) lasted at least 3 min but not 20 min ( compare lanes 6 7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-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

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) β2AR was IPed before IB for α11.2 (top part of IB), GluA1 (middle part of IB), and β2AR (bottom part of IB), as indicated.(B,C) For quantification, α11.2 and GluA1 IB signals were normalized to β2AR (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (A, lanes 2-6) lasted at least 3 min but not 20 min (compare lanes 6 and 7).


Forebrain slices from WT mice were treated with vehicle ( water ; lanes 1 , 5 ; numbers on bottom ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) a second application of ISO for 5 min ( lane 6 ) , before solubilization ultracentrifugation . ( D ) α11 . 2 GluA1 were concurrently IPed from same samples as in ( A ) by simultaneous addition of anti - α11 . 2 - GluA1 antibodies before probing stripping / re - probing upper part of IB for pS1928 , pS1700 , total α11 . 2 ( top three panels ) middle part for pS845 , pS831 , total GluA1 ( bottom three panels ) . ( E - H ) For , pS1928 pS1700 IB signals were normalized to total α11 . 2 pS845 pS831 signals to total GluA1 ( * * p < 0 . 01 , ANOVA ) . ISO - induced displacement of β2AR from α11 . 2 ( see A ) rendered α11 . 2 ( but not GluA1 ) refractory to re - phosphorylation of S1928 S1700 upon a second ISO application of α11 . 2 ( D , compare lanes 5 6 ) .

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

Forebrain slices from WT mice were treated with vehicle (water; lanes 1,5; numbers on bottom) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(D) α11.2 and GluA1 were concurrently IPed from same samples as in (A) by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 (top three panels) and middle part for pS845, pS831, and total GluA1 (bottom three panels).(E-H) For quantification, pS1928 and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ANOVA).ISO-induced displacement of the β2AR from α11.2 (see A) rendered α11.2 (but not GluA1) refractory to re-phosphorylation of S1928 and S1700 upon a second ISO application of α11.2 (D, compare lanes 5 and 6).


Forebrain slices from S1928A KI mice were treated with vehicle ( lanes 1 , 5 ) or 10 M ISO for 5 min , followed , if indicated , by 1 , 3 , or 20 min wash out of ISO ( lanes 3 - 7 ) a second application of ISO for 5 min ( lane 6 ) , before solubilization ultracentrifugation . ( A ) α11 . 2 GluA1 were concurrently IPed from same samples by simultaneous addition of anti - α11 . 2 - GluA1 antibodies before probing stripping / re - probing upper part of IB for pS1928 , pS1700 , total α11 . 2 middle part for pS845 , pS831 , total GluA1 . In S1928A KI mice , S1700 re - phosphorylation after a 3 min wash out of ISO was not blunted ( lane 6 ) in contrast to WT mice but rather augmented ( compare to lane 5 ) . ( B - E ) For , pS1928 ( virtually absent ) pS1700 IB signals were normalized to total α11 . 2 pS845 pS831 signals to total GluA1 ( * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ) .

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

Forebrain slices from S1928A KI mice were treated with vehicle (lanes 1,5) or 10 M ISO for 5 min, followed, if indicated, by 1, 3, or 20 min wash out of ISO (lanes 3-7) and a second application of ISO for 5 min (lane 6), before solubilization and ultracentrifugation.(A) α11.2 and GluA1 were concurrently IPed from same samples by simultaneous addition of anti-α11.2 and -GluA1 antibodies before probing and stripping/re-probing upper part of IB for pS1928, pS1700, and total α11.2 and middle part for pS845, pS831, and total GluA1. In S1928A KI mice, S1700 re-phosphorylation after a 3 min wash out of ISO was not blunted (lane 6) in contrast to WT mice but rather augmented (compare to lane 5).(B-E) For quantification, pS1928 (virtually absent) and pS1700 IB signals were normalized to total α11.2 and pS845 and pS831 signals to total GluA1 (**p<0.01, ***p<0.001, ANOVA).


( A ) Representative single channel recordings from hippocampal neurons at 7 - 14 DIV with 500 nM BayK - 8644 , 1 µM ω - conotoxin GVIA , 1 µM MVIIC in patch pipette upon depolarization from - 80 to - 30 , - 20 , - 10 , 0 mV . right panel depicts single - channel current - voltage relationship . Mean amplitude of unitary currents for different potential studied are - 2 . 07 pA ( - 30 mV ) , - 1 . 75 ( - 20 mV ) , - 1 . 53 ( - 10 mV ) - 1 . 25 ( 0 mV ; n = 5 patches per potential ) . Solid represents best - fit of data using a linear equation ( R2 = 0 . 92 ) revealing a slope conductance for these channels of 27 ± 2 pS .

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

(A) Representative single channel recordings from hippocampal neurons at 7-14 DIV with 500 nM BayK-8644, 1 µM ω-conotoxin GVIA, and 1 µM MVIIC in patch pipette upon depolarization from -80 to -30, -20, -10, and 0 mV. The right panel depicts the single-channel current-voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are -2.07 pA (-30 mV), -1.75 (-20 mV), -1.53 (-10 mV) and -1.25 (0 mV; n=5 patches per test potential). Solid line represents best-fit of data using a linear equation (R2=0.92) revealing a slope conductance for these channels of 27 ± 2 pS.


( B - D ) Representative single channel traces summary plot upon depolarization from - 80 to 0 mV under control conditions in presence of ISO in patch pipette . Cultures were pre - incubated for 15 min with 10 M 11R - PKI if indicated . patch pipette contained either vehicle for control , nifedipine ( nif ; 1M ) , ISO ( 1M ) , or ISO plus nifedipine , which blocked currents . ISO - induced increase in NPo was prevented by 11R - PKI .

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(B-D) Representative single channel traces and summary plot upon depolarization from -80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre-incubated for 15 min with 10 M 11R-PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1M), ISO (1M), or ISO plus nifedipine, which blocked all currents. The ISO-induced increase in NPo was prevented by 11R-PKI.


( E - H ) Representative single channel currents upon depolarization from - 80 to 0 mV summary plot after pretreatment of whole cultures with ISO . Cultures were pre - incubated with vehicle ( H2O , mock wash , E ) or 1 M ISO for 5 min ( F , G ) washed for 3 ( E , F ) or 10 min ( G ) before forming - attached patch with ISO present in patch pipette . upregulation of NPo to ~ 0 . 4 ( cf . C , D ) occurred only if neurons were pretreated with vehicle instead of ISO ( E , H ; mock wash ) or if ISO wash out duration was 10 min ( G , H ) but not if wash out was only 3 min ( F , H ) .

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

(E-H) Representative single channel currents upon depolarization from -80 to 0 mV and summary plot after pretreatment of whole cultures with ISO. Cultures were pre-incubated with vehicle (H2O, mock wash, E) or 1 M ISO for 5 min (F,G) and washed for 3 (E,F) or 10 min (G) before forming the cell-attached patch with ISO present in the patch pipette. The upregulation of NPo to ~0.4 (cf. C,D) occurred only if neurons were pretreated with vehicle instead of ISO (E,H; mock wash) or if ISO wash out duration was 10 min (G,H) but not if wash out was only 3 min (F,H).


( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , IP of β2AR ( A - C ) or simultaneously α11 . 2 GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( A ) Myr - Pep 2 displaced α11 . 2 ( lane 5 vs . 3 , top of blot ) but not GluA1 ( middle , same blot ) from β2AR ( bottom , same blot ) ; inverse was true for Myr - DSPL ( lane 1 vs . 3 ) . ( B , C ) For , α11 . 2 GluA1 immunosignals were normalized to β2AR signals ( * * p < 0 . 01 , * * * p < 0 . 001 , One Way ANOVA ) .

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

(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA).


( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , IP of β2AR ( A - C ) or simultaneously α11 . 2 GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( D ) Myr - Pep2 blunted ISO - induced phosphorylation of α11 . 2 S1700 ( lane 6 vs . 4 , top of blot ) but not GluA1 S845 ( middle , same blot ) ; inverse was true for Myr - DSPL ( lane 2 vs . 4 ) . ( E , F ) For , pS1700 pS845 immunosignals were normalized to α11 . 2 GluA1 signals , respectively ( * * * p < 0 . 001 , One Way ANOVA ) .

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

(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA).


( G ) Representative - attached recordings from hippocampal neurons as in Figure 7 . In interleafed experiments cultures were pre - incubated for 30 min with 10 M Myr - Pep2 or scrambled Myr - Pep2 ( Myr - Pep2scr ) . patch pipette contained either vehicle ( H2O ; control ) or 1 µM ISO . ISO induced increase in NPo was prevented by Myr - Pep2 but not Myr - Pep2scr . Arrows indicate 0 - current level ( i . e . closed channel ) . ( H ) Summary plot for ( G ) . For statistical , NPo value was determined for each recording pooled under each condition for comparison ( n = 10 - 12 patches ; * p < 0 . 05 , One Way ANOVA with Tukey post - hoc ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).


Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before after either a 5Hz / 3 min ( A - C ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani bars perfusion with 1 µM ISO . Inserts show sample traces immediately before ( left ) ~ 30 min after ( right ) tetani . ( A - C ) Litter - matched WT but not conditional Cav1 . 2 KO mice showed PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) . ( D - F ) WT as well as Cav1 . 2 KO mice showed NMDAR - dependent 100 Hz LTP .

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

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(A-C) Litter-matched WT but not conditional Cav1.2 KO mice showed PTT-LTP (**p < 0.001, one-way ANOVA).(D-F) WT as well as Cav1.2 KO mice showed NMDAR-dependent 100 Hz LTP.


Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before after either a 5Hz / 3 min ( A - C , G - I ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani bars perfusion with 1 µM ISO 10 µM of Myr - Pep2 or Myr - Pep2scr . Inserts show sample traces immediately before ( left ) ~ 30 min after ( right ) tetani . ( G - I ) Myr - Pep2 but not Myr - Pep2scr blocked PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) .

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

Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C, G-I) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr-Pep2 or Myr-Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(G-I) Myr-Pep2 but not Myr-Pep2scr blocked PTT-LTP (**p < 0.001, one-way ANOVA).


Left : Immunofluorescence of cGAS in nucleus ( DAPI ) cytosol ( phalloidin ) in BMDMos cultured at low / high density Scale bar : 50 μm . Right : Corresponding of cGAS from 6 different fields with n > 50 .

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Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured at low/high density Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Left : Immunofluorescence of cGAS in nucleus ( DAPI ) cytosol ( phalloidin ) in BMDMos cultured with / without serum Scale bar : 50 μm . Right : Corresponding of cGAS from 6 different fields with n > 50 .

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Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without serum Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Immunofluorescence of cGAS in nucleus ( DAPI ) cytosol ( phalloidin ) in BMDMos cultured with / without Aphidicolin ( Aphi ) ( C ) . Scale bar : 50 μm . Right : Corresponding of cGAS from 6 different fields with n > 50 .

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Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without Aphidicolin (Aphi) (C). Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Immunoblot estimation of cGAS in / subcellular fraction of BMDMos cultured under indicated conditions . Lamin B α - Tubulin are markers respectively .

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Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively.


Flow cytometric analysis of cell cycle of BMDMos


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . Scale bar : 10 μm ( A ) .

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Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). Scale bar: 10 μm (A).


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . average MNs / . Graphs show Mean ± ( n = 3 independent experiments ) representing six different microscopic fields with over 200 .

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Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). The average MNs/cell. Graphs show Mean ± SEM (n=3 independent experiments) representing six different microscopic fields with over 200 cells.


IFNB1 response in HEK293 stimulated with transfected plasmid . Mean ± of n = 3 independent experiments .

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IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n=3 independent experiments.


Micronuclei ( indicated by arrow head ) cGAS in WT cGAS - / - BMDMos exposed to γ - irradiation ( 10 Gy ) . Scale bar : 10 μm .

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Micronuclei (indicated by arrow head) and cGAS staining in WT and cGAS-/- BMDMos exposed to γ-irradiation (10 Gy). Scale bar: 10 μm.


Average MNs / in BMDMos . MN graphs show Mean ± ( n = 3 independent experiments ) representing eight different microscopic fields with over 200 .

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Average MNs/cell in BMDMos. MN graphs show Mean ± SEM (n=3 independent experiments) representing eight different microscopic fields with over 200 cells.


in WT cGAS - / - BMDMos that were first synchronized at G2 / M , then γ - irradiated ( 10 Gy ) followed by release at indicated time points . Mean ± SD , x biological triplicates ( n = 3 ) per treatment group are shown .

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Cell death in WT and cGAS-/- BMDMos that were first synchronized at G2/M, then γ-irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ±SD, x biological triplicates (n=3) per treatment group are shown.


BMDMos from cGAS - / - mice exhibit enhanced repair efficiency than those from WT Sting - / - mice . ( A ) Representative comet tails of WT , cGAS - / - Sting - / - BMDMos exposed to γ - irradiation ( IR : 10 Gy ) on ice then incubated at 37 ̊ C for indicated duration . ( B ) Corresponding of comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments .

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BMDMos from cGAS-/- mice exhibit enhanced DNA repair efficiency than those from WT and Sting-/- mice. (A) Representative comet tails of WT, cGAS-/- and Sting-/-BMDMos exposed to γ-irradiation (IR: 10 Gy) on ice then incubated at 37 ̊C for indicated duration. (B) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments.


cGAS promotes micronuclei generation in BMDMos independently of STING . Confocal microscopic visualization of micronuclei ( indicated by arrow head ) cGAS in WT , cGAS - / - Sting - / - BMDMos exposed to γ - irradiation ( 10Gy ) . Scale bar : 10 μm .

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cGAS promotes micronuclei generation in BMDMos independently of STING. Confocal microscopic visualization of micronuclei (indicated by arrow head) and cGAS staining in WT, cGAS-/- and Sting-/- BMDMos exposed to γ-irradiation (10Gy). Scale bar: 10 μm.


cGAS promotes micronuclei generation in BMDMos independently of STING . Average MNs / in corresponding representative . Bar graphs show mean values from eight different microscopic fields with over 200 .

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cGAS promotes micronuclei generation in BMDMos independently of STING. Average MNs/cell in corresponding representative images. Bar graphs show mean values from eight different microscopic fields with over 200 cells.


γ - irradiation - induced in WT , cGAS - / -

γ-irradiation-induced cell death in WT, cGAS-/- BMDMos


γ - irradiation - induced in Sting - / - cGAS - / - Sting - / - BMDMos ( F ) .

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γ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).


Kinetics of in vivo depletion of indicated bone marrow in WT mice ( n = 3 ) after γ - irradiation ( 9 Gy ) .

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Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy).


Bone marrow in WT ( n = 3 ) cGAS - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in WT (n=3) and cGAS-/- (n=3) mice 10 hours post γ-irradiation.


Bone marrow in Sting - / - ( n = 3 ) cGAS - / - Sting - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.


Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS . Immunoblot inserts in depict knockdown efficiency of cGAS Histone H1 . 2 .

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Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1.2.


Results showing effect of hcGAS , hcGAS △ cGAMP , hcGAS △ or hcGAS △ Oligo on HR in HEK293 . Corresponding immunoblot inserts depict cGAS expression .

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Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


Results showing effect of hcGAS , hcGAS △ cGAMP , hcGAS △ or hcGAS △ Oligo on NHEJ in HEK293 . Corresponding immunoblot inserts depict cGAS expression .

Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


cGAS is not recruited to DSB sites : Confocal microscopic of GFP - NLS - or GFP - hcGAS - expressing U2OS - DSB reporter incubated ( or not ) with Shield - 1 4 - OHT to induce expression translocation of mCherry - LacI - FokI ( red ) to specific DSB sites . Scale bar : 10μm . arrowheads indicate DSB sites .

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cGAS is not recruited to DSB sites: Confocal microscopic images of GFP-NLS- or GFP-hcGAS-expressing U2OS-DSB reporter cells incubated (or not) with Shield-1 and 4-OHT to induce the expression and translocation of mCherry-LacI-FokI (red) to specific DSB sites. Scale bar: 10μm. The arrowheads indicate DSB sites.


cGAS does not colocalize with γ - H2AX at DSB sites : GFP - NLS - or GFP - hcGAS - expressing HEK293 exposed ( or not ) to γ - irradiation ( IR : 10 Gy ) then stained for γ - H2AX . Scale bar : 10 μm .

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cGAS does not colocalize with γ-H2AX at DSB sites: GFP-NLS- or GFP-hcGAS- expressing HEK293 cells exposed (or not) to γ-irradiation (IR: 10 Gy) then stained for γ-H2AX. Scale bar: 10 μm.


cGAS is mainly - bound remains unaltered upon γ - irradiation . ( cyto ) fractions of γ - irradiated ( 10 Gy , 30 min ) BMDMos analyzed for cGAS indicated molecules .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic (cyto) and nuclear fractions of γ-irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules.


cGAS is mainly - bound remains unaltered upon γ - irradiation . , soluble fractions from BMDMos were immunoblotted for cGAS indicated .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic, soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins.


cGAS co - isolates with repair because of bound bridges . Nuclease digestion abrogates co - isolation of cGAS repair : Lysates of control ( - IR ) γ - irradiated ( + IR , 10 Gy , 30 min ) GFP - hcGAS expressing HEK293 were treated ( or not ) with Benzonase before cGAS immunoprecipitation for indicated . Agarose gel of in corresponding lysates in ( E ) .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Nuclease digestion abrogates the co-isolation of cGAS and DNA repair proteins: Lysates of control (-IR) and γ-irradiated (+IR, 10 Gy, 30 min) GFP-hcGAS expressing HEK293 cells were treated (or not) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins. Agarose gel analysis of DNA in corresponding cell lysates in (E).


cGAS co - isolates with repair because of bound bridges . Co - isolation of cGAS repair depends on its binding to : cGAS pulldowns along with lysate inputs of control γ - irradiated HEK293 expressing GFP - hcGAS or GFP - hcGASΔDNA probed for indicated .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Co-isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ-irradiated HEK293 cells expressing GFP-hcGAS or GFP-hcGASΔDNA probed for indicated proteins.


Confocal of γ - irradiated GFP - NLS - , GFP - hcGAS - expressing HEK293 stained for RAD51 ( red ) with or without γ - irradiation . Scale bar : 10 μm .

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Confocal images of γ-irradiated GFP-NLS-, GFP-hcGAS-expressing HEK293 cells stained for RAD51 (red) with or without γ-irradiation. Scale bar: 10 μm.


Pre - incubation of dsDNA with mcGAS - cat prevents D - loop formation by human RAD51 , but does not affect RAD1 activity once RAD51 filaments are bound to dsDNA . of D - loop formed in each reaction ( left ) was graphed as average of triplicates ± SD .

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Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD.


Pre - incubation of template dsDNA with hcGAScat blocks subsequent D - loop formation . of D - loop formation ( below ) was graphed as average of triplicates ± SD .

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Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.


Negative - stain electron of cGAS - dsDNA complexes following incubation of dsDNA with indicated cGAS variants . Scale bar : 100 nm .

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Negative-stain electron micrographs of cGAS-dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation when pre - incubated with dsDNA . of D - loop formed in each reaction ( left ) graphed as average of triplicates ± SD .

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Effect of indicated hcGAS variants on D-loop formation when pre-incubated with dsDNA. Percentage of D-loop formed in each reaction (left) graphed as the average of triplicates± SD.


DR - GFP showing that hcGASΔDNA - Y215E is impaired in HR inhibition .

DR-GFP assay showing that hcGASΔDNA-Y215E is impaired in HR inhibition.


hcGASΔDNA - Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24 .

hcGASΔDNA-Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24.


hcGASΔDNA - Y215E hcGASΔcGAMP are defective in synthase activity .

hcGASΔDNA-Y215E and hcGASΔcGAMP are defective in synthase activity.


Negative - stain electron showing that hcGAScat - ΔDNA - Y215E is defective in inducing cGAS - dsDNA complexes . Scale bar : 100 nm .

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Negative-stain electron micrographs showing that hcGAScat-ΔDNA-Y215E is defective in inducing cGAS-dsDNA complexes. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation .

Effect of indicated hcGAS variants on D-loop formation.


( E ) Representative photos of iWAT , eWAT BAT from Ftoflox / flox FtoAKO mice fed a HFD . Scale bar , 1 cm . ( F ) Representative H & E of iWAT , eWAT BAT from Ftoflox / flox FtoAKO mice fed a HFD . Scale bar , 200 μm . (

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(E) Representative photos of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 1 cm. (F) Representative H&E staining of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 200 μm. (


( I - K ) Serum of glucose ( I ) , triglyceride ( J ) free fatty acid ( FFA ) ( K ) in Ftoflox / flox FtoAKO mice fed a HFD followed by fasting . Data information : data were presented as mean ± SD ( n = 6 ) . Statistical analyses were performed using Student t ‐ . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(I-K) Serum levels of glucose (I), triglyceride (J) and free fatty acid (FFA) (K) in Ftoflox/flox and FtoAKO mice fed a HFD followed by fasting. Data information: The data were presented as the mean ± SD (n = 6). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( A ) O2 consumption of 16 - week - old Ftoflox / flox FtoAKO mice fed a HFD . gray areas in graphs indicate day night , respectively . ( B ) CO2 generation of 16 - week - old Ftoflox / flox FtoAKO mice fed a HFD . gray areas in graphs indicate day night , respectively . ( Data information : data were presented as mean ± SD of n = 6 mice per group or triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(A) O2 consumption of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. (B) CO2 generation of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. ( Data information: The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) qPCR of genes involved in thermogenesis , mitochondrial biogenesis adipogenesis in iWAT of Ftoflox / flox FtoAKO mice .

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(F) qPCR analysis of genes involved in thermogenesis, mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox/flox and FtoAKO mice.


( G ) Western blot of of UCP1 , PPARGC1A PPARG in iWAT of Ftoflox / flox FtoAKO mice .

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(G) Western blot analysis of the protein levels of UCP1, PPARGC1A and PPARG in iWAT of Ftoflox/flox and FtoAKO mice.


( I ) Immunofluorescence detection of UCP1 in iWAT from Ftoflox / flox FtoAKO mice following 6 days cold exposure ( 4 ° C ) . Scale bar , 20 μm .

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(I) Immunofluorescence detection of UCP1 in iWAT from Ftoflox/flox and FtoAKO mice following 6 days cold exposure (4°C). Scale bar, 20 μm.


( M ) Western blot of UCP1 , PPARGC1A PPARG in differentiated beige adipocytes described in ( J ) .

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(M) Western blot analysis of UCP1, PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in (J).


( N ) Immunofluorescent of UCP1 in differentiated beige adipocytes described in ( J ) . Scale bar , 20 μm .

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(N) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (J). Scale bar, 20 μm.


( G ) Western blot of of FTO , HIF1A , UCP1 , PPARGC1A PPARG in differentiated beige adipocytes described in ( F ) .

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(G) Western blot analysis of the protein levels of FTO, HIF1A, UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (F).


( C ) O2 consumption rate ( OCR ) in differentiated beige adipocytes from 3T3 - L1 overexpressing Vec or HIF1A plasmid . average basal maximal respiration rates are shown in right panel . Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(C) The O2 consumption rate (OCR) in differentiated beige adipocytes from 3T3-L1 cells overexpressing Vec or HIF1A plasmid. The average basal and maximal respiration rates are shown in right panel. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( G ) qPCR of genes involved in thermogenesis in differentiated beige adipocytes described in ( C ) . ( H ) qPCR of genes involved in thermogenesis in differentiated beige adipocytes described in ( D ) . ( Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C). (H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D). ( Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( I ) Immunofluorescent of UCP1 in differentiated beige adipocytes described in ( D ) . Scale bar , 20 μm .

(I) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (D). Scale bar, 20 μm.


( C ) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl siFto 3T3 - L1 . Blue boxes represent exons , blue lines represent introns . m6A peaks were in dashed red box .

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(C) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A peaks were in the dashed red box.


( D ) Methylated immunoprecipitation ( MeRIP ) - qPCR of m6A of Hif1a mRNA in siCtrl siFto 3T3 - L1 . Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(D) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


( H ) Western blot of of HIF1A , UCP1 , PPARGC1A , PPARG GFP in 3T3 - L1 infected with lentiviruses pCDHpuro expressing Vec , WT - or MUT - HIF1A .

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(H) Western blot analysis of the protein levels of HIF1A, UCP1, PPARGC1A, PPARG and GFP in 3T3-L1 cells infected with lentiviruses pCDHpuro expressing Vec, WT- or MUT-HIF1A.


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(A) Western blot analysis of the protein levels of YTHDF1, YTHDF2 and HIF1A in siCtrl, siYthdf1 and siYthdf2 3T3-L1 cells.


( G ) Polysome profiling of 3T3 - L1 overexpressing Vec or YTHDC2 plasmid . ( H ) qPCR of mRNA of Hif1a in 40 - 80S or polysome fraction in 3T3 - L1 overexpressing Vec or YTHDC2 plasmid . ( I ) Polysome profiling of siCtrl siYthdc2 3T3 - L1 . ( J ) qPCR of mRNA of Hif1a in 40 - 80S or polysome fraction in siCtrl siYthdc2 3T3 - L1 . Data information : data were presented as mean ± SD of triplicate ( n = 3 ) . Statistical analyses were performed using Student t ‐ . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(G) Polysome profiling of 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (H) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (I) Polysome profiling of siCtrl and siYthdc2 3T3-L1 cells. (J) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in siCtrl and siYthdc2 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) in response to different of KCl stress . Points reflect measured values , lines correspond to best model fit .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) in response to different levels of KCl stress. Points reflect the measured values, and lines correspond to best model fit.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) at 0 . 4M KCl .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) at 0.4M KCl.


Representation of experiments shown in ( B , C ) in parameter space . Connected shows progression in response to increasing KCl inferred tonparameter ( Y axis ) vs . KCl ( X axis ) of KCl gradient experiment inferred total open time - toff − ton ( Y axis ) vs . inferred production rate ( X axis ) of KCl gradient

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Representation of the experiments shown in (B,C) in parameter space. Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter (Y axis) vs. KCl concentration (X axis) of the KCl gradient experiment The inferred total open time - toff− ton(Y axis) vs. the inferred production rate (X axis) of the KCl gradient experiment


Summary of Hsp12 - GFP dynamics single mutant screen . 68 mutants were divided into three groups : increased / decreased / did not change total GFP produced . Each group was independently hierarchically clustered . values shown are Z - Scores of each parameter : ton - response onset time ; production rate ; total open time - of transcriptional window ( toff − ton ) ; Basal GFP - GFP measured before exposure to stress ; Delta GFP - amount of GFP produced throughout experiment ( Max GFP - Basal GFP ) .

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Summary of Hsp12-GFP dynamics single mutant screen. 68 mutants were divided into three groups: increased/decreased/did not change the total GFP produced. Each group was independently hierarchically clustered. The values shown are the Z-Scores of each parameter: ton- response onset time; production rate; total open time - length of transcriptional window (toff − ton); Basal GFP - GFP levels measured before exposure to stress; Delta GFP - amount of GFP produced throughout the experiment (Max GFP - Basal GFP).


mRNA of HSP12 ( Y axis ) as a function of time ( X axis ) in six representative strains .

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mRNA levels of HSP12 (Y axis) as a function of time (X axis) in six representative strains.


Comparison of to . X axis is sum over HSP12 mRNA ( see D ) . Y axis is maximum over Hsp12 - GFP values . red indicates best linear fit .

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Comparison of RNA levels to protein levels. X axis is the sum over the HSP12 mRNA counts (see D). Y axis is the maximum over the Hsp12-GFP values. The red line indicates the best linear fit.


Comparing total open time parameter inferred from mRNA data ( X axis ) to one inferred from data ( Y axis ) .

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Comparing the total open time parameter inferred from mRNA data (X axis) to the one inferred from protein data (Y axis).


Time - lapse microscopy of Hsp12 - GFP in response to stress ( 0 . 4M KCl ) . Examples of raw ( top ) traces of ( bottom ) . Traces are colored according to their basal GFP level .

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Time-lapse microscopy of Hsp12-GFP in response to stress (0.4M KCl). Examples of raw images (top) and traces of individual cells (bottom). Traces are colored according to their basal GFP level.


Scatter plot showing production rate estimated from single traces vs . production rate estimated from population average . Each point represents a different yeast .

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Scatter plot showing the production rate estimated from single traces vs. the production rate estimated from the population average. Each point represents a different yeast strain.


Estimated parameters ( median over repeats ) of ~ 1600 double mutant strains . color represents total Hsp12 - GFP produced . same amount of GFP can be produced with multiple parameter combinations . ellipses illustrate iso - expression areas in graph . Three examples of strains ( marked in B ) with nearly same total expression but dramatically different dynamics .

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Estimated parameters (median over repeats) of all ~1600 double mutant strains. The color represents the total Hsp12-GFP produced. The same amount of GFP can be produced with multiple parameter combinations. The ellipses illustrate iso-expression areas in the graph. Three examples of individual strains (marked in B) with nearly the same total expression but dramatically different dynamics.


Illustration of genetic interaction trends of mutants . For each single mutant we plot median of double mutants that contain it ( purple dots ) . gray dots are double mutant strains ( B ) .

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Illustration of the genetic interaction trends of individual mutants. For each single mutant we plot the median of all the double mutants that contain it (purple dots). The gray dots are all individual double mutant strains (B).


Illustration of interactions of Δhog1 in production rate parameter . Shown production rate of single mutant ( X axis ) versus production rate of double mutant with Δhog1 ( Y axis ) . gray marks Hog1 single mutant . Points close to gray are ones where value of double mutant is close to value of Δhog1 defined as epistasis of Δhog1 ( pink dots ) .

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Illustration of the interactions of Δhog1 in the production rate parameter. Shown the production rate of the single mutant (X axis) versus the production rate of the double mutant with Δhog1 (Y axis). The gray line marks the Hog1 single mutant levels. Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 (pink dots).


Illustration of interactions of Δhog1 for total open time parameter .

Illustration of the interactions of Δhog1 for the total open time parameter.


Epistatic interactions of Δmck1 in production rate

Epistatic interactions of Δmck1 in the production rate parameter


Epistatic interactions of Δmck1 in total open time

Epistatic interactions of Δmck1 in the total open time parameter


Upper : HSP12 mRNA in response to osmotic stress ( 0 . 4M KCl ) . Lower : Estimate of Msn2 / 4 dependent production of HSP12 mRNA over time ( difference between WT Δmsn2Δmsn4 production profiles , Figure EV4A , Methods ) .

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Upper: HSP12 mRNA in response to osmotic stress (0.4M KCl). Lower: Estimate of Msn2/4 dependent production of HSP12 mRNA over time (difference between WT and Δmsn2Δmsn4 production profiles, Figure EV4A, Methods).


Δmck1 effect on response of stress genes . integral over mRNA values in WT ( X axis ) vs . integral in Δmck1 ( Y values ) . inset shows integral for HSP12 gene .

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Δmck1 effect on the response of all stress genes. The integral over mRNA values in WT strain (X axis) vs. the integral in the Δmck1 strain (Y values). The inset shows the integral for the HSP12 gene.


showing expression of stress responsive genes in WT two mutants over eight timepoints ( See 2C ) . Each gene is presented relative to its pre - stress level normalized by median over WT values to visualize dynamic range in genes . genes are separated into MSN dependent independent genes ( Figure EV4B ) . MSN - dependent genes are sorted on onset time to three groups ( Early , Average Late onset ) .

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Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints (See 2C). Each gene is presented relative to its pre-stress level and normalized by the median over WT values to visualize the dynamic range in all genes. The genes are separated into MSN dependent and independent genes (Figure EV4B). The MSN-dependent genes are sorted based on the onset time to three groups (Early, Average and Late onset).


behaviour of early late onset genes . Similar to ( C ) but showing average over genes in each group .

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The behaviour of early and late onset genes. Similar to (C) but showing the average over the genes in each group.


Msn2 - GFP localization following stress ( 0 . 4M KCl ) in selected strains . Top : of ( Y axis ) over time ( X axis ) . Bottom : of number of import events in live - time - lapse microscopy at two relevant periods .

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Msn2-GFP localization following stress (0.4M KCl) in selected strains. Top: The percentage of nuclear cells (Y axis) over time (X axis). Bottom: count of the number of nuclear import events in live-cell time-lapse microscopy at the two relevant periods.


( A ) Representative of a PDGFB tumour : H & E on right to identify tumour , an adjacent section stained for Iba1 ( red ) , p - S6 ( green ) DAPI ( blue ) at centre region selection for on left ( non - tumour in blue , tumour edge in orange tumour in blue ) .

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(A) Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue).


( B ) Representative of , edge adjacent non - tumour brain tissue for Iba1 , p - S6 DAPI in Pten - / - p53 - / - ( right ) GL261 ( left ) tumours . of Iba1 + co - expressing p - S6 in three defined regions . Five high grade glioma models were analysed - GL261 ( n = 3 ) , PDGFB ( n = 3 ) , Ntv - a ; PDGFB + shp53 ( n = 2 ) , Pten - / - p53 - / - ( n = 3 ) , Pten - / - p53 - / - Idh1mut ( n = 3 ) ( mean ± ; Two - Way ANOVA Tukey ) .

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(B) Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten-/-p53-/- (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high grade glioma models were analysed - GL261 (n=3), PDGFB (n=3), Ntv-a;PDGFB+shp53 (n=2), Pten-/-p53-/- (n=3), and Pten-/-p53-/-Idh1mut (n=3) (mean ±SEM; Two-Way ANOVA Tukey test).


( F , G ) Expression of ( F ) Rps6 ( G ) Eif4ebp1 in healthy versus GL261 microglia macrophage ( n = 3 ; mean ± , likelihood in edgeR ) .

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(F,G) Expression levels of (F) Rps6 and (G) Eif4ebp1 in healthy versus GL261 microglia and macrophage (n=3; mean ±SEM, likelihood ratio test in edgeR).


Signalling was analysed in microglia by immunoblotting of whole lysates collected at 4hr ( B normalised against non - phosphorylated vinculin analysed on same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr (B and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry was carried out in microglia for ( C ) p - S6 S240 / 244 Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Flow cytometry analysis was carried out in microglia for (C) p-S6 S240/244 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Signalling was analysed in microglia by immunoblotting of whole lysates collected at 4hr normalised against non - phosphorylated vinculin analysed on same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry was carried out in microglia for ( E ) p - 4EBP1 T37 / 46 Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

Flow cytometry analysis was carried out in microglia for (E) p-4EBP1 T37/46 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry was carried out in microglia for ( G ) p - AKT S473 ; Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin , was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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Flow cytometry analysis was carried out in microglia for (G) p-AKT S473; Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin, was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Signalling was analysed in microglia by immunoblotting of whole lysates collected at 0 . 5hr ( H ) normalised against non - phosphorylated vinculin analysed on same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 0.5hr (H) and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM +LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry was carried out in microglia for ( I ) p - AKT T308 . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

Flow cytometry analysis was carried out in microglia for (I) p-AKT T308. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+ LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


( B ) Survival for Cx3cr1 - Rheb1Δ / Δ ( n = 7 ) Rheb1fl / fl ( n = 7 ) mice . Chi square .

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(B) Survival analysis for Cx3cr1-Rheb1Δ/Δ (n=7) and Rheb1fl/fl (n=7) mice. Chi square test.


( C ) Representative of tumour volume with MRI of Rheb1fl / fl ( n = 8 ) compared to Cx3cr1 - Rheb1Δ / Δ ( n = 8 ) mice . ( mean ± ; Unpaired parametric t ) .

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(C) Representative images and quantification of the tumour volume with MRI of Rheb1fl/fl (n=8) compared to Cx3cr1-Rheb1Δ/Δ (n=8) mice. (mean ±SEM; Unpaired parametric t test).


( D ) H & E showing representative histological features ( overview high magnification of microvascular proliferation ) of Rheb1fl / fl Cx3cr1 - Rheb1Δ / Δ GL261 tumours . Scale bar is 125 μm ( top , H & E ) 80 μm ( other )

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(D) H&E staining showing representative histological features (overview and high magnification of microvascular proliferation) of Rheb1fl/fl and Cx3cr1-Rheb1Δ/Δ GL261 tumours. Scale bar is 125 μm (top, H&E) and 80 μm (all other images)


( E ) of GFP + CD45 - GL261 tumour in Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) Rheb1fl / fl ( n = 7 ) tumours , with representative FACS plot ( mean ± ; Unpaired parametric t ) .

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(E) Percentage of GFP+ CD45- GL261 tumour cells in Cx3cr1-Rheb1Δ/Δ (n=5) and Rheb1fl/fl (n=7) tumours, with representative FACS plot (mean ±SEM; Unpaired parametric t test).


( B ) of TAM - MG ( P2RY12 + CD49d - ) TAM - BMDM ( P2RY12 - CD49d + ) gated from CD45 + population in GL261 tumours from Rheb1fl / fl ( n = 4 ) Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) mice by flow cytometry ( representative flow cytometry plots on top at bottom ; mean ± ; Two - Way ANOVA Tukey ) .

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(B) Levels of TAM-MG (P2RY12+ CD49d-) and TAM-BMDM (P2RY12- CD49d+) gated from CD45+ population in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice by flow cytometry (representative flow cytometry plots on top and quantification at the bottom; mean ±SEM; Two-Way ANOVA Tukey test).


( C ) of CD8 CTL ( top ) CD4 Th ( CD4 ) in GL261 tumours from Rheb1fl / fl ( n = 4 ) Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) mice assessed by flow cytometry ( representative flow cytometry plots , left , right ; mean ± ; Unpaired t ) .

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(C) Percentage of CD8 CTL (top) and CD4 Th (CD4) cells in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice assessed by flow cytometry (representative flow cytometry plots, left and quantification, right; mean ±SEM; Unpaired t test).


( D ) Tumour tissues from Rheb1fl / fl ( n = 4 ) Cx3cr1 - Rheb1Δ / Δ ( n = 4 ) were stained for CD8 , CD4 FoxP3 . DAB was quantified as % of positive using Definiens software . Representative of stained tissues are shown . ( mean ± ; unpaired parametric t ) . Scale bar is 100µm .

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(D) Tumour tissues from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown. (mean ±SEM; unpaired parametric t test). Scale bar is 100µm.


( B ) of differential correlation between CIBERSORT fractions signatures enrichment scores ( ssGSEA ) for lymphocytes in Cx3cr1 - Rheb1Δ / Δ ( n = 3 ) vs Rheb1fl / fl ( n = 3 ) GL261 tumours .

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(B) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores (ssGSEA) for lymphocytes in the Cx3cr1-Rheb1Δ/Δ (n=3) vs Rheb1fl/fl (n=3) GL261 tumours.


( C , D ) CD4 Th ( C ) CD8 CTL ( D ) expression of Ki67 , perforin , granzyme b , IFNγ , in Cx3cr1 - Rheb1fl / fl ( n = 5 ) Rheb1Δ / Δ ( n = 5 ) GL261 tumours . Representative FACS plots show control samples ( tumour - derived stimulated in culture in absence of inhibitor cocktail - in orange ) compared to stimulated derived from Rheb1fl / fl ( fl / fl , red ) Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , blue ) GL261 tumours ( stimulated in culture with inhibitor cocktail ) ( mean ± ; Two - Way ANOVA Tukey ) .

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(C,D) CD4 Th cells (C) and CD8 CTL cells (D) expression of Ki67, perforin, granzyme b, and IFNγ, in Cx3cr1-Rheb1fl/fl (n=5) and Rheb1Δ/Δ (n=5) GL261 tumours. Representative FACS plots show control samples (tumour-derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange) compared to stimulated cells derived from Rheb1fl/fl (fl/fl, red) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, blue) GL261 tumours (cells stimulated in culture with protein inhibitor cocktail) (mean ±SEM; Two-Way ANOVA Tukey test).


( E , F ) Expression of CD44 ( E ) CD62L ( F ) by CD4 Th CD8 CTL in Cx3cr1 - Rheb1fl / fl ( n = 5 ; fl / fl , red ) Rheb1Δ / Δ ( n = 5 ; Δ / Δ , blue ) GL261 tumours with representative FACS plots show control samples ( mean ± ; Two - Way ANOVA Tukey ) .

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(E,F) Expression of CD44 (E) and CD62L (F) by CD4 Th and CD8 CTL in Cx3cr1-Rheb1fl/fl (n=5; fl/fl, red) and Rheb1Δ/Δ (n=5; Δ/Δ, blue) GL261 tumours with representative FACS plots show control samples (mean ±SEM; Two-Way ANOVA Tukey test).


( B ) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to Log fold change ( Log ( FC ) ) .

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(B) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change (Log(FC)).


( C ) Upstream regulators as identified by IPA software from differentially regulated genes .

(C) Upstream regulators as identified by the IPA software from differentially regulated genes.


( E ) Microglia were pre - treated with Torin , or medium as indicated then stimulated with mNSC - CM , mGICPten - / - ; p53 - / - - CM , or GL261 - CM . p - NF - ĸB ( p - P65 ) p - STAT3 were analysed by immunoblotting of whole lysates collected at 4hr .

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(E) Microglia were pre-treated with Torin, or medium as indicated and then stimulated with mNSC-CM, mGICPten-/-;p53-/--CM, or GL261-CM. p-NF-ĸB (p-P65) and p-STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr.


( F ) Immunofluorescence of NF - ĸB ( P65 ) in conditioned microglia . of translocation of NF - κB from . Units represent % of voxels in NF - κB channel colocalised with DAPI ( n = 3 ; mean ± ; One - Way ANOVA Tukey - right ) . Scale bar is 50µm .

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(F) Immunofluorescence images of NF-ĸB (P65) in conditioned microglia. Quantification of nuclear translocation of NF-κB from staining. Units represent the % of voxels in the NF-κB channel colocalised with DAPI (n=3; mean ±SEM; One-Way ANOVA Tukey test - right). Scale bar is 50µm.


( G ) Production of Il - 12 ( Il - 12p40 ) , Tnf , Il6 , Il10 by conditioned microglia was determined by qPCR . Each treatment was normalised to housekeeping gene unconditioned control . ( n = 3 ; mean ± ; Two - Way ANOVA Tukey ) .

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(G) Production of Il-12 (Il-12p40), Tnf, Il6, and Il10 by conditioned microglia was determined by qPCR. Each treatment was normalised to housekeeping gene and the unconditioned control. (n=3; mean ±SEM; Two-Way ANOVA Tukey test).


( H , I ) MFI of pSTAT3 in TAM - MG ( CD45 + P2RY12 + CD49d - ) ( H ) p - NF - κB ( p - P65 ) in TAM - BMDM ( CD45 + P2RY12 - CD49d + ) ( I ) from Rheb1fl / fl ( fl / fl , blue , n = 4 ) Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , red , n = 5 ) GL261 tumours . Representative FACS plots are displayed for each type ( mean ± , Two - Way ANOVA Tukey ) .

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(H,I) MFI levels of pSTAT3 in TAM-MG (CD45+ P2RY12+ CD49d-) (H) and p-NF-κB (p-P65) in TAM-BMDM (CD45+ P2RY12- CD49d+) (I) from Rheb1fl/fl (fl/fl, blue, n=4) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, red, n=5) GL261 tumours. Representative FACS plots are displayed for each cell type (mean ±SEM, Two-Way ANOVA Tukey test).


( A ) Correlation between ssGSEA enrichment scores for mTOR signature versus TAM - MG or TAM - BMDM signatures in TCGA GBM transcriptomic data . Comparison carried out on IDH - wild - type samples , in a subgroup specific manner according to Wang ' s classifier . of circle is indicative of Rsquare value bold outline represents a p - value ≤ 0 . 05 .

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(A) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in TCGA GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples, and in a subgroup specific manner according to Wang's classifier. Size of circle is indicative of Rsquare value and bold outline represents a p-value ≤0.05.


( B ) Separation of IDH - wildtype GBM samples between those displaying high mTOR microglia enrichment ( + ve correlation , group 1 in orange ) those without this signature ( group 2 in green ) .

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(B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green).


( C ) CIBERSORT fractions calculated from TPM of TCGA IDH - wild type GBM samples from group 1 compared to those from group 2 .

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(C) CIBERSORT cell fractions calculated from the TPM of TCGA IDH-wild type GBM samples from group 1 compared to those from group 2.


( F ) by flow cytometry of EPSC , YS - EBs matched - iMGL with indicated markers ( n = 3 ; mean ± ) .

(F) Staining by flow cytometry of EPSC, YS-EBs and matched-iMGL with indicated markers (n=3; mean ±SEM).


( G , H ) Mature iMGL were immunostained for ( G ) PU1 CD11b ( H ) Iba1 TREM2 . Scale bar 20µm .

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(G,H) Mature iMGL were immunostained for (G) PU1 and CD11b and (H) Iba1 and TREM2. Scale bar 20µm.


( I - K ) ( I ) P - S6 , ( J ) p - STAT3 , ( K ) p - NF - ĸB ( p - P65 ) were analysed by flow cytometry in iMGL , pre - treated with Torin , as indicated then stimulated with matched EPSC - or hGIC - CM . ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) .

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(I-K) (I) P-S6, (J) p-STAT3, and (K) p-NF-ĸB (p-P65) were analysed by flow cytometry in iMGL, pre-treated with Torin, as indicated and then stimulated with matched patient EPSC- or hGIC-CM. (n=3; mean ±SEM, Two-Way ANOVA Tukey test).


( A ) AH109 was co ‐ transformed with pGBKT7 containing C ‐ terminal ( CT ) domain of mAtg9 ( bait ) pGADT7 , or pACT2 , containing CT domain of p38IP ( prey ) . interaction was then visualized by growth of co ‐ transformed yeast on SD ‐ Trp / Leu / His plates for 3 days at 30 ° C .

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(A) The AH109 strain was co‐transformed with pGBKT7 containing the C‐terminal (CT) domain of mAtg9 (bait) and pGADT7, or pACT2, containing the CT domain of p38IP (prey). The interaction was then visualized by growth of co‐transformed yeast on SD‐Trp/Leu/His plates for 3 days at 30°C.


( C ) HEK293A were transfected with RFP - mAtg9 HA - p38IP . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody co ‐ immunoprecipitated RFP - mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP was detected with an anti ‐ HA antibody . Asterisk indicates a non ‐ specific band .

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(C) HEK293A cells were transfected with RFP-mAtg9 and HA-p38IP. HA-p38IP was immunoprecipitated with an anti‐HA antibody and the co‐immunoprecipitated RFP-mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP was detected with an anti‐HA antibody. Asterisk indicates a non‐specific band.


Indirect immunofluorescence of mAtg9 with ( D ) HA - full ‐ p38IP ( HA - FL ) , ( E ) HA - C ‐ terminal p38IP ( HA - CT ) , ( F ) HA - N ‐ terminal p38IP ( HA - NT ) . Bars = 5 μm . co ‐ localization seen in ( D ) ( E ) was not because of overlap of randomly distributed vesicles . Cross ‐ correlation function ( CCF ) ( van Steensel et al , 1996 ) showed a peak maximum Rp value around ΔX = 0 , indicating that co ‐ localization between mAtg9 p38IP is positively correlated non random ( Supplementary Figure S1 ) .

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Indirect immunofluorescence analysis of mAtg9 with (D)HA-full‐length p38IP (HA-FL), (E)HA-C‐terminal p38IP (HA-CT), and (F) HA-N‐terminal p38IP (HA-NT). Bars=5 μm. The co‐localization seen in (D) and (E) was not because of overlap of randomly distributed vesicles. Cross‐correlation function (CCF) analysis ( van Steensel et al, 1996) showed a peak and maximum Rp value around ΔX=0, indicating that co‐localization between mAtg9 and p38IP is positively correlated and non random ( Supplementary Figure S1).


( A ) HEK293A were transfected with HA - p38IP alone or co ‐ transfected with HA - p38IP RFP - mAtg9 , homogenized subjected to centrifugation , resulting post ‐ supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into pellet cytosol . Equal amounts were resolved by SDS - PAGE immunoblotted with anti ‐ mannose ‐ 6 ‐ phosphate receptor ( MPR ) antibody as a control for ‐ associated , anti ‐ superoxide dismutase ( SOD ) antibody as a control for , anti ‐ mAtg9 antibody , anti ‐ HA antibody . HA - p38IP were quantified using ImageJ software plotted relative to HA - p38IP in PNS ( n = 4 ) ( * * P = 0 . 0051 ) .

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(A) HEK293A cells were transfected with HA-p38IP alone or co‐transfected with HA-p38IP and RFP-mAtg9, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol. Equal protein amounts were resolved by SDS-PAGE and immunoblotted with anti‐mannose‐6‐phosphate receptor (MPR) antibody as a control for membrane‐associated proteins, anti‐superoxide dismutase (SOD) antibody as a control for cytosolic proteins, anti‐mAtg9 antibody, and anti‐HA antibody. HA-p38IP levels were quantified using ImageJ software and plotted relative to HA-p38IP in the PNS (n=4) (**P=0.0051).


( B ) HEK293 were depleted of mAtg9 using siRNA , then transfected with HA - p38IP incubated in full medium or EBSS ( data not shown ) . asterisk indicates mAtg9 ‐ depleted . Bar is 5 μM . of versus in control depleted of mAtg9 under fed starvation conditions was analysed using ImageJ software ( * * * P = 0 . 0001 , Students t ‐ )

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(B) HEK293 cells were depleted of mAtg9 using siRNA, then transfected with HA-p38IP and incubated in full medium or EBSS (data not shown). The asterisk indicates mAtg9‐depleted cells. Bar is 5 μM. The intensity of the nuclear versus cytosolic fluorescence in control and cells depleted of mAtg9 under fed and starvation conditions was analysed using ImageJ software (***P=0.0001, Students t‐test)


( C ) HEK293A were transfected with HA - p38IP processed for indirect immunofluorescence . HA - p38IP was detected with an anti ‐ HA antibody endogenous mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP localizes to nucleus in full medium or EBSS . HA - p38IP co ‐ localizes with mAtg9 in periphery of ( arrowheads ) . Bars ( upper panels ) 5 μM , ( lower panels ) 1 μM .

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(C) HEK293A cells were transfected with HA-p38IP and processed for indirect immunofluorescence. HA-p38IP was detected with an anti‐HA antibody and endogenous mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP localizes to the nucleus and cytoplasmic puncta in full medium or EBSS. HA-p38IP co‐localizes with mAtg9 in the periphery of the cell (arrowheads). Bars (upper panels) 5 μM, (lower panels) 1 μM.


( D ) Anti ‐ mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 transfected with HA - p38IP alone or with RFP - mAtg9 HA - p38IP . A total of 5 % of lysates or immunoprecipitates were probed with anti ‐ HA or anti ‐ mAtg9 antibodies . left right bottom panels were obtained from same immunoblot , but exposed for different times to allow endogenous mAtg9 to be visualized better .

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(D) Anti‐mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 cells transfected with HA-p38IP alone or with RFP-mAtg9 and HA-p38IP. A total of 5% of lysates or immunoprecipitates were probed with anti‐HA or anti‐mAtg9 antibodies. The left and right hand bottom panels were obtained from the same immunoblot, but exposed for different times to allow the endogenous mAtg9 to be visualized better.


( E ) HEK293 were transfected with control or p38IP siRNA . At 72 h after transfection , were incubated in either full medium or EBSS for 2 h , then fixed immunostained to detect endogenous mAtg9 localization . mAtg9 localization was quantified by visually scoring for either a juxta ‐ or dispersed phenotype . Bars = 5 μm ( data are represented as mean ± s . e . m . of 200 , * P = 0 . 0413 , Student ' s t ‐ ) .

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(E) HEK293 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. mAtg9 localization was quantified by visually scoring cells for either a juxta‐nuclear or dispersed phenotype. Bars=5 μm (data are represented as mean±s.e.m. of 200 cells, *P=0.0413, Student's t‐test).


( A ) 293 / GFP - LC3 were transfected with control or p38IP siRNA . At 72 h after transfection , were incubated in either full medium ( FM ) , full medium with leupeptin ( FM Leu ) , EBSS ( ES ) , or EBSS with leupeptin ( ES Leu ) for 2 h . FM Leu ( data not shown ) is identical to FM alone . of GFP - LC3 ‐ positive autophagosome number was performed by counting in a blinded experiment . Bars = 5 μm ( data are represented as mean ± s . e . m . of 60 , * * * P = 0 . 0002 , Students t ‐ ) .

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(A) 293/GFP-LC3 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium (FM), full medium with leupeptin (FM Leu), EBSS (ES), or EBSS with leupeptin (ES Leu) for 2 h. FM Leu (data not shown) is identical to FM alone. Quantification of GFP-LC3‐positive autophagosome number was performed by counting in a blinded experiment. Bars=5 μm (data are represented as mean±s.e.m. of 60 cells, ***P=0.0002, Students t‐test).


( B ) HEK293A were transfected with siRNA control , siRNA for p38IP , siRNA for ULK1 . lysates were analysed by SDS - PAGE for endogenous LC3 lipidation using an anti ‐ LC3 antibody , or immunoblotted with anti ‐ ULK1 antibody .

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(B) HEK293A cells were transfected with siRNA control, siRNA for p38IP, and siRNA for ULK1. Cell lysates were analysed by SDS-PAGE for endogenous LC3 lipidation using an anti‐LC3 antibody, or immunoblotted with anti‐ULK1 antibody.


( C ) HeLa were transfected with siRNA control , siRNA for p38IP . After incubation for 2 h as in ( A ) , samples were immunoblotted with anti ‐ LC3 anti ‐ actin antibodies . Endogenous LC3II / LC3I were quantified presented as arbitrary units . In ( B ) , n = 4 , * * * P = 0 . 0001 * P = 0 . 0324 , Students t ‐ . In ( C ) , data are representative of two experiments .

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(C) HeLa cells were transfected with siRNA control, and siRNA for p38IP. After incubation for 2 h as in (A), samples were immunoblotted with anti‐LC3 and anti‐actin antibodies. Endogenous LC3II/LC3I levels were quantified and the ratio presented as arbitrary units. In (B), n=4, ***P=0.0001 and *P=0.0324, Students t‐test. In (C), data are representative of two experiments.


( D ) 293 / GFP - LC3 were transfected with siRNA control or siRNA p38IP , incubated in full medium , or for 2 4 h in EBSS . Samples were immunoblotted with anti ‐ p62 antibodies , actin as a loading control .

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(D) 293/GFP-LC3 cells were transfected with siRNA control or siRNA p38IP, and incubated in full medium, or for 2 and 4 h in EBSS. Samples were immunoblotted with anti‐p62 antibodies, and actin as a loading control.


( E ) HEK293A were transfected with HA - p38IP or myc - ULK1 C ‐ terminal domain ( CTD ) . After 24 h , were labelled with [ 14C ] valine as described in Material Methods section , incubated in either full medium or EBSS for 2 h . were then collected analysed for long ‐ lived degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS mock versus EBSS HA - p38IP ( * * * P = 0 . 036 ) ; EBSS mock versus ULK1 CTD ( * * * P = 0 . 0001 ) ; Students t ‐ ) .

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(E) HEK293A cells were transfected with HA-p38IP or myc-ULK1 C‐terminal domain (CTD). After 24 h, cells were labelled with [14C]valine as described in Material and Methods section, and incubated in either full medium or EBSS for 2 h. Cells were then collected and analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS mock versus EBSS HA-p38IP (***P=0.036); EBSS mock versus ULK1 CTD (***P=0.0001); Students t‐test).


( A ) 293 / GFP - LC3 were treated with 10 μM anisomycin for 30 min , or exposed to UV irradiation for 3 min followed by a 40 ‐ min recovery incubation in full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . were then fixed visualized by confocal microscopy . GFP - LC3 ‐ positive structures per were quantified as in Figure 3A . Bars = 5 μm . Data are represented as mean ± s . e . m . n = 60 , mock versus anisomycin EBSS ( * * * P = 0 . 0001 ) ; mock versus UV EBSS ( * * * P = 0 . 0001 ) ; mock versus anisomycin EBSS with leupeptin ( * * * P = 0 . 0001 ) ; mock versus EBSS with leupeptin UV ( * * * P = 0 . 0001 ) . analysed using Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293/GFP-LC3 cells were treated with 10 μM anisomycin for 30 min, or exposed to UV irradiation for 3 min followed by a 40‐min recovery and incubation in full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells were then fixed and visualized by confocal microscopy. GFP-LC3‐positive structures per cell were quantified as in Figure 3A. Bars=5 μm. Data are represented as mean±s.e.m. n=60 cells, mock versus anisomycin EBSS (***P=0.0001); mock versus UV EBSS (***P=0.0001); mock versus anisomycin EBSS with leupeptin (***P=0.0001); mock versus EBSS with leupeptin UV (***P=0.0001). All analysed using Student's t-test.


( B ) HEK293A were pretreated with 10 μM anisomycin for 30 min , or UV irradiation for 3 min followed by a 40 ‐ min recovery , before incubation in EBSS for 2 h . were analysed as in Figure 2E . Bars = 5 μm . Data are represented as mean ± s . e . m . of 60 , EBSS mock versus EBSS anisomycin ( * P = 0 . 0142 ) ; EBSS mock versus EBSS UV ( * P = 0 . 0150 ) ; Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were pretreated with 10 μM anisomycin for 30 min, or UV irradiation for 3 min followed by a 40‐min recovery, before incubation in EBSS for 2 h. Images were analysed as in Figure 2E. Bars=5 μm. Data are represented as mean±s.e.m. of 60 cells, EBSS mock versus EBSS anisomycin (*P=0.0142); EBSS mock versus EBSS UV (*P=0.0150); Student's t‐test.


( A ) 293 / GFP - LC3 were pre ‐ treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS . lysates were analysed by SDS - PAGE for GFP - LC3 lipidation using an anti ‐ LC3 antibody . was also probed with anti ‐ β tubulin . GFP - LC3 lipidation was quantified as amount of GFP - LC3II / GFP - LC3I ( data are represented as mean ± s . e . m . of triplicates , P = 0 . 0091 , Student ' s t ‐ ) .

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

(A) 293/GFP-LC3 cells were pre‐treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS. Cell lysates were analysed by SDS-PAGE for GFP-LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐β tubulin. The GFP-LC3 lipidation was quantified as the amount of GFP-LC3II/GFP-LC3I (data are represented as mean±s.e.m. of triplicates, P=0.0091, Student's t‐test).


( B ) HEK293A labelled with [ 14C ] valine , as described in Material Methods section , were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium , EBSS , or EBSS with leupeptin for 2 h . were then analysed for long ‐ lived degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS untreated versus anisomycin treated ( P = 0 . 0363 ) ; EBSS untreated versus leupeptin treated ( P = 0 . 0001 ) , Student ' s t ‐ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells labelled with [14C]valine, as described in Material and Methods section, were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium, EBSS, or EBSS with leupeptin for 2 h. Cells were then analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS untreated versus anisomycin treated (P=0.0363); EBSS untreated versus leupeptin treated (P=0.0001), Student's t‐test).


( C ) HEK293 were transfected with control siRNA or siRNA for p38IP . After 24 ‐ h incubation , control or p38IP ‐ depleted were pre ‐ treated with anisomycin , followed by a 2 ‐ h incubation with full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin . lysates were analysed with anti ‐ p38α or anti ‐ LC3 antibodies . LC3II / LC3I were quantified presented normalized to control , untreated incubated in EBSS . Data are representative of two experiments .

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

(C) HEK293 cells were transfected with control siRNA or siRNA for p38IP. After 24‐h incubation, control or p38IP‐depleted cells were pre‐treated with anisomycin, followed by a 2‐h incubation with full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin. Cell lysates were analysed with anti‐p38α or anti‐LC3 antibodies. LC3II/LC3I levels were quantified and presented normalized to control, untreated cells incubated in EBSS. Data are representative of two experiments.