Figure 3 ( A ) Representative western blot of soluble HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation - incompetent mutants with the indicated kinases for 48 hour in HEK293T cells . ( B ) The graph indicates the fold change in HTT in samples like illustrated in A , relative to empty vector controls normalized to GAPDH . ( C ) Representative immunoblot of the HTT ( ab - MAB5492 ) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation - incompetent mutants with the indicated kinases for 48 hour in HEK293T cells . ( D ) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C . ( E ) Representative immunofluorescence images of HTT aggregates ( eGFP ) and TBK1 ( ab - anti - MYC ) upon coexpression of HTTex1 72Q eGFP or its phosphorylation - incompetent mutants with the indicated kinases for 48 hour in HEK293T cells ( scale bar 20μm ) . ( F ) Quantification of the percentage of co - transfected cells presenting aggregates upon expression of the indicated kinase , from the experiments like in E . ( G ) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E . ( H ) Representative western blots of HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour ( to enable the formation of aggregates before the kinase expression ) ; with TBK1 ; cells were lysed at the indicated time after kinase transfection . ( I ) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H .

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

Figure 3(A) Representative western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) upon coexpression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (B) The graph indicates the fold change in HTT in samples like illustrated in A, relative to empty vector controls normalized to GAPDH. (C) Representative immunoblot of the HTT (ab-MAB5492) after filter retardation assay from the insoluble cellular fraction upon expression of HTTex1 16Q eGFP or 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells. (D) Quantification of the fold change in HTT aggregates compared to EV expressing HTTex1 16Q from the experiments like in C. (E) Representative immunofluorescence images of HTT aggregates (eGFP) and TBK1 (ab-anti-MYC) upon coexpression of HTTex1 72Q eGFP or its phosphorylation-incompetent mutants with the indicated kinases for 48 hour in HEK293T cells (scale bar 20μm).(F) Quantification of the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in E. (G) Quantification of the percentage of cells presenting nuclear aggregates from the experiments like in E. (H) Representative western blots of HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) in soluble fraction and aggregates from HEK293T cells transfected first with HTTex1 72Q eGFP and then after 48 hour (to enable the formation of aggregates before the kinase expression); with TBK1; cells were lysed at the indicated time after kinase transfection. (I) Quantification of the percentage of cells presenting aggregates and kinase expression from the experiments in H.


Figure 4 ( A Quantification of cell death by a nuclear condensation assay , cells were fixed and the nuclei were stained with Hoechst dye , cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead . ( A ) The percentage of cell death at DIV14 , after co - transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 ( n = 3 ) , B ) Quantification of cell death by a nuclear condensation assay , cells were fixed and the nuclei were stained with Hoechst dye , cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead . ( B ) The percentage of cell death at DIV7 , after co - transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 ( n = 8 ) . ( C ) Quantification of cell death by a TUNEL assay in rat primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid . The cells were fixed at DIV14 , and the percentage of TUNEL - positive neurons was counted and plotted ( n = 3 ) . ( D ) The percentage of cell death . , quantification by a nuclear condensation assay in mouse primary cortical neurons co - transfected with the indicated HTT N586 and kinases at DIV5 , cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7 . Cells were fixed , and the nuclei were stained with Hoechst dye ( n = 3 ) . ( E ) Quantification of cell death by a TUNEL assay in mouse primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid . The cells were fixed on DIV14 , and the percentage of TUNEL - positive neurons was counted and plotted ( n = 3 ) .

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

Figure 4(A Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (A) The percentage of cell death at DIV14, after co-transfection of indicated kinases and HTTex1 plasmid in rat primary striatal neurons at DIV9 (n=3),B) Quantification of cell death by a nuclear condensation assay, cells were fixed and the nuclei were stained with Hoechst dye, cells with a nuclear intensity higher than the average intensity plus two standard deviations are considered dead. (B) The percentage of cell death at DIV7, after co-transfection of indicated kinases and HTT N586 plasmid in mouse primary striatal neurons at DIV5 (n=8).(C) Quantification of cell death by a TUNEL assay in rat primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed at DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).(D) The percentage of cell death., quantification by a nuclear condensation assay in mouse primary cortical neurons co-transfected with the indicated HTT N586 and kinases at DIV5, cells were treated with TBK1 inhibitor MRT 68601 at DIV5 till DIV7. Cells were fixed, and the nuclei were stained with Hoechst dye (n=3).(E) Quantification of cell death by a TUNEL assay in mouse primary striatal neurons transfected with the indicated kinases at DIV9 with the indicated HTT plasmid. The cells were fixed on DIV14, and the percentage of TUNEL-positive neurons was counted and plotted (n=3).


Figure 5 ( A ) Representative immunofluorescence image of C . elegans overexpressing HTT N513 Q15 and HTT N513 Q128 ( scale bar 20μm ) . Top panel complete worm , lower panel head region . ( B ) Western blot showing HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT pT3 ( ab - CHDI - 90001528 - 1 ) levels in C . elegans overexpressing HTT N513 Q15 and HTT N513 Q128 . ( C ) Quantification of the fold changes in ratios of HTT pS13 and pT3 to total HTT in C . elegans overexpressing HTT N513 Q15 and HTT N513 Q128 from the experiments like in B ( n = 3 ) . ( D ) Representative western blots showing the HTT ( ab - MAB5492 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) levels from the detergent soluble fraction and HTT aggregates ( ab - MAB5492 ) from the detergent insoluble fraction in three different transgenic C . elegans lines overexpressing TBK1 or the TBK1 KD . ( E ) The bottom graph indicates the fold change in the HTT pS13 ( ab - CHDI - 90001039 - 1 ) ratios to total HTT ( ab - MAB5492 ) compared to those in the kinase - dead mutant line 1 from the experiments like in D . The top graph indicates the fold change in HTT aggregates ( ab - MAB5492 ) compared to that in the kinase - dead mutant the experiments like in D ( n = 3 ) . ( F ) Mobility of the Q15 and Q128 C . elegans lines upon overexpression of TBK1 or TBK1 KD ( n = 26 - 29 ) . ( G ) Survival graph of the Q15 and Q128 C . elegans lines upon overexpression of TBK1 or TBK1 KD ( n = 30 ) . ( H ) Representative western blots showing HTT ( ab - MAB5492 ) and HTT pS13 ( ab - CHDI - 90001039 - 1 ) from the detergent soluble fraction and HTT aggregates ( ab - MAB5492 ) from the detergent insoluble fraction in TBK1 orthologue ( ikke - 1 or unc - 51 ) RNAi - treated C . elegans lines expressing HTT N513 Q15 and HTT N513 Q128 . ( I ) Quantification of the fold change in the ratio of pS13 HTT to total HTT compared to non - targeting RNAi from the experiments like in H ( n = 4 ) .

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

Figure 5(A) Representative immunofluorescence image of C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 (scale bar 20μm). Top panel complete worm, lower panel head region.(B) Western blot showing HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT pT3 (ab-CHDI-90001528-1) levels in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128. (C) Quantification of the fold changes in ratios of HTT pS13 and pT3 to total HTT in C. elegans overexpressing HTT N513 Q15 and HTT N513 Q128 from the experiments like in B (n=3). (D) Representative western blots showing the HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) levels from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in three different transgenic C. elegans lines overexpressing TBK1 or the TBK1 KD. (E) The bottom graph indicates the fold change in the HTT pS13 (ab-CHDI-90001039-1) ratios to total HTT (ab-MAB5492) compared to those in the kinase-dead mutant line 1 from the experiments like in D. The top graph indicates the fold change in HTT aggregates (ab-MAB5492) compared to that in the kinase-dead mutant the experiments like in D (n=3). (F) Mobility of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n= 26-29).(G) Survival graph of the Q15 and Q128 C. elegans lines upon overexpression of TBK1 or TBK1 KD (n=30).(H) Representative western blots showing HTT (ab-MAB5492) and HTT pS13 (ab-CHDI-90001039-1) from the detergent soluble fraction and HTT aggregates (ab-MAB5492) from the detergent insoluble fraction in TBK1 orthologue (ikke-1 or unc-51) RNAi-treated C. elegans lines expressing HTT N513 Q15 and HTT N513 Q128. (I) Quantification of the fold change in the ratio of pS13 HTT to total HTT compared to non-targeting RNAi from the experiments like in H (n=4).


Figure 6 ( A ) Representative immunoblot of a filter retardation of HTT ( ab - MAB5492 ) from the insoluble cellular fraction ; HEK293T cells co - expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour , and for the last 16 hour , they were treated with the indicated proteasome inhibitor ( MG132 , 5 µM ) or autophagy inhibitor ( Baf A1 , 200 nM , NH4Cl , 10 mM , 3 - MA , 5 mM ) . ( B ) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A ( n = 3 ) . ( C ) Immunoblot of LC3 ( ab48394 ) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour ; for the last hour , Baf A1 ( 500 nM ) was added as indicated . ( D ) Fold change in LC3 - II levels ( lower band ) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C ( n = 3 ) . ( E ) Immunoblot of LC3 ( ab48394 ) from soluble rat primary neuronal cells overexpressing lentivirus - mediated TBK1 and TBK1 KD for 96 hour . For the last 1 or 4 hour , Baf A1 ( 500 nM ) was added as indicated . ( F ) Quantification of the fold change in LC3 - II levels ( lower band ) compared to the kinase dead mutant , which was untreated , normalized to GAPDH from the blots like in E ( n = 3 ) .

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

Figure 6(A) Representative immunoblot of a filter retardation of HTT (ab-MAB5492) from the insoluble cellular fraction; HEK293T cells co-expressed HTTex1 72Q eGFP and TBK1 or TBK1 KD for 48 hour, and for the last 16 hour, they were treated with the indicated proteasome inhibitor (MG132, 5 µM) or autophagy inhibitor (Baf A1, 200 nM, NH4Cl, 10 mM, 3-MA,5 mM). (B) Quantification of the fold change in HTT aggregates compared to TBK1 KD treated with DMSO from the blots like in A (n=3).(C) Immunoblot of LC3 (ab48394) from soluble HEK293 cellular fractions overexpressing TBK1 or TBK1 KD for 24 hour; for the last hour, Baf A1 (500 nM) was added as indicated. (D) Fold change in LC3-II levels (lower band) relative to the respective untreated kinase dead mutant normalized to actin from the blots like in C (n=3). (E) Immunoblot of LC3 (ab48394) from soluble rat primary neuronal cells overexpressing lentivirus-mediated TBK1 and TBK1 KD for 96 hour. For the last 1 or 4 hour, Baf A1 (500 nM) was added as indicated. (F) Quantification of the fold change in LC3-II levels (lower band) compared to the kinase dead mutant, which was untreated, normalized to GAPDH from the blots like in E (n=3).


Figure 7 ( A ) Western blot of soluble HTT ( ab - MAB5492 ) , HTT pS13 ( ab - CHDI - 90001039 - 1 ) and HTT aggregates ( ab - MAB5492 ) , upon coexpression of the HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A / S16A variant with TBK1 KD , TBK1 or TBK1 Δ690 - 713 for 48 hour in HEK293 cells . ( B ) The graph indicates the calculated fold changes in HTT , HTT pS13 ratio to total HTT and HTT aggregates all compared to the levels in TBK1 KD from the experiments like in A ( bottom panel normalized to Tubulin , n = 3 ) . ( C ) Representative immunofluorescence images of eGFP HTTex1 72Q or its phosphorylation - deficient variant S13A / S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690 - 713 ( ab - anti - Myc ) with for 48 hour in HEK293 cells ( scale bar 20μm ) . ( D ) The graph indicates the percentage of co - transfected cells presenting aggregates upon expression of the indicated kinase , from the experiments like in C ( n = 3 ) .

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

Figure 7(A) Western blot of soluble HTT (ab-MAB5492), HTT pS13 (ab-CHDI-90001039-1) and HTT aggregates (ab-MAB5492), upon coexpression of the HTTex1 72Q eGFP or HTTex1 72Q eGFP S13A/S16A variant with TBK1 KD, TBK1 or TBK1 Δ690-713 for 48 hour in HEK293 cells. (B) The graph indicates the calculated fold changes in HTT, HTT pS13 ratio to total HTT and HTT aggregates all compared to the levels in TBK1 KD from the experiments like in A (bottom panel normalized to Tubulin, n=3). (C) Representative immunofluorescence images of eGFP HTTex1 72Q or its phosphorylation-deficient variant S13A/S16A upon coexpression with TBK1 KD or TBK1 or TBK1 Δ690-713 (ab-anti-Myc) with for 48 hour in HEK293 cells (scale bar 20μm). (D) The graph indicates the percentage of co-transfected cells presenting aggregates upon expression of the indicated kinase, from the experiments like in C (n=3).


Figure 1 ( A ) Representative single immunofluorescent staining for dsDNA at 6 h , 24 h , and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain . Scale bars , 5 μm . ( B ) Representative images of double - immunofluorescent stained for 53BP - 1 and dsDNA ( upper panel ) , Iba - 1 and dsDNA ( middle panel ) , and for GFAP and dsDNA ( lower panel ) . Scale bars , 5 μm . ( C ) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO . ( D ) Quantitative analysis for Western blot analysis . ( E ) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri - ischemic area as well as in the corresponding regions of sham control brains . Scale bars , 50 μm , 20 μm in insets . ( F ) Quantitative analysis of cGAS immunohistochemical staining . n = 6 mice per group . * * P < 0 . 01 , two - tailed unpaired Student ' s t test . ( G ) Representative double immunofluorescent stainings for cGAS ( red ) and 53BP1 ( green ) . n = 6 mice per group .

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

Figure 1(A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm.(B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm.(C) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO. (D) Quantitative analysis for Western blot analysis.(E) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50 μm, 20 μm in insets. (F) Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test.(G) Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n=6 mice per group.


Figure 2Mice received daily intraperitoneal ( IP ) injections of A151 ( 300 μg ) or an equal volume of vehicle for 3 consecutive days after MCAO induction . ( A ) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT - PCR . n = 6 mice per group . * * ( B ) The expression levels of cGAS , STING , and NF - κB proteins in the brains of indicated groups were measured using Western blot analysis . ( C ) Bar graph shows the relative protein expression levels of cGAS , STING , and NF - κB in the brains of indicated groups . n = 6 mice per group . ( D ) The mRNA expression levels of the AIM2 inflammasome components ( AIM2 , ASC , and caspase - 1 ) , effector of pyroptosis GSDMD , IL - 1β , and IL - 18 after MCAO were determined using qRT - PCR . n = 6 mice per group . ( E ) The expression of AIM2 inflammasome components , GSDMD , and IL - 1β in the brains of indicated groups was measured using Western blot analysis . ( F ) Bar graph shows the relative protein expression levels of AIM2 inflammasome components , GSDMD , and IL - 1β in the brains of indicated groups . n = 6 mice per group .

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

Figure 2Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. **(B) The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. (C) Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group.(D) The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT-PCR. n = 6 mice per group.(E) The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. (F) Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group.


Figure 3 ( A ) Representative images of immunohistochemical staining of IL - 1β , caspase - 1 , and GSDMD in the brains of indicated group . Scale bars , 50 μm . ( B ) Quantitative analysis for immunohistochemical staining . n = 6 mice per group . ( C ) Representative images of double - immunofluorescent stained for GFAP and GSDMD , for Iba - 1 and GSDMD , and for NeuN and GSDMD in post - ischemic brains after tMCAO . ( D ) Representative images of double - immunofluorescent stained for Iba - 1 and GSDMD , for Iba - 1 and caspase - 1 , and for Iba - 1 and IL - 1β of sham + vehicle , MCAO + vehicle , and MCAO + A151 groups . Scale bars , 50 μm . ( E ) Bar graph shows the relative immunofluorescence intensity of GSDMD , caspase - 1 , and IL - 1β in the brains of indicated groups . n = 6 mice per group . DAPI staining : nuclei .

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

Figure 3(A) Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. (B) Quantitative analysis for immunohistochemical staining. n = 6 mice per group.(C) Representative images of double-immunofluorescent stained for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO.(D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. DAPI staining: nuclei.


Figure 4 ( A ) Expression of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 ( FL ) , and GSDMD in primary microglia lysates was detected using western blot . ( B ) Quantitative analysis for Western blot analysis of cGAS , STING , NF - κB , ASC , IL - 1β ( FL ) , caspase - 1 and GSDMD . ( C ) The protein levels of caspase - 1 p20 and IL - 1β ( p17 ) in the supernatant of cultured primary microglia were determined by western blot . ( D ) Quantitative analysis for Western blot analysis of caspapse - 1 and IL - 1β . * P < 0 . 05 , * * P < 0 . 01 compared with the untreated group , # P < 0 . 05 , # # P < 0 . 01 compared with the LPS + poly ( dA : dT ) group . ( E - F ) Immunofluorescence was conducted to detect the expression levels of cGAS , NF - κB , and pyroptosis associated molecules ( GSDMD , caspase - 1 , and IL - 1β ) in BV2 cells . n = 3 in each group . Scale bars , 50 μm .

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Figure 4(A) Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using western blot. (B) Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 and GSDMD.(C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group.(E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm.


Figure 5 ( A ) Gating strategy of brain - infiltrating immune cells including CD4 + T cells ( CD3 + CD4 + ) , CD8 + T cells ( CD3 + CD8 + ) , B cells ( CD3 - CD19 + ) , NK cells ( CD3 - NK1 . 1 + ) , monocyte / macrophages ( CD11b + CD45highF4 / 80 + ) , neutrophils ( CD11b + CD45highLy6G + ) , and microglia ( CD11b + CD45int ) and their expression of IL ( interleukin ) - 6 , TNF - α ( tumor necrosis factor - α ) , TGF - β ( transforming growth factor - β ) , and IL - 4 . FMO , fluorescence minus one . ( B ) Quantitative analysis of the counts of brain - infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment . ( C ) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment . n = 6 mice per group . ( D ) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group . * ( E ) Bar graph shows percentages of microglia expressing IL - 6 , TNF - α , TGF - β , and IL - 10 in the brains of MCAO mice receiving indicated treatment . n = 6 mice per group . ( F ) Representative images of immunofluorescent staining for ly6G in peri - infarct areas of MCAO mice receiving indicated treatment . ( G ) Bar graph shows the number of ly6G + neutrophils . n = 6 mice per group . ( H ) Representative images of immunofluorescent staining for Iba - 1 in peri - infarct areas of MCAO mice receiving indicated treatment . ( I ) Bar graph shows the number of Iba - 1 + microglia . n = 6 mice per group .

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Figure 5(A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *(E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group.(F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group.(H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group.


Figure 6 ( B ) Neurological tests were performed to evaluate the motor , sensory , and balance functions in mice receiving A151 , or vehicle at days 1 , 3 , 7 , and 14 after MCAO . n = 10 mice per group . ( C ) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO , the infarct area is shown in white . ( D ) Bar graph shows percentages of infarct volume of indicated groups . n = 10 mice per group . * ( E ) Flow cytometry plots and ( F ) summarized results show percentages of Annexin V + PI + cells in the brains of MCAO mice receiving A151 or vehicle . n = 6 mice per group . ( G ) Representative histology images for TUNEL staining . ( H ) Bar gragh shows percentages of TUNEL - positive cells . n = 6 mice per group .

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Figure 6(B) Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving A151, or vehicle at days 1, 3, 7, and 14 after MCAO. n = 10 mice per group.(C) Representative TTC staining of brain sections from MCAO mice on day 3 after MCAO, the infarct area is shown in white.(D) Bar graph shows percentages of infarct volume of indicated groups. n = 10 mice per group. *(E) Flow cytometry plots and (F) summarized results show percentages of Annexin V+ PI+ cells in the brains of MCAO mice receiving A151 or vehicle. n = 6 mice per group.(G) Representative histology images for TUNEL staining. (H) Bar gragh shows percentages of TUNEL-positive cells. n = 6 mice per group.


Figure 7 ( A ) Representative double immunofluorescent stainings for Iba - 1 and cGAS , and for GFAP and cGAS . Scale bars , 10 μm . ( D ) Representative genotyping analysis for WT , cGAS - KD , and cGAS - KO mice . ( F ) Representative images of TTC staining of brain sections from vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice on day 3 after tMCAO . Three representative rostrocaudal levels of TTC staining are shown . ( G ) Bar graph shows percentages of infarct volume of indicated group . n = 6 mice per group . ( H ) Bar graph shows modified Neurological Severity Score ( mNSS ) in vehicle - treated WT MCAO mice , vehicle - treated cGAS - KO mice , and A151 - treated cGAS - KO mice n = 10 mice per group .

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Figure 7(A) Representative double immunofluorescent stainings for Iba-1 and cGAS, and for GFAP and cGAS. Scale bars, 10 μm.(D) Representative genotyping analysis for WT, cGAS-KD, and cGAS-KO mice.(F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group.


Figure 1 ( A ) Kinetics of cell numbers in Lineage - EPCR + CD150 + CD48 - ( L - ESLAM ) HSCs , Lineage - EPCR + CD150 + CD48 + ( CD150 + CD48 + ) progenitor cells , and Lineage - EPCR + CD150 - CD48 + ( CD150 - CD48 + ) progenitor cells after the administration of 5 - FU ( 250 mg / kg , i . v . ) . The small graph is an enlargement of 0 to 8 days after the administration of 5 - FU . ( * or * * ) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days , respectively .

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Figure 1(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively.


Figure 3 ( A ) A principal component analysis ( PCA ) using the ATAC - seq data of HSCs in the steady state ( untreated ) , early phase ( 5 - FU 6d ) , or late phase ( 5 - FU 10d ) .

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Figure 3(A) A principal component analysis (PCA) using the ATAC-seq data of HSCs in the steady state (untreated), early phase (5-FU 6d), or late phase (5-FU 10d).


Figure 4mRNA ( C ) in Acly in HSCs after the administration of 5 - FU . protein ( D ) , and phosphorylation levels of Ser455 ( E ) in Acly in HSCs after the administration of 5 - FU . The levels of intracellular Glu ( G ) in HSCs obtained from untreated or 5 - FU - treated mice . total acetylated lysine ( I ) in HSCs obtained from untreated or 5 - FU - treated mice .

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Figure 4mRNA (C) in Acly in HSCs after the administration of 5-FU.protein (D), and phosphorylation levels of Ser455 (E) in Acly in HSCs after the administration of 5-FU.The levels of intracellular Glu (G) in HSCs obtained from untreated or 5-FU-treated mice.total acetylated lysine (I) in HSCs obtained from untreated or 5-FU-treated mice.


Figure 5 ( B - C ) The effects of CTPI - 2 ( B ) or BMS303141 ( BMS ) ( C ) on H3K27ac in HSCs in the early phase after the administration of 5 - FU . ( D - E ) The effects of CTPI - 2 ( D ) , BMS303141 ( BMS ) ( E ) , A - 485 ( D ) , and TV - 3664 ( E ) on HSC expansion in the early phase after the administration of 5 - FU . Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells .

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Figure 5(B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU.(D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells.


Figure 7Global H3K27ac ( A ) , Acly levels ( B ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU . acetylated lysine ( C ) , and ΔΨm ( D ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU .

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Figure 7Global H3K27ac (A), Acly levels (B) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.


Figure 8 ( C ) The frequencies of the CD150 + CD48 - and CD48 + fractions within the lineage - EPCR + fraction after a 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ; 100 μM ) , SB204990 ( SB ; 100 μM ) , an alternative Acly inhibitor , or CTPI - 2 ( 50 μM ) . ( D and E ) Total cell number ( D ) or CD48 + cell number ( E ) after the 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ) , SB204990 ( SB ) , or CTPI - 2 . Numbers within the graph represent the inhibitory effects of each treatment ( fold decrease by an inhibitor treatment ) .

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Figure 8(C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). (D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment).


f1 ( a ) Mitochondrial tubules in HeLa cells ( top , TMRE stained ) exceeded the dimensions of that for autophagosomes ( labelled by EGFP - LC3B ) under either normal ( middle , same field as TMRE image ) or starvation conditions ( right ) . ( c ) Illuminating a selective region ( white dotted circle , top ) within a KR - dMito / EBFP2 - parkin - expressing HeLa cell led to the immediate loss of KR fluorescence ( top two panels ) . Third to fifth row : magnified view of the photo - impaired region , EBFP2 - parkin translocated onto impaired mitochondrial tubules synchronously ( 38 - 60 min after 559 illumination , white arrows ) . Scale bars ( all panels ) , 10 μm .

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f1(a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right).(c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38-60 min after 559 illumination, white arrows). Scale bars (all panels), 10 μm.


f2 ( a ) A HeLa cell expressing KR - dMito , EGFP - LC3B and EBFP2 - parkin ( cyan ) were 559 nm illuminated ( white dotted circle region ; immediate loss of KR fluorescence ) . ( b - d ) Magnified view of the photo - inactivated region in a 34 - 93 min after 559 nm illumination . ( b ) Induction of parkin - mediated mitophagy on a single , long mitochondria tubule ( white arrows ) . ( c ) LC3B - coated structures were recruited onto distinct spots on the photo - impaired mitochondrial tubule ( white arrows : the location of the original impaired single mitochondrial tubule ) . ( d ) White dotted line region outlined in c , LC3 structures segregated from the main parkin - labelled cluster , carrying away bits of the parkin - labelled mitochondria with them . Two individual events are labelled in the image sequence with white and yellow arrows , respectively . Scale bars ( all panels ) , 5 μm .

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f2(a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b-d) Magnified view of the photo-inactivated region in a 34-93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 μm.


f3 ( a ) A HeLa cell expressing KR - dMito ( red ) , EGFP - DFCP1 ( green ) and EBFP2 - parkin ( cyan ) were illuminated with 559 nm at the white dotted circle region , leading to the generation of a long parkin - labelled mitochondria tubule ( right , magnified view of the light - activated region ) for autophagic turnover ( white arrow ) . Scale bars , 10 μm . ( b ) After the generation of parkin - labelled mitochondrial tubules following treatments in a , discrete DFCP1 foci ( green , middle panel , ER - associated omegasomes ) formed along the parkin - labelled mitochondria ( white arrows indicate DFCP1 spots that colocalized with parkin ) . Bottom panel : merged image of parkin and DFCP1 . Scale bar , 10 μm . ( c ) Time - lapse imaging showing DCFP1 puncta formation on parkin - labelled mitochondria . A HeLa cell expressing KR - dMito , EGFP - DFCP1 ( green ) and EBFP2 - parkin ( pseudo - coloured red ) were 559 nm illuminated , leading to the generation of parkin - labelled mitochondria tubules . Time - lapse imaging ( 1 frame per min ) revealed that DFCP1 puncta ( green ) formed transiently at discrete sites on parkin - labelled mitochondrial tubules ( red ) . White arrows mark two DFCP1 puncta ( ER - associated omegasomes ) formation / disassembly events directly on parkin - labelled tubules . Scale bar , 5 μm . ( d ) A COS - 7 cell expressing EBFP2 - parkin ( cyan ) , AcGFP - sec61β ( green ) and TagRFP - DFCP1 ( red ) were treated with 10 μM CCCP for 4 h and imaged . Omegasomes ( DFCP1 ) appeared where ER ( sec61β ) and impaired mitochondria ( parkin ) overlap . Scale bar , 10 μm . ( e ) Histogram of the lifetime of individual DFCP1 foci on parkin - labelled mitochondria in CCCP - treated HeLa cells expressing TagRFP - DFCP1 , EGFP - LC3B and EBFP2 - parkin . ( f ) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin - labelled mitochondria in HeLa cells expressing KR - dMito , EGFP - LC3B and EBFP2 - parkin . Each ' + ' marks the times measured in individual HeLa cells ( the red ' □ ' ( square ) denotes the average from five cells ) . P = 0 . 0172 ( Student ' s t - test ) .

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f3(a) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 μm.(b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 μm.(c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 μm.(d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm.(e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin.(f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each '+' marks the times measured in individual HeLa cells (the red '□' (square) denotes the average from five cells). P=0.0172 (Student's t-test).


f4 ( a ) A COS - 7 cell expressing EBFP2 - Ub ( red ) , AcGFP - sec61β ( green ) and mcherry - parkin ( cyan ) was treated with 10 μM CCCP for 1 h and imaged . Impaired mitochondria ( parkin ) displayed preferentially ubiquitinated foci ( white arrows ) at selected regions that overlap with the ER ( sec61β ) . ( b ) HeLa cells expressing KR - dMito ( red ) , EGFP - LC3 ( green ) and EBFP2 - parkin ( cyan ) were illuminated with 559 nm at the white dotted square regions , generating parkin - labelled mitochondria ( bottom right , magnified view of the light - activated regions ) . ( c ) Magnified view of the photo - inactivated region in b 80 min after illumination ( post - stained with anti - ubiquitin antibody ) . LC3 and ubiquitin colocalized at discrete sites along parkin - labelled mitochondria ( white arrows ) . ( d ) A MG - 132 treated HeLa cell expressing KR - dMito , EBFP2 - parkin ( green ) and EGFP - LC3B ( red ) was 559 nm illuminated , leading to the appearance of parkin - labelled mitochondria ( white arrows one to three indicated structures , top panels ) . The parkin - labelled structures did not fragment nor became engulfed by LC3 structures ( bottom panels ) in the presence MG - 132 . Scale bars ( all panels ) , 5 μm .

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

f4(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β).(b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows).(d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.


Figure 1B - Western blot verifying the expression of the CTD variants RPB1 - 52R and RPB1 - 25R in U2OS cells in whole cell extracts . U1 snRNP was used as a loading control . C - Changes in RNA synthesis upon CTD shortening . MA plot showing RNA synthesis changes in TT - seq datasets upon CTD shortening in U2OS cells in steady state conditions . RPB1 - 52R cells are used as a control and the data was normalized using spike - in counts . 16214 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red . 473 genes were significantly upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) and 704 genes were significantly downregulated ( adjusted p value < 0 . 05 , log2 fold change ≤ 1 ) . D - Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts ( 12014 transcripts ) based on TT - seq and RNA - seq datasets in RPB1 - 52R and RPB1 - 25R cells . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . E - Box plot of estimated RNA degradation rates of expressed RefSeq transcripts ( 12014 transcripts ) based on TT - seq and RNA - seq datasets in RPB1 - 52R and RPB1 - 25R cells . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . F - Box plot of estimated RNA half - lives of expressed RefSeq transcripts ( 12014 transcripts ) based on TT - seq and RNA - seq datasets in RPB1 - 52R and RPB1 - 25R cells . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

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

Figure 1B- Western blot verifying the expression of the CTD variants RPB1-52R and RPB1-25R in U2OS cells in whole cell extracts. U1 snRNP was used as a loading control.C- Changes in RNA synthesis upon CTD shortening. MA plot showing RNA synthesis changes in TT-seq datasets upon CTD shortening in U2OS cells in steady state conditions. RPB1-52R cells are used as a control and the data was normalized using spike-in counts. 16214 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red. 473 genes were significantly upregulated (adjusted p value < 0.05, log2 fold change >=1) and 704 genes were significantly downregulated (adjusted p value < 0.05, log2 fold change ≤1).D- Box plot of estimated RNA synthesis rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.E- Box plot of estimated RNA degradation rates of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.F- Box plot of estimated RNA half-lives of expressed RefSeq transcripts (12014 transcripts) based on TT-seq and RNA-seq datasets in RPB1-52R and RPB1-25R cells. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


Figure 2A - Box plots showing ratios of spliced TT - seq reads over total unspliced and spliced TT - seq reads for all constitutive 5 ' SSs and 3 ' SSs detected in major RNA isoforms in RPB1 - 52R and RPB1 - 25R cells . p value was calculated using Mann - Whitney U test . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . B - D - Box plots showing ratios of spliced TT - seq reads over total unspliced and spliced TT - seq reads in RPB1 - 52R and RPB1 - 25R cells for all constitutive 5 ' SSs and 3 ' SSs detected in major RNA isoforms based on intron position in the transcript : ( B ) first intron junction , ( C ) intermediate intron junctions and ( D ) last intron junctions . p values were calculated using Mann - Whitney U test . Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . E - Differential mRNA isoform usage upon CTD shortening . Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1 - 52R and RPB1 - 25R cells using long read sequencing . 47416 isoforms were detected , RPB1 - 52R condition was used as a control . 335 isoforms show differential expression ( p value < 0 . 05 , Fischer ' s exact test ) .

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

Figure 2A- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms in RPB1-52R and RPB1-25R cells. p value was calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. B-D- Box plots showing ratios of spliced TT-seq reads over total unspliced and spliced TT-seq reads in RPB1-52R and RPB1-25R cells for all constitutive 5'SSs and 3'SSs detected in major RNA isoforms based on intron position in the transcript: (B) first intron junction, (C) intermediate intron junctions and (D) last intron junctions. p values were calculated using Mann-Whitney U test. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. E- Differential mRNA isoform usage upon CTD shortening. Scatter plot showing read counts for mRNA isoforms detected in the chromatin fraction of RPB1-52R and RPB1-25R cells using long read sequencing. 47416 isoforms were detected, RPB1-52R condition was used as a control. 335 isoforms show differential expression (p value < 0.05, Fischer's exact test).


Figure 3A - CTD shortening leads to a decreased Pol II occupancy in promoter - proximal region . Median coverage plot of PRO - seq signal at genes encoding major isoforms . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed . B , C - Pol II pause position remains unchanged upon CTD shortening . Histogram of estimated positions of paused Pol II in RPB1 - 52R and RPB1 - 25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp . D - Median coverage plot of TT - seq signal at genes encoding major isoforms in RPB1 - 52R and RPB1 - 25R cells . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed . E - Box plot showing productive transcription initiation rate in RPB1 - 52R and RPB1 - 25R cells for genes encoding major isoforms . p value = 0 . 13 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . F - Box plot showing Pol II pausing duration in RPB1 - 52R and RPB1 - 25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp . p value < 2 . 2e - 16 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . G - Median scaled coverage plot of TT - seq signal at genes encoding major isoforms . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed . H - Pol II velocity remains unchanged upon CTD shortening . Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . I - Median coverage plot of TT - seq signal centered at PAS at genes encoding major isoforms in RPB1 - 52R and RPB1 - 25R cells . Solid lines represent median signal and shaded area refer to 95 % bootstrap confidence intervals . Two independent biological replicates were analyzed . J - Box plot showing length of the poly ( A ) tail in RPB1 - 52R and RPB1 - 25R cells measured using Nanopore sequening data . p value < 2 . 2e - 16 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . K - Box plot showing distance from the polyadenylation site ( PAS ) to the transcription termination site ( TTS ) in RPB1 - 52R and RPB1 - 25R cells for 1958 genes encoding major isoforms . p value = 0 . 8 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range .

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

Figure 3A- CTD shortening leads to a decreased Pol II occupancy in promoter-proximal region. Median coverage plot of PRO-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.B,C- Pol II pause position remains unchanged upon CTD shortening. Histogram of estimated positions of paused Pol II in RPB1-52R and RPB1-25R cells at genes encoding major isoforms with constitutive first exon longer than 100 bp.D- Median coverage plot of TT-seq signal at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.E- Box plot showing productive transcription initiation rate in RPB1-52R and RPB1-25R cells for genes encoding major isoforms. p value=0.13 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.F- Box plot showing Pol II pausing duration in RPB1-52R and RPB1-25R cells for genes encoding major isoforms with constitutive first exon longer than 100 bp. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.G- Median scaled coverage plot of TT-seq signal at genes encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.H- Pol II velocity remains unchanged upon CTD shortening. Metagene profile of transcription elongation velocity at 4480 genes longer than 10 kb encoding major isoforms. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals.I- Median coverage plot of TT-seq signal centered at PAS at genes encoding major isoforms in RPB1-52R and RPB1-25R cells. Solid lines represent median signal and shaded area refer to 95% bootstrap confidence intervals. Two independent biological replicates were analyzed.J - Box plot showing length of the poly(A) tail in RPB1-52R and RPB1-25R cells measured using Nanopore sequening data. p value<2.2e-16 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.K- Box plot showing distance from the polyadenylation site (PAS) to the transcription termination site (TTS) in RPB1-52R and RPB1-25R cells for 1958 genes encoding major isoforms. p value= 0.8 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range.


Figure 4B - Box plots showing RNA synthesis levels ( RPKs ) of putative eRNAs annotated using TT - seq data in RPB1 - 52R and RPB1 - 25R cells . p value = 9 . 27e - 103 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . C - Box plots showing lengths of putative eRNAs annotated using TT - seq data in RPB1 - 52R and RPB1 - 25R cells . p value = 0 . 000519 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1 - 52R and RPB1 - 25R cells .

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

Figure 4B- Box plots showing RNA synthesis levels (RPKs) of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =9.27e-103 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.C- Box plots showing lengths of putative eRNAs annotated using TT-seq data in RPB1-52R and RPB1-25R cells. p value =0.000519 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.D - Histogram showing a distribution of number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells.


Figure 5A - Changes in RNA synthesis in response to 15 min of TPA treatment . MA plot showing RNA synthesis changes in TT - seq datasets upon 15 min treatment with TPA ( 200 nM ) in RPB1 - 52R and RPB1 - 25R cells . DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes and adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) . B - Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1 - 52R and RPB1 - 25R cells ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) . P value = 0 . 02257 ( Mann - Whitney test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . C - Changes in RNA synthesis in response to 30 min of TPA treatment . MA plot showing RNA synthesis changes in TT - seq datasets upon 30 min treatment with TPA ( 200 nM ) in RPB1 - 52R and RPB1 - 25R cells . DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization . 14636 expressed genes annotated in RefSeq were analyzed . Differentially expressed genes are in red ( adjusted p value < 0 . 05 , log2 fold change > = 1 for upregulated genes and adjusted p value < 0 . 05 , log2 fold change ≤ 1 for downregulated genes ) D - Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1 - 52R and RPB1 - 25R cells ( adjusted p value < 0 . 05 , log2 fold chage > = 1 ) . P value = 0 . 001749 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

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

Figure 5A- Changes in RNA synthesis in response to 15 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 15 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes ). B- Box plots showing log2 fold change of genes significantly upregulated upon 15 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold change >=1). P value =0.02257 (Mann-Whitney test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Changes in RNA synthesis in response to 30 min of TPA treatment. MA plot showing RNA synthesis changes in TT-seq datasets upon 30 min treatment with TPA (200 nM) in RPB1-52R and RPB1-25R cells. DMSO treatment of the respective cell line was used as a control and the data was normalized using library size normalization. 14636 expressed genes annotated in RefSeq were analyzed. Differentially expressed genes are in red (adjusted p value < 0.05, log2 fold change >=1 for upregulated genes and adjusted p value < 0.05, log2 fold change ≤1 for downregulated genes) D- Box plots showing log2 fold change in RNA synthesis of genes significantly upregulated upon 30 min treatment with 200 nM TPA in RPB1-52R and RPB1-25R cells (adjusted p value < 0.05, log2 fold chage >=1). P value =0.001749 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


Figure 6A - Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1 - 52R and RPB1 - 25R cells upon 15 and 30 min TPA treatment ( 200 nM ) as well as in the DMSO controls . B - Box plots showing RNA synthesis levels ( RPK ) of putative eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R and RPB1 - 25R cells upon 15 min of TPA treatment ( 200 nM ) . p value = 0 . 000732 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed . C - Box plots showing RNA synthesis levels ( RPK ) of putative enhancer eRNAs paired with genes upregulated ( adjusted p value < 0 . 05 , log2 fold change > = 1 ) in RPB1 - 52R and RPB1 - 25R cells upon 30 min of TPA treatment ( 200 nM ) . p value = 2 . 77e - 5 ( Mann - Whitney U test ) Box limits are the first and third quartiles , the band inside the box is the median . The ends of the whiskers extend the box by 1 . 5 times the interquartile range . Two independent biological replicates were analyzed .

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

Figure 6A- Histogram showing the distribution of a number of putative eRNAs paired to genes in RPB1-52R and RPB1-25R cells upon 15 and 30 min TPA treatment (200 nM) as well as in the DMSO controls.B- Box plots showing RNA synthesis levels (RPK) of putative eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 15 min of TPA treatment (200 nM). p value = 0.000732 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed. C- Box plots showing RNA synthesis levels (RPK) of putative enhancer eRNAs paired with genes upregulated (adjusted p value < 0.05, log2 fold change >=1) in RPB1-52R and RPB1-25R cells upon 30 min of TPA treatment (200 nM). p value = 2.77e-5 (Mann-Whitney U test) Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Two independent biological replicates were analyzed.


Figure 1The Dyf phenotype of the ts - che - 3 mutant was confirmed , with animals grown at the permissive temperature ( 15ºC ) showing the ability to uptake dye , while those raised at the restrictive temperature ( 25ºC ) are Dyf . Scale bar , 20 µmUsing the homodimeric kinesin OSM - 3 as a marker for IFT , proper localisation is seen in the ts - che - 3 mutant at the permissive temperature , with the strongest signal at the basal body ( bb ) . At the restrictive temperature , most of the OSM - 3 signal is found within the axoneme of the short , bulbous cilia . Scale bar is 4 µmThe ts - che - 3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature , indicating defective chemosensation . The osm - 9 mutant is included as a positive control . n = 50 animals ; error bars are standard

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

Figure 1The Dyf phenotype of the ts-che-3 mutant was confirmed, with animals grown at the permissive temperature (15ºC) showing the ability to uptake dye, while those raised at the restrictive temperature (25ºC) are Dyf. Scale bar, 20 µmUsing the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µmThe ts-che-3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm-9 mutant is included as a positive control. n = 50 animals; error bars are standard error


Figure 2Three TZ markers ( NPHP - 4 , CEP - 290 and MKS - 6 ) are shown at both the permissive ( 15ºC ) and restrictive ( 25ºC ) temperatures in the ts - che - 3 mutant . At the permissive temperature , all three markers localise to the TZ , while at the restrictive temperature exhibit ectopic localisation ( leakage ) distally into the ciliary axoneme . While NPHP - 4 and MKS - 6 show significant accumulations , CEP - 290 is grossly normal . At the permissive temperature , both IFT co - markers ( CHE - 13 and XBX - 1 ) display strong localisation to the basal body ( bb ) and along the axoneme . At the restrictive temperature , the IFT markers show accumulation in the axoneme . Accumulations emphasized using asterisksTo determine if ciliary compartmentalisation could be restored , young ts - che - 3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature . The TZ proteins reassembled at the TZ , with loss of the ectopic accumulations , and IFT protein localisation was also restored - strongest at the basal body , similar to animals grown at the permissive temperature . ( c ) After 24 hours at the restrictive temperature , TZ proteins appear to begin to accumulate distally within the axoneme in ts - che - 3 mutants . This is seen , in small amounts , for NPHP - 4 and CEP - 290 but with stronger ectopic localisation for MKS - 6 . The IFT proteins CHE - 13 and XBX - 1 also show accumulations in the axoneme after 24 hours at the restrictive temperature . Data information : For ( A ) , ( B ) , and ( C ) , fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right . Each point in the plot represents one pixel along the centre of the basal body ( BB ) , transition zone ( TZ ) and middle segment ( MS ) regions . Dotted areas ( three pixels ) in the MS were used to quantify ectopic localisation ( TZ markers ) or accumulation ( IFT markers ) for statistical analyses . n = 10 cilia ( 4 - 7 animals ) , Kruskal - Wallis test , * P < 0 . 05 , * * P < 0 . 01 . tz , transition zone ; bb , basal body ; scale bars are 4

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

Figure 2Three TZ markers (NPHP-4, CEP-290 and MKS-6) are shown at both the permissive (15ºC) and restrictive (25ºC) temperatures in the ts-che-3 mutant. At the permissive temperature, all three markers localise to the TZ, while at the restrictive temperature exhibit ectopic localisation (leakage) distally into the ciliary axoneme. While NPHP-4 and MKS-6 show significant accumulations, CEP-290 is grossly normal. At the permissive temperature, both IFT co-markers (CHE-13 and XBX-1) display strong localisation to the basal body (bb) and along the axoneme. At the restrictive temperature, the IFT markers show accumulation in the axoneme. Accumulations emphasized using asterisksTo determine if ciliary compartmentalisation could be restored, young ts-che-3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored-strongest at the basal body, similar to animals grown at the permissive temperature. (c) After 24 hours at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts-che-3 mutants. This is seen, in small amounts, for NPHP-4 and CEP-290 but with stronger ectopic localisation for MKS-6. The IFT proteins CHE-13 and XBX-1 also show accumulations in the axoneme after 24 hours at the restrictive temperature. Data information: For (A), (B), and (C), fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4-7 animals), Kruskal-Wallis test, *P<0.05, **P<0.01. tz, transition zone; bb, basal body; scale bars are 4 µm


Figure 3In the ts - che - 3 mutant grown at the permissive temperature ( 15ºC ) , both RPI - 2 / RP2 and TRAM - 1 / TRAM1 are localised to the PCMC , just proximal to the ciliary axoneme . At the restrictive temperature , TRAM - 1 shows leakage into the axoneme with its strongest localisation at the PCMC . Intriguingly , RPI - 2 now displays strong localisation to the axoneme , indicating that in the absence of IFT , RPI - 2 may be targeted into the cilium . When ts - che - 3 mutant animals grown at the restrictive temperature ( 25ºC ) are shifted at young adult to the permissive ( 15ºC ) for 24 hours , both TRAM - 1 and RPI - 2 exhibit restoration of their PCMC localisation . Consistent with other TZ markers , both MKS - 2 and MKSR - 1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature . Also consistent with what was observed for other markers , after 24 hours at the permissive temperature , animals grown at the restrictive temperature show loss of the ectopic localisation . Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right . Each point in the plot represents one pixel along the centre of the basal body ( BB ) , transition zone ( TZ ) and middle segment ( MS ) regions . Dotted areas ( three pixels ) in the MS were used to quantify ectopic localisation ( MKS - 2 and MKSR - 1 ) or accumulation ( RPI - 2 and TRAM - 1 ) for statistical analyses . n = 10 cilia ( 4 - 6 animals ) , Kruskal - Wallis test , * P < 0 . 05 , * * P < 0 . 01 ; scale bars are 4 µmLoss of CHE - 3 function results in short , bulbous cilia containing electron dense accumulations and abnormal membrane - microtubule connections . Shown are transmission electron micrograph ( TEM ) cross sections of an amphid channel cilium in wild - type and che - 3 ( e1124 ) null mutant animals . Representative images of wild - type cilia show intact middle segment ( containing doublet microtubules ) and transition zone ( with Y - shaped links ) ( left top and bottom panels ) regions . Representative images of che - 3 ( e1124 ) cilia reveal apparently intact transition zones , with visible Y - link structures , but enlarged ciliary ends filled with electron - dense accumulations , which often appear vesicular . The bulbous structures reveal doublet microtubules associated with the membrane , and occasionally ectopic microtubule - to - membrane connections , which sometimes appear similar to transition zone Y - links in the region just distal to the seemingly " normal " TZ structure . Schematics show longitudinal ( left images ) and cross section ( right images ) representations of wild - type and che - 3 ( e1124 ) cilia . tz , transition zone ; pcmc , periciliary membrane compartment ; scale bars are as indicated in ( nm )

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

Figure 3In the ts-che-3 mutant grown at the permissive temperature (15ºC), both RPI-2/RP2 and TRAM-1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM-1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI-2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI-2 may be targeted into the cilium. When ts-che-3 mutant animals grown at the restrictive temperature (25ºC) are shifted at young adult to the permissive (15ºC) for 24 hours, both TRAM-1 and RPI-2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS-2 and MKSR-1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 hours at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS-2 and MKSR-1) or accumulation (RPI-2 and TRAM-1) for statistical analyses. n = 10 cilia (4-6 animals), Kruskal-Wallis test, *P<0.05, **P<0.01; scale bars are 4 µmLoss of CHE-3 function results in short, bulbous cilia containing electron dense accumulations and abnormal membrane-microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild-type and che-3(e1124) null mutant animals. Representative images of wild-type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y-shaped links) (left top and bottom panels) regions. Representative images of che-3(e1124) cilia reveal apparently intact transition zones, with visible Y-link structures, but enlarged ciliary ends filled with electron-dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule-to-membrane connections, which sometimes appear similar to transition zone Y-links in the region just distal to the seemingly "normal" TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild-type and che-3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for


Figure 4Using the IFT marker BBS - 7 , cilia lengths were measured for wild - type and the ts - che - 3 mutant animals raised at the permissive ( 15ºC ) and shifted to the restrictive temperature ( 25ºC ) for three time points . After 12 hours some accumulations ( indicated by asterisks in the fluorescence images ) are observed within the cilia but there is not a significant reduction in cilium length . After 18 and 24 hours , significant reduction in ciliary length is observed and large accumulations of BBS - 7 is seen within short bulbous cilia , similar to what is observed in ts - che - 3 animals raised at the restrictive temperature . n = 30 - 34 cilia , one per animal ; Kruskal - Wallis test , * * P < 0 . 01 ; error bars are mean absolute deviation ; scale bars are 4 µmIFT velocities were measured for wild - type or ts - che - 3 mutant animals ( grown at permissive temperature ) and imaged at the permissive temperature , restrictive temperature or after 3 hours at the restrictive temperature . Compared to wild - type , anterograde velocities in the ts - che - 3 dynein mutant are similar , while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments . Interestingly , the ts - che - 3 IFT dynein mutant appears to not only have slow retrograde IFT trains , but the trains also appear much larger and less frequent . Initially upon the temperature shift ( < 15min ) IFT rates are much faster for both wild - type and ts - che - 3 dynein mutant , although retrograde remains much slower in the mutant . After 3 hours at the restrictive temperature , IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to

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

Figure 4Using the IFT marker BBS-7, cilia lengths were measured for wild-type and the ts-che-3 mutant animals raised at the permissive (15ºC) and shifted to the restrictive temperature (25ºC) for three time points. After 12 hours some accumulations (indicated by asterisks in the fluorescence images) are observed within the cilia but there is not a significant reduction in cilium length. After 18 and 24 hours, significant reduction in ciliary length is observed and large accumulations of BBS-7 is seen within short bulbous cilia, similar to what is observed in ts-che-3 animals raised at the restrictive temperature. n = 30-34 cilia, one per animal; Kruskal-Wallis test, **P<0.01; error bars are mean absolute deviation; scale bars are 4 µmIFT velocities were measured for wild-type or ts-che-3 mutant animals (grown at permissive temperature) and imaged at the permissive temperature, restrictive temperature or after 3 hours at the restrictive temperature. Compared to wild-type, anterograde velocities in the ts-che-3 dynein mutant are similar, while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments. Interestingly, the ts-che-3 IFT dynein mutant appears to not only have slow retrograde IFT trains, but the trains also appear much larger and less frequent. Initially upon the temperature shift (<15min) IFT rates are much faster for both wild-type and ts-che-3 dynein mutant, although retrograde remains much slower in the mutant. After 3 hours at the restrictive temperature, IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to 25ºC


Figure 5To assess the effects of IFT restoration on TZ assembly , ts - che - 3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development ( days 1 , 4 and 8 of adulthood ) . The TZ markers MKS - 6 ( a ) and NPHP - 4 ( b ) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood . After a shift to the permissive temperature for 24 hours , day 2 adults show " repaired " TZ protein localisation resembling that of wild - type animals . For both markers , Day 4 adults ( maintained at the restrictive temperature ) show less ectopic TZ protein localisation than Day 1 adults . The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 ( to day 5 ) of adulthood compared to that observed in day 1 ( to day 2 ) adults . For MKS - 6 , but not NPHP - 4 , day 8 adults ( maintained at the restrictive temperature ) also display less ectopic accumulation than day 1 adults . The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers , indicating that this repair is most effective in day 1 adults . In summary , as the adults age , ectopic TZ protein localisation is reduced , while the ability to correct this phenotype by restoring IFT function

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

Figure 5To assess the effects of IFT restoration on TZ assembly, ts-che-3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development (days 1, 4 and 8 of adulthood). The TZ markers MKS-6 (a) and NPHP-4 (b) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood. After a shift to the permissive temperature for 24 hours, day 2 adults show "repaired" TZ protein localisation resembling that of wild-type animals. For both markers, Day 4 adults (maintained at the restrictive temperature) show less ectopic TZ protein localisation than Day 1 adults. The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 (to day 5) of adulthood compared to that observed in day 1 (to day 2) adults. For MKS-6, but not NPHP-4, day 8 adults (maintained at the restrictive temperature) also display less ectopic accumulation than day 1 adults. The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers, indicating that this repair is most effective in day 1 adults. In summary, as the adults age, ectopic TZ protein localisation is reduced, while the ability to correct this phenotype by restoring IFT function al