Calbindin of sections showing axons of Purkinje as in ( A ) . While wild type spastin - / - show normal axons , Ccp1 - / - Ccp1 - / - spastin - / - mice show axonal swellings ( arrow heads ) in remaining Purkinje . Scale bars : 20 µm .

B-GENEPROD O O O B-SUBCELLULAR O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O

Calbindin staining of sections showing axons of Purkinje cells as in (A). While wild type and spastin-/- show normal axons, Ccp1-/- and Ccp1-/-spastin-/- mice show axonal swellings (arrow heads) in the remaining Purkinje cells. Scale bars: 20 µm.


Immunoblot of extracts from Ccp1flox / floxCcp6flox / flox hippocampal neurons without , or after transduction with lentivirus expressing either GFP or GFP - 2A - cre after 4 days in culture . Note specific increase of polyglutamylation ( polyE ) in neurons transduced with GFP - 2A - cre lentivirus .

B-EXP_ASSAY O O O B-GENEPROD O O O O B-TISSUE B-CELL_TYPE O O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD B-ORGANISM O

Immunoblot of cell extracts from Ccp1flox/floxCcp6flox/flox hippocampal neurons without, or after transduction with lentivirus expressing either GFP or GFP-2A-cre after 4 days in culture. Note the specific increase of polyglutamylation (polyE) in neurons transduced with GFP-2A-cre lentivirus.


kymographs of one representative set of experiments . Mitochondria movements ( Movie S1 ) were plotted in kymographs analyzed as described in ( A ) . Scale bars : 60 sec , 20 µm . Analyses of ( D ) run ( E ) speed distribution , represented as scatter dot plots with each point representing a single run extracted from kymographs shown in ( C ) . Each group represents pool of measurement of multiple neurons made in experiment . black indicates median of distribution with whiskers at interquartile range . Numerical value of medians is indicated below each plot . ( F ) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to total number of mitochondria during recording time of 60 s . Values ( below plots ) from one set of experiment ( C ) show a decrease of about 50 % in Ccp1 - / - Ccp6 - / - conditions , while ( G ) mitochondria density is unaltered . ( H - K ) Statistical analyses of mitochondria transport parameters of experiments shown in ( D - G ; Fig . EV4A - D ) . Each bar represents mean ( ± ) of medians of single experiments for run ( H ) speed ( I ) distributions , average ( ± ) mitochondria motility ( J ) density ( K ) of seven experimental sets . Values of fold change between control Ccp1 - / - Ccp6 - / - conditions are indicated below graphs . Green bars are for anterograde , red for retrograde transport events . Significance was tested using unpaired t - . Average run ( H ) as well as speed ( I ) showed no significant differences in these two parameters between control Ccp1 - / - Ccp6 - / - conditions . ( J ) average of overall motility reveals an about 50 % decrease in overall mitochondria motility , while mitochondria density ( K ) is unaltered between control Ccp1 - / - Ccp6 - / - conditions .

B-EXP_ASSAY O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O O O 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-EXP_ASSAY O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O 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-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O 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-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O 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-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O

All kymographs of one representative set of experiments. Mitochondria movements (Movie S1) were plotted in kymographs and analyzed as described in (A). Scale bars: 60 sec, 20 µm. Analyses of (D) run length and (E) speed distribution, represented as scatter dot plots with each point representing a single run extracted from the kymographs shown in (C). Each group represents the pool of measurement of multiple neurons made in the experiment. The black line indicates the median of the distribution with whiskers at interquartile range. Numerical value of the medians is indicated below each plot. (F) Overall motility of mitochondria represented as fraction of time mitochondria spent in movement relative to the total number of mitochondria during the recording time of 60 s. Values (below plots) from one set of experiment (C) show a decrease of about 50% in Ccp1-/-Ccp6-/- conditions, while (G) mitochondria density is unaltered. (H-K) Statistical analyses of mitochondria transport parameters of experiments shown in (D-G; Fig. EV4A-D). Each bar represents the mean (±SEM) of medians of single experiments for run length (H) and speed (I) distributions, and average (±SEM) mitochondria motility (J) and density (K) of all seven experimental sets. Values of fold change between control and Ccp1-/-Ccp6-/- conditions are indicated below the graphs. Green bars are for anterograde, and red for retrograde transport events. Significance was tested using unpaired t-test. Average run length (H) as well as speed (I) showed no significant differences in these two parameters between control and Ccp1-/-Ccp6-/- conditions. (J) The average of overall motility reveals an about 50% decrease in overall mitochondria motility, while mitochondria density (K) is unaltered between control and Ccp1-/-Ccp6-/- conditions.


( A ) Optically evoked auditory brainstem response in an AAV - CatCh - injected gerbil in response to a 1 ms of ~ 35 mW ( mean of 1000 stimulus presentations at 10 Hz repetition rate ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

(A) Optically evoked auditory brainstem response in an AAV-CatCh-injected gerbil in response to a 1 ms laser pulse of ~35 mW (mean of 1000 stimulus presentations at 10 Hz repetition rate).


( B - D ) Catch - eYFP - expressing SGNs in apical cochlear turn identified by parvalbumin expression ( B ) CatCh - eYFP ( C ) . inlay ( D ´ ) of merged immunostaining ( D ) indicates lack of CatCh - EYFP signal in inner hair ( dashed ) . Scale bar : 20 µm .

O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O

(B-D) Catch-eYFP-expressing SGNs in the apical cochlear turn identified by parvalbumin expression (B) and CatCh-eYFP (C). The inlay (D´) of the merged immunostaining (D) indicates the lack of CatCh-EYFP signal in inner hair cells (dashed white line). Scale bar: 20 µm.


( I ) oCI inserted into cochlea ( dashed black lines ) via a cochleostomy in basal cochlear turn ( black , solid ) . round window niche is highlighted by a dashed , round window by a solid . SA : stapedial artery .

O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O

(I) oCI inserted into the cochlea (dashed black lines) via a cochleostomy in the basal cochlear turn (black, solid line). The round window niche is highlighted by a dashed white line, the round window by a solid white line. SA: stapedial artery.


( J ) 3D X - ray tomography reconstruction of a 16 - channel oCI implanted in a gerbil cochlea via round window . Cables μLEDs are marked in blue , most apical µLED is indicated by black arrowhead ; basilar is marked in green . µLEDs have a of 60 × 60 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O

(J) 3D X-ray tomography reconstruction of a 16-channel oCI implanted in a gerbil cochlea via the round window. Cables and μLEDs are marked in blue, the most apical µLED is indicated by the black arrowhead; the basilar membrane is marked in green. µLEDs have a size of 60×60 µm.


( A - D ) Exemplar STCs in response to SGN illumination with a single μLED ( A ) , a block of four neighboring μLEDs ( B ) μLEDs of an oCI inserted via round window in a CatCh - transduced gerbil ( C ) with µLEDs in a wildtype gerbil ( WT ; D ) . μLEDs were spaced by 250 μm .

O O O O O O O O O O B-CELL_TYPE O O O O 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-GENEPROD O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O

(A-D) Exemplar STCs in response to SGN illumination with a single μLED (A), a block of four neighboring μLEDs (B) and all μLEDs of an oCI inserted via the round window in a CatCh-transduced gerbil (C) and with all µLEDs in a wildtype gerbil (WT; D). μLEDs were spaced by 250 μm.


( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) after deafening ( right ; star : threshold ) . ( F ) Optically evoked auditory brainstem responses before ( left ) after deafening ( right ) , elicited by ~ 35 mW pulses of 1 ms duration , delivered by a - coupled fiber ( 1000 stimuli presented at 10 Hz ) , recorded from same . Optogenetic activation of auditory system was still possible after deafening . Differences in amplitude waveform before after deafening are likely due to different positioning of recording electrodes optical fiber .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O 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-DISEASE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE O O O O O O O O O O O O O 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-TISSUE I-TISSUE O O O O B-DISEASE O O O O O O O B-DISEASE O O O O O O O O O O O O

(E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold). (F) Optically evoked auditory brainstem responses before (left) and after deafening (right), elicited by ~35 mW pulses of 1 ms duration, delivered by a laser-coupled fiber (1000 stimuli presented at 10 Hz), recorded from the same animal. Optogenetic activation of the auditory system was still possible after deafening. Differences in amplitude and waveform before and after deafening are likely due to different positioning of recording electrodes and the optical fiber.


( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) after deafening ( right ; star : threshold ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O 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-DISEASE O O O O O O O O

(E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold).


( G - H ) Exemplar STCs in response to SGN illumination with µLEDs in a CatCh - transduced , kanamycin - deafened gerbil before ( G ) after ( H ) sacrifice .

O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-DISEASE B-ORGANISM O O O O O O O O O O

(G-H) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh-transduced, kanamycin-deafened gerbil before (G) and after (H) sacrifice.


( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , 16 ( ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , with a - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) post - hoc multiple comparison ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . : p = 0 . 0096 ; vs . fiber : p = 0 . 0016 ; CatCh vs . WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with µLEDs has also been performed in WT ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) in sacrificed CatCh - transduced ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened . ( J ) Number of active electrodes ( d ´ > 1 ; mean ± standard deviation ) upon oCI stimulation with 1 ( n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , 16 µLEDs ( n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) , with optical fiber stimulation ( n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 30 . 18 , 4 degrees of freedom , p = 1 . 42 * 10 - 21 ) post - hoc multiple comparison ; indiv . vs . block : p = 6 . 8 * 10 - 5 ; block vs . : p = 0 . 006 ; vs . fiber : p = 0 . 25 ; CatCh vs . WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . Responses to oCI stimulation with µLEDs were also observed in hearing WT ( n = 3 oCIs in 2 gerbils ) . No active electrodes were found in response to 4 / 7 oCI stimulations in 2 / 2 hearing / deaf wildtype gerbils ) . ( K ) Thresholds ( d ´ = 1 , measured at best electrode ( BE ) ) for ICC activation evoked with 16 active µLEDs on an oCI ( n = 24 / 9 in N = 9 / 3 hearing / deaf gerbils ) with a - coupled optical fiber ( n = 9 / 3 in N = 9 / 3 hearing / deaf gerbils ; p = 0 . 012 , Wilcoxon rank sum ) . Central horizontal indicates median , lower , upper horizontal lines indicate minimum maximum values , box edges indicate 25th 75th percentile , respectively .

O O O O O O B-TISSUE O O O O O O O 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-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O 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-DISEASE B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-DISEASE B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O 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-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-DISEASE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals. (J) Number of active electrodes (d´ > 1; mean ± standard deviation) upon oCI stimulation with 1 (n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 µLEDs (n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils), and with optical fiber stimulation (n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 30.18, 4 degrees of freedom, p = 1.42*10-21) and post-hoc multiple comparison test; indiv. vs. block: p = 6.8*10-5; block vs. all: p = 0.006; all vs. fiber: p = 0.25; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). Responses to oCI stimulation with all µLEDs were also observed in hearing WT animals (n = 3 oCIs in 2 gerbils). No active electrodes were found in response to 4/7 oCI stimulations in 2/2 hearing/deaf wildtype gerbils). (K) Thresholds (d´ = 1, measured at the best electrode (BE)) for ICC activation evoked with 16 active µLEDs on an oCI (n = 24/9 in N = 9/3 hearing/deaf gerbils) and with a laser-coupled optical fiber (n = 9/3 in N = 9/3 hearing/deaf gerbils; p = 0.012, Wilcoxon rank sum test). Central horizontal line indicates the median, lower, upper horizontal lines indicate the minimum and maximum values, and box edges indicate the 25th and 75th percentile, respectively.


( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , 16 ( ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , with a - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) post - hoc multiple comparison ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . : p = 0 . 0096 ; vs . fiber : p = 0 . 0016 ; CatCh vs . WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with µLEDs has also been performed in WT ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) in sacrificed CatCh - transduced ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened .

O O O O O O B-TISSUE O O O O O O O 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-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O 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-DISEASE B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-DISEASE B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O O O B-DISEASE O

(I) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals.


( A ) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via round window , from apical to basal direction ( A - D ) . markers define best electrode ( BE ) , measurement of spread of excitation ( SoE ) is indicated in left panel . In STCs where more than one peak was detected , dorsal - ventral - most electrodes have been considered as boundaries of STC in order to avoid underestimation of SoE .

O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window, from apical to basal direction (A-D). White markers define the best electrode (BE), and measurement of the spread of excitation (SoE) is indicated in the left panel. In STCs where more than one peak was detected, the dorsal- and ventral-most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE.


( C ) Spectral spread of excitation upon SGN stimulation with µLED - oCIs ( blocks of 4 µLEDs , solid lines ; mean ± standard error of mean ) , acoustic ( black dashed ) , - coupled optical ( blue dashed ) , as well as mono - bipolar electrical stimulation with a clinical - style eCI ( dashed dotted red lines , respectively ) . Average data from stimulation via optical eCIs are replotted from Dieter et al ( Dieter et al , 2019 ) .

O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Spectral spread of excitation upon SGN stimulation with µLED-based oCIs (blocks of 4 µLEDs, solid lines; mean ± standard error of the mean), acoustic (black dashed line), laser-coupled optical fibers (blue dashed line), as well as mono- and bipolar electrical stimulation with a clinical-style eCI (dashed and dotted red lines, respectively). Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al (Dieter et al, 2019).


Figure 1b . Genotyping of AS WT CDK12 clones . Ethidium bromide - stained agarose gel visualizing PCR products from genomic of AS ( AS - PCR ) WT ( WT - PCR ) CDK12 HCT116 their digest with BslI enzyme ( indicated as AS - BslI WT - BslI ) . Primer positions BslI restriction sites are depicted at Expanded View Fig . 1a . Numbers on left right indicate marker fragment sizes , respectively .

O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD B-CELL_LINE O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively.


Figure 1d . Effect of CDK12 inhibition on phosphorylation of CTD of RNAPII . Western blot analyses of by indicated antibodies in AS CDK12 HCT116 treated with 5 µM 3 - MB - PP1 for indicated times . Long short exp . = long ( 4 - 14 min ) short ( 10 - 60 s ) exposures , respectively . FUS tubulin are loading controls . A representative image from 3 replicates is shown .

O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O 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-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O

Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown.


Figure 1e , f . Inhibition of CDK12 in AS CDK12 HCT116 results in down - regulation of CDK12 - dependent HR genes . Graph shows RT - qPCR of relative of mRNAs of described genes in AS CDK12 HCT116 ( e ) WT CDK12 HCT116 ( f ) treated for indicated times with 3 - MB - PP1 . mRNA were normalized to HPRT1 mRNA expression mRNA of untreated control ( CTRL ) were set to 1 . n = 3 replicates , error bars indicate standard error of mean ( ) .

O O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD B-CELL_LINE O O O O B-GENEPROD B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM).


Figure 2b . CDK12 kinase activity is needed for G1 / S progression in arrested by serum starvation . Flow cytometry profiles of control ( - 3 - MB - PP1 ) or inhibitor ( + 3 - MB - PP1 ) treated from experiment depicted in Fig . 2a . red arrow points to onset of G1 / S progression defect in 3 - MB - PP1 - treated . To better visualize G1 / S delay in presence of inhibitor 24 h time point is also shown . n = 3 replicates ; representative result is shown .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O 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 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation . Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown.


Figure 2b . CDK12 kinase activity is needed for G1 / S progression in arrested by serum

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE

Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation


Figure 2d . CDK12 peak in G0 / G1 phase of cycle . Western blots show of at indicated time points after release of serum starved AS CDK12 HCT116 . Corresponding cycle phases are depicted above time points . A representative Western blot from 3 replicates is shown .

O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE O O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Figure 2d. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from 3 replicates is shown.


Figure 2f , g . Inhibition of CDK12 in early G1 perturbs normal cycle progression . of ( % ) in cycle phases from flow cytometry profiles of propidium iodide ( f ) BrdU ( g ) labeled upon addition of 3 - MB - PP1 at indicated time points after serum addition in experiment depicted in Fig . 2e . CTRL in Fig . 2g = control sample without 3 - MB - PP1 . n = 3 replicates , representative result is shown .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O

Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown.


Figure 2h . Short - term CDK12 inhibition does not activate damage checkpoints . Western blot analyses of phosphorylation of depicted damage response markers upon inhibition of CDK12 for indicated times . CPT corresponds to 5 µM camptothecin . A representative Western blot from 3 replicates is shown . FUS is a loading control .

O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O

Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control.


Figure 3a . CDK12 inhibition results in differential expression of a subset of genes . Comparison of log2 fold - changes versus log2 mean expression in 3 ' end - seq data shows differentially regulated genes after inhibition of CDK12 . Down - ( log2 fold - change < - 1 ) up - regulated ( log2 fold - change > 1 ) genes are shown in blue red , respectively .

O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O 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. CDK12 inhibition results in differential expression of a subset of genes. Comparison of log2 fold-changes versus log2 mean expression in 3'end RNA-seq data shows differentially regulated genes after inhibition of CDK12. Down- (log2 fold-change < -1) and up-regulated (log2 fold-change >1) genes are shown in blue and red, respectively.


Figure 3d . Validation of - seq for select replication genes by RT - qPCR . Graph shows relative of mRNAs of described genes in serum arrested released ( 0 h G0 / G1 ) AS CDK12 HCT116 either treated ( 3 - MB - PP1 ) or not ( CTRL ) with inhibitor for indicated times after release . mRNA were normalized to B2M mRNA expression mRNA for each gene at time of release ( 0 h ) was set as 1 . n = 3 replicates , error bars indicate .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 3d. Validation of RNA-seq for select DNA replication genes by RT-qPCR. Graph shows relative levels of mRNAs of described genes in serum arrested and released (0 h G0/G1) AS CDK12 HCT116 cells either treated (3-MB-PP1) or not (CTRL) with the inhibitor for indicated times after the release. mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release (0 h) was set as 1. n=3 replicates, error bars indicate SEM.


Figure 3e . of replication factors are dependent on CDK12 kinase activity . Western blot analyses of expression by depicted antibodies in serum synchronized released ( 0 h ) either treated or not with 3 - MB - PP1 for indicated times after release . FUS is a loading control . A representative Western blot of 3 replicates is shown .

O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Figure 3e. Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity. Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released (0 h) cells either treated or not with 3-MB-PP1 for the indicated times after the release. FUS is a loading control. A representative Western blot of 3 replicates is shown.


Figure 3f . CDK12 inhibition affects loading of CDC6 CDT1 replication factors to . Western blotting analyses of association of indicated replication factors in serum synchronized released AS CDK12 HCT116 treated or not with 3 - MB - PP1 for indicated times . Histone H2A serves as a loading control of fractions . A = asynchronous , 0 h = time of release . A representative Western blot of 3 replicates is shown .

O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O B-GENEPROD B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Figure 3f. CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin. Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3-MB-PP1 for the indicated times. Histone H2A serves as a loading control of chromatin fractions. A=asynchronous cells, 0 h=time of release. A representative Western blot of 3 replicates is shown.


Removal of CDK12 inhibitor in early G1 / S rescues replication gene expression cycle progression . RT - qPCR of replication gene mRNA RT - qPCR were performed 7 , 12 15 h post - release , respectively . CTRL = control samples without 3 - MB - PP1 . In b , n = 3 error bars indicate . In c , d representative from 3 biological replicates are shown .

O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR of replication gene mRNA RT-qPCR were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.


Removal of CDK12 inhibitor in early G1 / S rescues cycle progression . Western blotting of , Western blotting were performed 7 , 12 15 h post - release , respectively . CTRL = control samples without 3 - MB - PP1 . In b , n = 3 error bars indicate . In c , d representative from 3 biological replicates are shown .

O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

Removal of CDK12 inhibitor in early G1/S rescues cell cycle progression. Western blotting of , protein levels Western blotting were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.


Removal of CDK12 inhibitor in early G1 / S rescues replication cycle progression . flow cytometry analyses of cycle progression , flow cytometry analyses were performed 7 , 12 15 h post - release , respectively . CTRL = control samples without 3 - MB - PP1 . In b , n = 3 error bars indicate . In c , d representative from 3 biological replicates are shown .

O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

Removal of CDK12 inhibitor in early G1/S rescues replication and cell cycle progression. flow cytometry analyses of cell cycle progression, flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.


Figure 4e . Rescued loading of CDC6 CDT1 on after removal of CDK12 inhibitor . Western blot analyses of fractions of serum starved AS CDK12 HCT116 treated with 3 - MB - PP1 for 6 or 9 h or with inhibitor washed off after 1 h of treatment . CTRL corresponds to not treated with inhibitor at time of serum addition . were harvested either 6 or 9 h after serum addition . Histone H2A serves as a loading control of fractions studied replication factors are indicated . A representative image of three replicates is shown .

O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O B-GENEPROD B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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-TISSUE O O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown.


Figure 4f . Inhibition of CDK12 kinase activity in cycling leads to decreased numbers of actively replicating . Asynchronous AS CDK12 HCT116 were grown for 24 48 h in presence or absence of 3 - MB - PP1 replicating BrdU - stained were quantified by FACS analyses . CTRL = control samples without 3 - MB - PP1 . A representative image of 3 replicates is shown .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown.


Figure 4g , h . Prolonged CDK12 inhibition causes chromosomal aberrations in . Specific chromosomal aberrations in treated with 3 - MB - PP1 ( 24 or 48 h ) , 4 mM hydroxyurea ( 5 h ) or control solvent ( CTRL ) were identified by microscopy . A representative image from 3 biological replicates is shown ( g ) . Total numbers of chromosomal aberrations per hundred of representative replicate in g is quantified ( h ) .

O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).


Figure 5b Genes down - regulated in - seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3 ´ ends Metagene analyses of RNAPII ChIP - seq data Each transcript was divided into two parts with fixed ( transcription start site ( TSS ) - 3kb to + 1 . 5kb transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) a central part with variable corresponding to of gene ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) average coverage for each bin was calculated for each transcript in each sample . curve for each transcript was normalized to a sum of one then averaged first across genes second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS TTS . color track at bottom of each subfigure indicate significance of paired Wilcoxon comparing normalized transcript coverages for each bin between untreated ( CTRL ) treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5b Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3´ ends Metagene analyses of RNAPII ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.


Genes down - regulated in - seq after CDK12 inhibition have higher relative occupancy of P - Ser2 in their gene bodies . Metagene analyses of P - Ser2 ChIP - seq data Each transcript was divided into two parts with fixed ( transcription start site ( TSS ) - 3kb to + 1 . 5kb transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) a central part with variable corresponding to of gene ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) average coverage for each bin was calculated for each transcript in each sample . curve for each transcript was normalized to a sum of one then averaged first across genes second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS TTS . color track at bottom of each subfigure indicate significance of paired Wilcoxon comparing normalized transcript coverages for each bin between untreated ( CTRL ) treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of P-Ser2 ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.


Figure 5d Examples of genes whose transcription processivity expression is dependent on CDK12 kinase activity . - seq data on respective strand RNAPII , P - Ser2 , P - Ser5 SPT6 ChIP - seq data for MED13 genes from either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read were normalized to total number of mapped reads per sample averaged between replicates . Blue red boxes below - seq data indicate 90 % in control CDK12 inhibited samples , respectively .

O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O

Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for MED13 genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance in control and CDK12 inhibited samples, respectively.


Figure 5d e . Examples of genes whose transcription processivity expression is dependent on CDK12 kinase activity . - seq data on respective strand RNAPII , P - Ser2 , P - Ser5 SPT6 ChIP - seq data for UBE3C genes from either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read were normalized to total number of mapped reads per sample averaged between replicates . Blue red boxes below - seq data indicate 90 % ( see Fig . 7d 7e corresponding text ) in control CDK12 inhibited samples , respectively .

O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O

Figure 5d e. Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for UBE3C genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance (see Fig. 7d and 7e and corresponding text) in control and CDK12 inhibited samples, respectively.


Figure 7a . Longer genes tend to have a larger fraction of differentially used exons . Box plot shows fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles on genomic ( including exons introns ) of their longest transcripts . n = 3 replicates .

O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

Figure 7a. Longer genes tend to have a larger fraction of differentially used exons. Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length (including exons and introns) of their longest transcripts. n=3 replicates.


Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative of pre - mRNAs of described genes in AS CDK12 HCT116 either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA were normalized to samples not treated with DRB ( Unt ) for which value was set as 1 . n = 3 independent experiments , error bars correspond to . Positions of primers ( designed to span exon - intron junctions ) their from transcription start site in kb are indicated in gene structures shown above graphs . Figure 8c . Proposed model . Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity functions that are especially dependent on optimal expression of these genes . situation in with normal aberrant CDK12 kinase activity is depicted . CDK12 ( green oval ) phosphorylates ( P ) unknown substrate ( s ) ( orange oval ) , possibly including CTD ( blue ) , which results in optimal elongation processivity ( blue arrow ) of RNAPII ( blue oval ) for CDK12 - sensitive genes . Full , functional mRNAs are synthesized ( upper panel ) . Inhibition of CDK12 leads to hyperphosphorylation ( capital P ) of Ser2 ( S2 ) in bodies of CDK12 - sensitive genes which is associated with slower elongation premature termination . Shorter , aberrant mRNAs are made ( lower panel ) . mRNAs are depicted as black lines .

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O 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 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs. Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines.


Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative of pre - mRNAs of described genes in AS CDK12 HCT116 either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA were normalized to samples not treated with DRB ( Unt ) for which value was set as 1 . n = 3 independent experiments , error bars correspond to . Positions of primers ( designed to span exon - intron junctions ) their from transcription start site in kb are indicated in gene structures shown above graphs .

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs.


Comparison of SARS - CoV - 2 SARS - CoV replication kinetics in Vero E6 . ( a ) Growth curve showing release of infectious viral progeny into medium of infected Vero E6 ( m . o . i . 3 ) , as determined by plaque ( n = 4 ; mean ± sd is presented ) . ( b ) . Comparison of SARS - CoV - 2 Australia / VIC01 / 2020 SARS - CoV Frankfurt - 1 plaque phenotype in Vero E6 .

O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_LINE I-CELL_LINE O

Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells.(a) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells (m.o.i. 3), as determined by plaque assay (n = 4; mean ± sd is presented). (b). Comparison of SARS-CoV-2 Australia/VIC01/2020 and SARS-CoV Frankfurt-1 plaque phenotype in Vero E6 cells.


Kinetics of SARS - CoV - 2 SARS - CoV synthesis in infected Vero E6 . ( a ) Hybridization of viral mRNAs isolated from SARS - CoV - 2 - SARS - CoV - infected Vero E6 , separated in an agarose gel probed with a radiolabelled oligonucleotide recognizing genome subgenomic mRNAs of both . Subsequently , gel was re - hybridized to a probe specific for 18S ribosomal , which was used as a loading control . ( b ) of relative abundance of each of SARS - CoV - 2 SARS - CoV transcripts . Phosphorimager was performed for bands of samples isolated at 12 , 14 24 h p . i . , which yielded essentially identical relative abundances . table shows average of these three measurements . SARS - CoV - 2 mRNA sizes were calculated on basis of position of leader transcription - regulatory sequences ( ACGAAC ) in viral genome ( Sawicki Sawicki 1995 , Xu , Hu et al . 2003 ) .

O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE I-CELL_LINE O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetics of SARS-CoV-2 and SARS-CoV RNA synthesis in infected Vero E6 cells.(a) Hybridization analysis of viral mRNAs isolated from SARS-CoV-2- and SARS-CoV -infected Vero E6 cells, separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses. Subsequently, the gel was re-hybridized to a probe specific for 18S ribosomal RNA, which was used as a loading control. (b) Analysis of the relative abundance of each of the SARS-CoV-2 and SARS-CoV transcripts. Phosphorimager quantification was performed for the bands of the samples isolated at 12, 14 and 24 h p.i., which yielded essentially identical relative abundances. The table shows the average of these three measurements. SARS-CoV-2 mRNA sizes were calculated on the basis of the position of the leader and body transcription-regulatory sequences (ACGAAC) in the viral genome (Sawicki and Sawicki 1995, Xu, Hu et al. 2003).


Cross - reactivity of antisera raised against SARS - CoV structural non - structural . Selected antisera previously raised against SARS - CoV nsps structural cross - react with corresponding SARS - CoV - 2 . SARS - CoV - 2 - infected Vero E6 ( m . o . i . of 0 . 3 ) were fixed at 12 or 24 h p . i . For immunofluorescence microscopy , were ( double ) labelled with ( a ) a rabbit antiserum recognising nsp4 a mouse mAb recognising dsRNA ; ( b ) anti - nsp4 rabbit serum a mouse mAb directed against N ; ( c - e ) rabbit antisera recognising against nsp3 , nsp13 M , respectively . was stained with Hoechst 33258 . Bar , 20 µm .

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE I-CELL_LINE 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-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins.Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double)labelled with (a) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c-e) rabbit antisera recognising against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33258. Bar, 20 µm.


to screen for compounds that inhibit SARS - CoV - 2 replication . Inhibition of SARS - CoV - 2 replication ( coloured bars ) was tested in Vero E6 by developing a CPE - reduction evaluating several previously identified inhibitors of SARS - CoV , which was included for comparison ( grey bars ) . For each compound a two - fold serial dilution series in low - micromolar range was tested ; ( a ) Remdesivir , ( b ) chloroquine , ( c ) Alisporivir ( d ) pegylated interferon alpha - 2 . viability was assayed using CellTiter 96 ® Aqueous One Solution proliferation ( MTS ) . Compound toxicity ( solid ) was evaluated in parallel using mock - infected , compound - treated . graphs show results of 3 independent experiments , each performed using quadruplicate samples ( mean ± SD are shown ) .

O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O 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-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assay to screen for compounds that inhibit SARS-CoV-2 replication.Inhibition of SARS-CoV-2 replication (coloured bars) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey bars). For each compound a two-fold serial dilution series in the low-micromolar range was tested; (a) Remdesivir, (b) chloroquine, (c) Alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96® Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of 3 independent experiments, each performed using quadruplicate samples (mean ± SD are shown).


A . Maximum projections across Z stacks of example from indicated mESCs stained for L1 Tf ( red ) combined with immunostaining for L1 ORF1p ( green ) nuclei stained with DAPI ( blue ) . grey square marks position of inset . arrow heads point to foci where L1 ORF1p co - localize . Scatter plot shows number of co - localized L1 Tf - ORF1 foci in cytoplasm per ( n = 275 WT , 304 Dicer _ KO1 , 311 Dicer _ KO2 ) . Red arrow heads point to relatively larger sized L1 RNP foci . Bar graphs are mean values of of with large L1 Tf - ORF1 foci co - localizing in cytoplasm ( n = 3 technical replicates ) .

O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O

A. Maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA (red) combined with immunostaining for L1 ORF1p (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. White arrow heads point to cytoplasmic foci where L1 RNA and ORF1p co-localize. Scatter plot shows the number of co-localized L1 Tf-ORF1 foci in the cytoplasm per cell (n=275 WT, 304 Dicer_KO1, 311 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 RNP foci. Bar graphs are mean values of percentage of cells with large L1 Tf-ORF1 foci co-localizing in the cytoplasm (n=3 technical replicates).


B . Maximum projections across Z stacks of example from indicated mESCs immunostained for L1 ORF1p ( red ) , MOV10 ( green ) nuclei stained with DAPI ( blue ) . grey square marks position of inset in zoomed image . arrow heads point to foci where L1 ORF1p MOV10 co - localize . Scatter plot shows number of co - localized ORF1 - MOV10 foci in cytoplasm per ( n = 293 WT , 295 Dicer _ KO1 , 295 Dicer _ KO2 ) . Red arrow heads point to relatively larger sized L1 ORF1 - MOV10 foci . Bar graphs are mean values of of with large ORF1 - MOV10 foci co - localizing in cytoplasm ( n = 3 technical replicates ) .

O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O

B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (red), MOV10 (green) and nuclei stained with DAPI (blue). The grey square marks position of inset in the zoomed image. White arrow heads point to cytoplasmic foci where L1 ORF1p and MOV10 co-localize. Scatter plot shows the number of co-localized ORF1-MOV10 foci in the cytoplasm per cell (n= 293 WT, 295 Dicer_KO1, 295 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 ORF1-MOV10 foci. Bar graphs are mean values of percentage of cells with large ORF1-MOV10 foci co-localizing in the cytoplasm (n=3 technical replicates).


A . WT Dicer _ KO mESCs were treated with 0 . 5mM Sodium Arsenite ( NaAsO2 ) for 20 minutes or left untreated prior to fixation with formaldehyde . Maximum projections across Z stacks of example from indicated mESCs immunostained for G3BP1 ( red ) MOV10 ( green ) with nuclei stained with DAPI ( blue ) .

O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O

A. WT and Dicer_KO mESCs were treated with 0.5mM Sodium Arsenite (NaAsO2) for 20 minutes or left untreated prior to fixation with formaldehyde. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 (red) and MOV10 (green) with nuclei stained with DAPI (blue).


B . Representative western blots showing low AGO2 in Dicer _ KO as compared to WT mESCs ( right side ) . LAMINB1 served as loading control . On left , maximum projections across Z stacks of example from indicated mESCs stained for L1 Tf FISH ( red ) combined with immunostaining for a resident of P - bodies , DDX6 ( green ) nuclei stained with DAPI ( blue ) . grey square marks position of inset . Yellow arrow heads point to foci where L1 DDX6 co - localize .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

B. Representative western blots showing low AGO2 protein levels in Dicer_KO as compared to WT mESCs (right side). LAMINB1 served as loading control. On the left, maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH (red) combined with immunostaining for a resident protein of P-bodies, DDX6 (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co-localize.


A . Representative western blots showing L1 ORF1p MOV10 in indicated lines . Immunoblot with antibody recognizing TUBULIN coomassie stained membranes depict loading , asterisk marks position of non - specific band in ORF1 immunoblot ( n = 3 technical replicates ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O

A. Representative western blots showing L1 ORF1p and MOV10 levels in the indicated cell lines. Immunoblot with antibody recognizing TUBULIN and coomassie stained membranes depict the loading, asterisk marks position of non-specific band in the ORF1 immunoblot (n=3 technical replicates).


B . Maximum projections across Z stacks of example from indicated mESCs immunostained for L1 ORF1p ( green ) combined with FISH for L1 Tf ( red ) nuclei stained with DAPI ( blue ) ( n = 3 technical replicates ) .

O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O

B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (green) combined with RNA FISH for L1 Tf RNA (red) and nuclei stained with DAPI (blue) (n=3 technical replicates).


C . Representative of BlastR colonies stained with crystal violet blue of indicated lines is shown on left . were transfected with either mutant reporter plasmid ( L1N21A ) or retrotransposition competent reporter ( L1WT ) as shown in scheme with timeline for experiment on top . Bar graphs on right depict average number of BlastR colonies ( n = 3 biological replicates ) .

O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

C. Representative images of BlastR colonies stained with crystal violet blue of indicated cell lines is shown on the left. Cells were transfected with either mutant reporter plasmid (L1N21A) or retrotransposition competent reporter (L1WT) as shown in the scheme with timeline for the experiment on the top. Bar graphs on the right depict the average number of BlastR colonies (n=3 biological replicates).


A . Scheme of relative luminescence from luciferase assays validating post - transcriptional regulation of MOV10 by miRNAs in HEK293T ( n = 4 biological replicates ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O

A. Scheme and quantification of relative luminescence from luciferase assays validating post-transcriptional regulation of MOV10 by miRNAs in HEK293T cells (n=4 biological replicates).


B . Scheme of relative luminescence from luciferase assays in Hek293T to confirm specificity of post - transcriptional regulation of MOV10 by miRNAs with either wild - type ( WT ) or Mutant ( MUT ) reporters where MicroRNA Response Element ( MRE ) for depicted miRNAs in 3 ' UTR of MOV10 were mutated ( n = 3 biological replicates ) .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O 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-GENEPROD O O O O O O O O O O

B. Scheme and quantification of relative luminescence from luciferase assays in Hek293T cells to confirm specificity of post-transcriptional regulation of MOV10 by miRNAs with either wild-type (WT) or Mutant (MUT) reporters where the MicroRNA Response Element (MRE) for the depicted miRNAs in the 3'UTR of MOV10 were mutated (n=3 biological replicates).


C . Scheme representative for western blot corresponding to assess miRNA mediated regulation on MOV10 expression in mESCs ( n = 3 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O

C. Scheme and representative images for western blot analysis and corresponding quantification to assess miRNA mediated regulation on MOV10 expression in mESCs (n=3 biological replicates).


D . Scheme representative from colony forming assays for plasmid retrotransposition where miRNA mediated downregulation of MOV10 in Drosha _ KO mESCs is accompanied by an increase in L1 retrotransposition ( n = 3 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O

D. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis where miRNA mediated downregulation of MOV10 in Drosha_KO mESCs is accompanied by an increase in L1 retrotransposition (n=3 biological replicates).


A . Scheme of experiment representative along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression . are maximum projections across Z stacks from indicated mESCs stained for L1 ORF1p ( red ) combined with immunostaining for HA ( green ) to detect ectopically expressed MOV10 tagged with HA , nuclei stained with DAPI ( blue ) . arrow heads point to foci where L1 ORF1p HA - MOV10 co - localize . Data collected from 289 Ctrl , 275 L1UP Cl1 296 L1UP Cl2 mESCs ( n = 3 biological replicates ) .

O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

A. Scheme of experiment and representative images along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression. Images are maximum intensity projections across Z stacks from indicated mESCs stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10 tagged with HA, and nuclei stained with DAPI (blue). White arrow heads point to cytoplasmic foci where L1 ORF1p and HA-MOV10 co-localize. Data collected from 289 Ctrl, 275 L1UP Cl1 and 296 L1UP Cl2 mESCs (n=3 biological replicates).


B . Scheme representative from colony forming assays for plasmid retrotransposition in Ctrl L1UP mESCs comparing rate of retrotransposition upon transfection with Empty Vector ( EV ) or ectopic expression of HA - MOV10 ( n = 3 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

B. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis in Ctrl and L1UP mESCs comparing rate of retrotransposition upon transfection with Empty Vector (EV) or ectopic expression of HA-MOV10 (n=3 biological replicates).


A . Scheme of experiment representative showing RNP aggregate formation upon ectopic expression of full L1 HA - MOV10 in WT mESCs . are maximum projections across Z stacks stained for L1 ORF1p ( red ) combined with immunostaining for HA ( green ) to detect ectopically expressed MOV10 , nuclei stained with DAPI ( blue ) . bottom panel shows where accumulation of ORF1 - MOV10 was observed in relatively large foci compared to where co - localization of two was mostly in relatively small foci ( top panel ) , 68 - 119 transfected were analyzed per experiment ( n = 3 biological replicates ) .

O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of full length L1 and HA-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10, and nuclei stained with DAPI (blue). The bottom panel shows cells where accumulation of ORF1-MOV10 was observed in relatively large foci compared to cells where co-localization of the two proteins was mostly in relatively small foci (top panel), 68-119 transfected cells were analyzed per experiment (n=3 biological replicates).


B . Scheme of experiment representative showing RNP aggregate formation upon ectopic expression of HA - ORF1 or HA - ORF1 in conjunction with T7 - MOV10 in WT mESCs . are maximum projections across Z stacks stained for HA ( green ) T7 ( red ) to detect ectopically expressed ORF1p MOV10 respectively , nuclei stained with DAPI ( blue ) . 100 - 147 transfected were analyzed per experiment ( n = 3 biological replicates ) .

O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

B. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of HA-ORF1 or HA-ORF1 in conjunction with T7-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for HA (green) and T7 (red) to detect ectopically expressed ORF1p and MOV10 respectively, and nuclei stained with DAPI (blue). 100-147 transfected cells were analyzed per experiment (n=3 biological replicates).


Heatmaps of AIB1 - YAP co - activated , YAP activated , AIB1 - YAP corepressed , YAP repressed genes shown in panel ( A ) . Red blue indicate high low , respectively . - sequencing was performed in triplicate statistics in Panel ( A - B ) were calculated by EdgeR with Benjamini - Hochberg multiple correction in R .

O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

Heatmaps of AIB1-YAP co-activated, YAP activated, AIB1-YAP corepressed, and YAP repressed genes shown in panel (A). Red and blue indicate high and low levels, respectively. RNA-sequencing was performed in triplicate and statistics in Panel (A-B) were calculated by EdgeR with Benjamini-Hochberg multiple test correction in R.


Select genes are regulated by YAP AIB1 , others are YAP regulated independently of AIB1 . Endogenous gene expression changes assessed by RT - qPCR .

O O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

Select genes are regulated by YAP and AIB1, others are YAP regulated independently of AIB1. Endogenous gene expression changes assessed by RT-qPCR.


Significant genomic co - occupancy between AIB1 TEAD in normal breast breast cancer . Overlap of AIB1 TEAD4 ChIP - seq peaks in MCF10A MCF7 lines . Select peaks from MCF10A ChIP - seq shown . ChIP - sequencing performed in triplicate .

O O O O O O B-GENEPROD I-GENEPROD O O B-TISSUE B-DISEASE I-DISEASE O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

Significant genomic co-occupancy between AIB1 and TEAD in normal breast and breast cancer cells. Overlap of AIB1 and TEAD4 ChIP-seq peaks in MCF10A and MCF7 cell lines. Select peaks from MCF10A ChIP-seq shown. ChIP-sequencing performed in triplicate.


TEAD binding motifs are enriched within AIB1 occupied sites across genome . HOMER de novo motif of AIB1 ChIP - seq peaks in MCF10A MCF7 .

B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE O

TEAD binding motifs are enriched within AIB1 occupied sites across the genome. HOMER de novo motif analysis of AIB1 ChIP-seq peaks in MCF10A and MCF7 cells.


AIB1 modulates TEAD transcriptional activity in a YAP dependent manner . HEK293T were transfected with CTGF - promoter reporter expression vectors of indicated .

B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O

AIB1 modulates TEAD transcriptional activity in a YAP dependent manner. HEK293T cells were transfected with CTGF-promoter reporter and expression vectors of indicated proteins.


AIB1 is engaged with TEAD on co - activated target promoters in normal . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A . of precipitated assessed by qPCR .

B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY O

AIB1 is engaged with TEAD on co-activated target promoters in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.


AIB1 is engaged with TEAD on co - activated target promoters in early stage breast cancer . Sequential ChIP - reChIP performed with indicated antibodies in MCFDCIS . of precipitated assessed by qPCR .

B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-DISEASE I-DISEASE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY O

AIB1 is engaged with TEAD on co-activated target promoters in early stage breast cancer cells. Sequential ChIP-reChIP performed with indicated antibodies in MCFDCIS cells. Levels of precipitated chromatin assessed by qPCR.


ANCO1 is recruited to YAP - TEAD by AIB1 . Positive Proximity Ligation ( PLA ) signal ( foci , falsely colored 594nm signal ) shown between ANCO1 - TEAD , ANCO1 - YAP , AIB1 - TEAD , AIB1 - YAP , in control are lost in AIB1 knockdown . Representative of Proximity Ligation ( PLA ) between indicated in MCF10A shGFP ( control ) shAIB1 lines using antibodies ( species ) as indicated ; Scale bars shown indicate 25μm .

B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O

ANCO1 is recruited to YAP-TEAD by AIB1. Positive Proximity Ligation Assay (PLA) signal (white foci, falsely colored 594nm signal) shown between ANCO1-TEAD, ANCO1-YAP, AIB1-TEAD, and AIB1-YAP, in control cells are lost in AIB1 knockdown. Representative images of Proximity Ligation Assay (PLA) between indicated proteins in MCF10A shGFP (control) and shAIB1 cell lines using antibodies (species) as indicated; Scale bars shown indicate 25μm.


YAP overexpression represses , AIB1 knockdown dysregulates , ANCO1 knockdown amplifies genes at 1q21 . 3 locus in MCF10A MCFDCIS . Gene set enrichment of - seq data cDNA array data generated from MCF10A lines as indicated .

B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-CELL_LINE I-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O

YAP overexpression represses, AIB1 knockdown dysregulates, and ANCO1 knockdown amplifies genes at the 1q21.3 locus in MCF10A and MCFDCIS cells. Gene set enrichment analysis of RNA-seq data and cDNA array data generated from MCF10A cell lines as indicated.


YAP represses 1q21 . 3 localized genes . of endogenous S100 SPPR genes in MCF10A lines following overexpression of YAP / S94A ; were assessed by RT - qPCR .

B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

YAP represses 1q21.3 localized genes. Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP/S94A; levels were assessed by RT-qPCR.


ANCO1 knockdown increases expression of S100 SPPR genes in MCF10A MCFDCIS ; were assessed by RT - qPCR .

B-GENEPROD O O B-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells; levels were assessed by RT-qPCR.


YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown . of endogenous 1q21 . 3 genes were assessed in YAP or S94A - expressing , control or shANCO1 MCF10A by RT - qPCR .

B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-CELL_LINE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown. Levels of endogenous 1q21.3 genes were assessed in YAP or S94A-expressing, control or shANCO1 MCF10A cells by RT-qPCR.


TEAD is engaged at annotated enhancer regions within 1q21 . 3 . of TEAD4 ChIP - seq in MDA - MB - 231 ( GSE66081 ) , MCF7 , A549 ( GSE32465 ) at 1q21 . 3 with enhancers scored by GeneHancer . Enhancer regions of interest indicated between dotted lines .

B-GENEPROD O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O

TEAD is engaged at annotated enhancer regions within 1q21.3. Analysis of TEAD4 ChIP-seq in MDA-MB-231(GSE66081), MCF7, and A549 Cells (GSE32465) at 1q21.3 with enhancers scored by GeneHancer. Enhancer regions of interest indicated between dotted lines.


TEAD , YAP , AIB1 , ANCO1 are engaged at annotated enhancer regions within 1q21 . 3 . Immunoprecipitation ( ChIP ) performed with indicated antibodies in MCF10A .

B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-CELL_LINE O

TEAD, YAP, AIB1, and ANCO1 are engaged at annotated enhancer regions within 1q21.3. Chromatin Immunoprecipitation (ChIP) performed with indicated antibodies in MCF10A cells.


AIB1 is engaged with TEAD on superenhancer sites at 1q21 . 3 in normal . Sequential ChIP - reChIP performed with indicated antibodies in MCF10A . of precipitated assessed by qPCR .

B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY O

AIB1 is engaged with TEAD on superenhancer sites at 1q21.3 in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.


ANCO1 is recruited to superenhancers at 1q21 . 3 by AIB1 is significantly reduced at 1q21 . 3 following AIB1 knockdown . ChIP performed with ANCO1 antibodies in control or shAIB1 . of precipitated assessed by qPCR .

B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O

ANCO1 is recruited to superenhancers at 1q21.3 by AIB1 and is significantly reduced at 1q21.3 following AIB1 knockdown. ChIP performed with ANCO1 antibodies in control or shAIB1 cells. Levels of precipitated chromatin assessed by qPCR.


YAP driven aberrant sphere formation of MCF10A lines is ablated by AIB1 knockdown , but further enhanced by ANCO1 knockdown . Indicated lines were plated on Matrigel allowed to form mammospheres for 7 days . 10x 40x magnifications shown , Scale bar = 200μm .

B-GENEPROD O O B-SUBCELLULAR O O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown, but further enhanced by ANCO1 knockdown. Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days. 10x and 40x magnifications shown, Scale bar = 200μm.


of aberrant spheres per field displayed in panel ( A ) , n = number of spheres .

O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O

Quantification of aberrant spheres per field displayed in panel (A), n= number of spheres.


of ( arbitrary units ) of aberrant spheres per field displayed in panel ( A ) , relative to control YAP S94A shGFP spheres . n = number of spheres .

O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O

Quantification of size (arbitrary units) of aberrant spheres per field displayed in panel (A), relative to control YAP S94A shGFP spheres. n= number of spheres.


MCFDCIS xenograft tumors have increased expression of AIB1 - YAP , EMT , 1q21 . 3 genes during invasive transition . Schematic of experiment shown . Gene set enrichment of - seq data generated from MCFDCIS xenografts at indicated time points .

B-CELL_LINE B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE B-EXP_ASSAY O O O O O

MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.


Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions . Representative of H & E , p63 , ANCO1 stained MCFDCIS xenografts as DCIS invasive lesions . Yellow circles indicate DCIS lesions black arrows indicate invasive lesions . 20x magnification ; Scale bar = 100μm . of ANCO1 positivity in DCIS Invasive lesions shown in panel ( E ) .

O O O O O B-GENEPROD O O B-DISEASE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY B-CELL_LINE B-EXP_ASSAY O B-DISEASE O O O O O O B-DISEASE O O O O O O O O O O O O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O

Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions. Representative images of H&E, p63, and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions. Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions. 20x magnification; Scale bar = 100μm. Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel (E).


ANCO1 decrease in breast cancer . ANCO1 mRNA expression from normal tissue ( GTEX ) ( TCGA ) for breast samples .

B-GENEPROD O O B-DISEASE I-DISEASE O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE O O

ANCO1 levels decrease in breast cancer. ANCO1 mRNA expression from normal tissue (GTEX) and Tumor (TCGA) for breast samples.


Representative of ANCO1 in normal human breast DCIS lesions . DCIS lesions can be stratified to either ANCO1 negative ( center panel ) , ANCO1 positive ( right panel ) . Scale bar = 50μm .

O O B-GENEPROD O O B-ORGANISM B-TISSUE B-DISEASE O O B-DISEASE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O

Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions. DCIS lesions can be stratified to either ANCO1 negative (center panel), ANCO1 positive (right panel). Scale bar = 50μm.


High ANCO1 are a good prognostic indicator in Basal Breast Cancer in . KM plot showing progression free survival in with stratified ANCO1 expression . n = 360 .

O B-GENEPROD O O O O O O O B-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O

High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients. KM plot showing progression free survival in patients with stratified ANCO1 expression. n= 360 patients.


breast samples with reduced ANCO1 have high expression of 1q21 . 3 genes , without altered AIB1 - YAP co - activated gene expression . Dot plots showing TCGA Pan - Cancer data accessed via Xenabroswer . were stratified to high or low expression of ANCO1 by median expression .

B-DISEASE O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY O

Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21.3 genes, without altered AIB1-YAP co-activated gene expression. Dot plots showing TCGA Pan-Cancer data accessed via Xenabroswer. Patients were stratified to high or low expression of ANCO1 by median expression.


B : Log2 gene expression fold changes of significantly induced genes ( FDR = 1 % ) across different treatment scenarios . Gene induction of immediate , delayed late responding genes is sustained upon constant activation ( ON scenario ) transient upon two - hour activation ( ON / OFF scenario ) . Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes . Genes were ranked by their model - derived response time .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

B: Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two-hour pulse activation (ON/OFF scenario). Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes. Genes were ranked by their model-derived response time.


B : pERK2 log2 fold change upon sustained activation ( left ) deduced input function ( right ) used for model fitting . Average pERK2 log2 fold change upon 4OHT treatment equals 100 % signalling amplitude .

O O O O O 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-SMALL_MOLECULE O O O O O O O

B: pERK2 log2 fold change upon sustained activation (left) and deduced input function (right) used for model fitting. Average pERK2 log2 fold change upon 4OHT treatment equals 100% signalling amplitude.


C : Measured gene expression kinetics resulting maximum likelihood fit of gene expression model of significantly induced response genes ( FDR = 1 % ) . Gene expression is shown as of response amplitude . Mean error is calculated as mean of absolute residuals serving as a goodness of fit measure .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

C: Measured gene expression kinetics and the resulting maximum likelihood fit of the gene expression model of all significantly induced primary response genes (FDR = 1%). Gene expression is shown as percentage of response amplitude. Mean error is calculated as the mean of absolute residuals serving as a goodness of fit measure.