F Total cellular protein levels of PD - L1 ( top ) and GAPDH ( bottom ) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN - γ stimulation . Numbers indicate relative band intensities of PD - L1 protein normalized to GAPDH .

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F Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN-γ stimulation. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.


B Relative cell numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( black ) or polyclonal MLLT6 knockout ( grey ) cells treated with varying amounts of EpCAM - CD3 BiTE and CTLs ( Student ' s t - test ; * * p < 0 . 01 ; degrees of freedom ( df ) = 4 ) .

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B Relative cell numbers (± SD; n=3; biological replicates) of U2OS mock (black) or polyclonal MLLT6 knockout (grey) cells treated with varying amounts of EpCAM-CD3 BiTE and CTLs (Student's t-test; **p < 0.01; degrees of freedom (df)=4).


C Kaplan Meier survival plot ( left ) showing changes in survival ( percent ) of mock ( grey ) and MLLT6 knockout ( red ) cells treated with CTLs and EpCAM - CD3 BiTE over a course of 9 . 5 hours . Representative images ( right ) of time - lapse fluorescence microscopy following U2OS mock ( eGFP tagged , green ) and MLLT6 knockout ( mCherry tagged , red ) cells immediately ( top left and bottom left ) after adding CTLs and EpCAM - CD3 BiTE and 9 . 5 hours later ( top right and bottom right ) ( scale bars = 100 μm ) .

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C Kaplan Meier survival plot (left) showing changes in survival (percent) of mock (grey) and MLLT6 knockout (red) cells treated with CTLs and EpCAM-CD3 BiTE over a course of 9.5 hours. Representative images (right) of time-lapse fluorescence microscopy following U2OS mock (eGFP tagged, green) and MLLT6 knockout (mCherry tagged, red) cells immediately (top left and bottom left) after adding CTLs and EpCAM-CD3 BiTE and 9.5 hours later (top right and bottom right) (scale bars = 100 μm).


D Relative cell numbers ( left ) ( ± SD ; n = 3 ; biological replicates ) of mock ( black ) , PD - L1 ( dark grey ) or MLLT6 ( light grey ) knockout cells treated with BiTE or bi - specific antibodies and CTLs as indicated ( Student ' s t - test ; * * p < 0 . 01 ; df = 4 ) . Representative images ( right ) from bright field microscopy of polyclonal U2OS mock ( top ) , PD - L1 ( middle ) or MLLT6 ( bottom ) knockout cells treated without or with EpCAM - CD3 BiTE or bispecific antibodies .

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D Relative cell numbers (left) (± SD; n=3; biological replicates) of mock (black), PD-L1 (dark grey) or MLLT6 (light grey) knockout cells treated with BiTE or bi-specific antibodies and CTLs as indicated (Student's t-test; **p < 0.01; df=4). Representative images (right) from bright field microscopy of polyclonal U2OS mock (top), PD-L1 (middle) or MLLT6 (bottom) knockout cells treated without or with EpCAM-CD3 BiTE or bispecific antibodies.


E Relative normalized cell numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( left ) , MLLT6 ( middle ) or PD - L1 ( right ) knockout cells treated with EpCAM - CD3 BiTE and CTLs in the presence ( grey ) or absence ( black ) of IFN - γ ( Student ' s t - test ; * * p < 0 . 01 ; df = 4 ) .

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E Relative normalized cell numbers (± SD; n=3; biological replicates) of U2OS mock (left), MLLT6 (middle) or PD-L1 (right) knockout cells treated with EpCAM-CD3 BiTE and CTLs in the presence (grey) or absence (black) of IFN-γ (Student's t-test; **p < 0.01; df=4 ).


A Scatter plots of pairwise comparisons of transcript expression in U2OS cells . Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN - γ as indicated . Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance ( log10 of p - value , n = 3 ) with candidates nominated for further validation ( red ) .

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A Scatter plots of pairwise comparisons of transcript expression in U2OS cells. Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN-γ as indicated. Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance (log10 of p-value, n=3) with candidates nominated for further validation (red).


B Correlation plots describing log2 fold change of transcripts in RNA sequencing ( x axis ) and Nanostring ( y axis ) measurements in mock cells + / - IFN - γ ( left ) and MLLT6 knockout cells with IFN - γ ( right ) . Transcripts previously described to have a role in tumor immunity ( red ) and PD - L1 ( blue ) are represented on the plots as indicated .

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B Correlation plots describing log2 fold change of transcripts in RNA sequencing (x axis) and Nanostring (y axis) measurements in mock cells +/- IFN-γ (left) and MLLT6 knockout cells with IFN-γ (right). Transcripts previously described to have a role in tumor immunity (red) and PD-L1 (blue) are represented on the plots as indicated.


C Changes ( log2 fold ) in transcript expression ( left ) of select immune - related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN - γ and total protein levels measured by immunoblotting ( right ) . Numbers indicate band intensities normalized to GAPDH .

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C Changes (log2 fold) in transcript expression (left) of select immune-related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN-γ and total protein levels measured by immunoblotting (right). Numbers indicate band intensities normalized to GAPDH.


A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN - γ . Numbers indicate band intensities normalized to GAPDH .

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A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH.


B Immunoblot showing total protein levels of STAT1 isoforms ( α and β ) , IDO1 , GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase ( Rluc , negative control ) , total STAT1 , STAT1α and STAT1β treated with and without IFN - γ .

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B Immunoblot showing total protein levels of STAT1 isoforms (α and β), IDO1, GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase (Rluc, negative control), total STAT1, STAT1α and STAT1β treated with and without IFN-γ.


C . Representative images of calyx synapses from littermate control and Nrxn123 TKO mice . Brainstem sections were labeled with antibodies to vGluT1 ( red ) and Syt2 ( green ) . Scale bar , 10 μm . D . Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity ( normalized to control ) .

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C. Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm. D. Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control).


E - H . Representative traces of spontaneous EPSCs ( sEPSCs ) ( E ) , and summary graphs of the sEPSC frequency ( F ) , amplitude ( G ) , and kinetics ( H ) recorded from littermate control and Nrxn123 TKO mice .

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E-H. Representative traces of spontaneous EPSCs (sEPSCs)(E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.


A . Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s , recorded in a standard bath solution containing 2 mM Ca2 + . B . Pan - neurexin deletion suppresses synaptic transmission . Summary graphs show the amplitude ( left ) and charge transfer ( right ) of the first EPSC recorded in response to the paired stimuli . C . Pan - neurexin deletion more than doubles the paired - pulse ratio ( PPR ) , suggesting a large decrease in release probability . D . Pan - neurexin deletion decelerates the EPSC time course . Summary graphs show the rise time ( left ) and decay time constants ( right ) of the first EPSC in response to the paired stimuli .

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A. Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca2+. B. Pan-neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli. C. Pan-neurexin deletion more than doubles the paired-pulse ratio (PPR), suggesting a large decrease in release probability. D. Pan-neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli.


E - F . Pan - neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high - frequency stimulus train ( 100 Hz for 0 . 5 sec ) . Left , representative ESPC traces ; right , cumulative summary plot of the EPSC amplitudes during the train ( dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back - extrapolation to zero time , which is used to correct for vesicle replenishment during the train ) .

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E-F. Pan-neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high-frequency stimulus train (100 Hz for 0.5 sec). Left, representative ESPC traces; right, cumulative summary plot of the EPSC amplitudes during the train (dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back-extrapolation to zero time, which is used to correct for vesicle replenishment during the train).


G . Pan - neurexin deletion does not decrease the readily - releasable pool of vesicles , but lowers the initial release probability during a high - frequency stimulus train . Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily - releasable pool size ( left ) , and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability ( right ) .

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G. Pan-neurexin deletion does not decrease the readily-releasable pool of vesicles, but lowers the initial release probability during a high-frequency stimulus train. Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily-releasable pool size (left), and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability (right).


A - D . Same as Fig 2A - D , except that ACSF contained only 1 mM Ca2 + , which is close to the estimated physiological concentration of extracellular Ca2 + ( Forsberg et al . , 2019 ) .

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A-D. Same as Fig 2A-D, except that ACSF contained only 1 mM Ca2+, which is close to the estimated physiological concentration of extracellular Ca2+ (Forsberg et al., 2019).


E - H . Same as Fig 2A - D , except that the slice were incubated with 0 . 2 mM EGTA - AM for 30 mins before recording , which introduces EGTA into the presynaptic terminals .

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E-H. Same as Fig 2A-D, except that the slice were incubated with 0.2 mM EGTA-AM for 30 mins before recording, which introduces EGTA into the presynaptic terminals.


A . Example traces of presynaptic Ca2 + - currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV . B . Summary graphs of the I - V relationship for the peak Ca2 + - current amplitude ( left ) and of the peak Ca2 + - current density ( right ) .

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A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV. B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right).


C . Example traces ( left ) and summary graphs ( right ) of the Ca2 + - current activation and deactivation kinetics . Currents were induced by a 50 ms step depolarization from - 80 mV to + 10 mV .

C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV.


D . Example traces of Ca2 + - currents evoked by an action potential - equivalent depolarization ( APe , from - 80 mV to + 17 mV for 1 ms ; left ) , and summary graph of the APe - evoked Ca2 + - current peak amplitude and charge ( right ) .

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D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right).


E . Example traces of presynaptic Ca2 + - currents induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV before and after perfusion of 200 nM agatoxin ( Agtx ) , a selective P / Q - type Ca2 + channel blocker .

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E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker.


F . Summary graph of the relative contribution of P / Q - type Ca2 + channels to the total presynaptic Ca2 + currents , as quantified by the relative reduction in Ca2 + currents evoked by step depolarization to + 10 mV , in control and Nrxn123 TKO synapses .

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F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.


B . Example traces of the presynaptic capacitance and Ca2 + - currents at the calyx terminals induced by a 20 ms step depolarization from − 80 mV to + 10 mV with an extracellular solution containing 2 mM Ca2 + , and an internal pipette solution containing a standard 0 . 05 mM BAPTA . C . Summary graphs of the depolarization - induced capacitance jump that monitors synaptic vesicle exocytosis ( left ) , and the peak Ca2 + - current density ( right ) . D , E . Same as ( B , C ) , except that 0 . 5 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution . F , G . Same as ( B , C ) , except that 1 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution . H , I . Same as ( B , C ) , except that 10 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution .

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B. Example traces of the presynaptic capacitance and Ca2+-currents at the calyx terminals induced by a 20 ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca2+, and an internal pipette solution containing a standard 0.05 mM BAPTA. C. Summary graphs of the depolarization-induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca2+-current density (right). D, E. Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. F, G. Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. H, I. Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.


J , K . Summary plots of the relationship between the total capacitance jump ( J ) or between the Ca2 + - current density ( K ) and the concentration of EGTA in the patch pipette .

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J, K. Summary plots of the relationship between the total capacitance jump (J) or between the Ca2+-current density (K) and the concentration of EGTA in the patch pipette.


A . Representative traces of Ca2 + - currents and 4 - AP insensitive K + - currents evoked by step depolarizations ( from − 50 mV to + 10 mV in 10 mV increments ) , recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12 - P14 . B . Same as ( A ) but after addition of 200 nM iberiotoxin ( IbTx ) by perfusion . C . Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in ( B ) from currents shown in ( A ) . D . Summary graphs of the capacitance , Ca2 + - current density , and BK - current density .

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A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14. B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion. C. Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in (B) from currents shown in (A). D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density.


A . Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 ( green ) , CaV2 . 1 - type Ca2 + - channels ( red ) , and the active zone protein Bassoon ( purple ) . Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12 - 14 . Scale bar , 10 μm .

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A. Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 (green), CaV2.1-type Ca2+-channels (red), and the active zone protein Bassoon (purple). Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12-14. Scale bar, 10 μm.


B . Summary graphs of the vGluT1 , CaV2 . 1 , and Bassoon immunostaining intensity ( normalized to control ; CaV2 . 1 and Bassoon signals were quantified over the vGluT1 - positive area to measure active zone - localized proteins ) .

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B. Summary graphs of the vGluT1, CaV2.1, and Bassoon immunostaining intensity (normalized to control; CaV2.1 and Bassoon signals were quantified over the vGluT1-positive area to measure active zone-localized proteins).


C . Summary graphs of the diameter and total size of the vGluT1 - positive presynaptic area of the calyx of Held .

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C. Summary graphs of the diameter and total size of the vGluT1-positive presynaptic area of the calyx of Held.


100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect the total number of Ki - 67 + cells ( A ) ( S - phase ) in the hair matrix . Unpaired t - test performed using N of 9 - 12 HFs from 3 patients .

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100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of Ki-67+ cells (A) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect the total number of EdU + cells ( B ) ( S - phase ) in the hair matrix . Unpaired t - test performed using N of 9 - 12 HFs from 3 patients .

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100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


C ) 100 nM paclitaxel treatment ( 24h ) significantly ( p = < 0 . 0001 [ * * * * ] ) increased the number of mitotic phospho - histone H3 ( pH3 ) + cells in the hair matrix . Welch ' s t - test performed using N of 9 HFs from 3 patients .

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C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients.


D ) 100 nM docetaxel treatment ( 24h ) significantly ( p = 0 . 0004 [ * * * ] ) increases the number of pH3 + cells in the hair matrix . Unpaired t - test performed using N of 8 - 9 HFs from 3 patients .

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D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients.


E ) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on ( i ) Ki - 67 expression [ paclitaxel ] ; ( ii ) EdU incorporation and pH3 immunoreactivity [ paclitaxel ] ; ( iii ) pH3 immunoreactivity [ docetaxel ] . 20 µm scale . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


The presence of micronucleated cells in the hair matrix in paclitaxel ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U test performed using N of 12 - 13 HFs ( paclitaxel ) from 3 patients .

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The presence of micronucleated cells in the hair matrix in paclitaxel (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 12-13 HFs (paclitaxel) from 3 patients.


The presence of micronucleated cells in the hair matrix in docetaxel treated ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U test performed using N of 8 HFs ( docetaxel ) from 3 patients .

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The presence of micronucleated cells in the hair matrix in docetaxel treated (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 8 HFs (docetaxel) from 3 patients.


C ) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix ( lined ) and dermal papilla in untreated ( vehicle ) human HFs . 20 µm scale .

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C) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20 µm scale.


D ) Paclitaxel treatment ( 100 nM , 24h ) induces the formation of micronucleated bodies , as visualised by Hoechst 33342 staining ( arrows ) , localising to the proliferative region of the hair matrix . i - 20 µm scale ; ii - 10 µm scale .

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D) Paclitaxel treatment (100 nM, 24h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i - 20 µm scale; ii - 10 µm scale.


E ) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies ( arrows ) . 10 µm scale . Error bars are standard error .

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E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.


A ) Nascent RNA synthesis , as detected by ethynyl uridine ( EU ) incorporation , is blocked within clusters of nuclei in the hair matrix ( arrows ) following paclitaxel treatment ( 100 nM , 24h ) . 20 µm scale .

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A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale.


B ) Quantitative analysis highlights a significant ( p = < 0 . 0001 [ * * * * ] ) decrease in the number of EU + nuclei following 24h paclitaxel treatment . Welch ' s t - test performed using N of 11 - 12 hair follicles ( HFs ) from 3 patients .

B) Quantitative analysis highlights a significant (p =<0.0001 [****]) decrease in the number of EU+ nuclei following 24h paclitaxel treatment. Welch's t-test performed using N of 11-12 hair follicles (HFs) from 3 patients.


C ) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3 + cell population that accumulates in response to paclitaxel treatment 10 µm scale .

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C) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment 10 µm scale.


D ) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment . 20 µm scale .

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D) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment. 20 µm scale.


E ) 100 nM paclitaxel treatment significantly ( p = 0 . 0016 [ exact ] [ * * ] ) increases the number of cleaved caspase 3 + cells in the hair matrix after 24h . Mann Whitney U test performed using N of 16 - 18 HFs from 5 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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E) 100 nM paclitaxel treatment significantly (p = 0.0016 [exact] [**]) increases the number of cleaved caspase 3+ cells in the hair matrix after 24h. Mann Whitney U test performed using N of 16-18 HFs from 5 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


Ai ) Paclitaxel treatment promotes mitotic arrest ( orange arrows / pH3 + cells ) and apoptosis ( white arrows / caspase 3 + cells ) , within the K15 + bulge and K15 + proximal bulb outer root sheath ( pbORS ) stem / progenitor cell compartments of the human hair follicle ( HF ) . 20 µm scale . Aii ) High magnification montage demonstrating mitotic arrest ( orange arrow ) and apoptosis / caspase 3 positivity ( white arrow ) within the K15 + bulge . 10 µm scale . pbORS - proximal bulb outer root sheath .

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Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale. pbORS - proximal bulb outer root sheath.


B ) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15 + bulge following extended paclitaxel organ culture experiments 50 µm scale . EC - extended cultures .

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B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments 50 µm scale. EC - extended cultures.


C ) Graph showing significantly ( p = 0 . 029 [ * * ] ) increased K15 / caspase 3 double + cells within the bulge following extended paclitaxel HF organ cultures . Welch ' s t - test performed using N of 8 - 9 HFs from 3 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


D ) K15 + cells of the human HF bulge express Ki - 67 during extended organ culture experiments . Paclitaxel treatment did not significantly affect the number of bulge K15 / Ki - 67 double + cells . Unpaired t - test performed using N of 8 - 9 HFs from 3 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


E ) Representative double immunofluorescence images of elevated γH2A . X immunoreactivity within the K15 + bulge following extended paclitaxel organ culture experiments ( see methods ) . 50 µm scale . EC - extended cultures .

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E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.


F ) γH2A . X analysis showing a significant ( p = 0 . 065 [ * * ] ) increase in the number of cells with DNA double - strand breaks in the K15 + bulge following extended organ culture experiments . Mann Whitney U test performed using N of 5 - 6 HFs from 2 patients . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


B ) Palbociclib - only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki - 67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki - 67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6 - 9 HFs per condition from 3 patients . Ordinary one - way ANOVA with multiple comparisons performed . Adjusted p values = 0 . 0001 [ * * * ] and 0 . 0001 [ * * * * ] respectively . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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B) Palbociclib-only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki-67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki-67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6-9 HFs per condition from 3 patients. Ordinary one-way ANOVA with multiple comparisons performed. Adjusted p values = 0.0001[***] and 0.0001 [****] respectively. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


C ) Data confirming that paclitaxel treatment significantly increases ( adjusted p value = 0 . 0001 [ * * * ] ) the number of pH3 + cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre - and co - incubated with palbociclib . Analysis performed using N of 6 - 9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


D ) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment ( adjusted p value = 0 . 0056 [ * * ] ) . Paclitaxel treated hair follicles pre - and co - incubated with palbociclib do not show enhanced cleaved caspase 3 expression , contrasting with paclitaxel - only treatment . Ordinary one - way ANOVA with multiple comparisons performed using N of 4 - 5 HFs per condition . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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D) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (adjusted p value = 0.0056 [**]). Paclitaxel treated hair follicles pre- and co-incubated with palbociclib do not show enhanced cleaved caspase 3 expression, contrasting with paclitaxel-only treatment. Ordinary one-way ANOVA with multiple comparisons performed using N of 4-5 HFs per condition. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


E ) Immunofluorescence data representing how palbociclib and / or paclitaxel affect ( i ) Ki - 67 expression , ( ii ) pH3 immunoreactivity and ( iii ) cleaved caspase 3 expression . 20 µm scale .

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E) Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki-67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase 3 expression. 20 µm scale.


B ) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle . DNA synthesis in the hair matrix resumed in the palbociclib - only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 13 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard error .

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B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


C ) A trending increase in the number of cleaved caspase 3 + cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout , but this was not significant , whereas paclitaxel - only treatment significantly increased the number of cleaved caspase 3 + cells in the hair matrix ( adjusted p value = 0 . 0003 [ * * * ] ) . Palbociclib - only treatment did not increase the number of cleaved caspase 3 + cells in the hair matrix following drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard error .

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C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


D ) HFs treated with paclitaxel alone showed a significant ( adjusted p value = 0 . 0316 [ * ] ) increase in the number of pH3 + cells in the matrix after washout . A significant ( adjusted p value = 0 . 0278 [ * ] ) increase in the number pH3 + cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout . Palbociclib treatment alone showed no significant change in the number of pH3 + hair matrix keratinocytes after drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard error .

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D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


E ) Representative fluorescence images of EdU and pH3 / cleaved caspase 3 staining in paclitaxel - only and palbociclib plus paclitaxel dual treated HFs following drug washout . Where DNA synthesis has resumed following reversal of the G1 arrest , paclitaxel cytotoxicity is emergent . HS - hair shaft ; " wo " - washout . 20 µm scale .

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E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. HS - hair shaft; "wo" - washout. 20 µm scale.


B ) Paclitaxel treatment did not affect the number of EdU + cells within the HF outer root sheath ( ORS ) following washout . In contrast , palbociclib treated HFs lacked EdU incorporation following drug washout ( adjusted p value = 0 . 0001 [ * * * ] ) . The number of EdU + cells was significantly decreased in the outer root sheath ( ORS ) of HFs dual treated with both paclitaxel and palbociclib ( adjusted p value = 0 . 0021 [ * * ] ) . Ordinary one - way ANOVA with multiple comparisons performed using N of 12 - 13 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard

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B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


C ) HFs treated with paclitaxel showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in the number of cleaved caspase 3 + cells in the ORS following drug washout . Palbociclib - only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3 + ORS cells . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard

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C) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


D ) Paclitaxel - only treated HFs showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in the number of pH3 + ORS cells following drug washout . Palbociclib treatment alone had no significant effect on the number of pH3 + ORS cells . There was a trending , yet non - significant , increase in the number of pH3 + ORS cells in HFs dual treated with palbociclib plus paclitaxel . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 patients . Values plotted represent the mean total number of cells counted per HF analysed . Error bars are standard

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D) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


E ) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout , whereby DNA synthesis has not resumed . ; wo - washout . 50 µm scale ;

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E) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. ; wo - washout. 50 µm scale;


F ) Representative images of pH3 / caspase 3 staining within the ORS of paclitaxel - only and palbociclib plus paclitaxel treated HFs following drug washout . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 50 µm

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F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 50 µm scale


Immunofluorescence images illustrating how palbociclib blocks the paclitaxel - induced accumulation of cleaved caspase 3 + ( G ) cells in the K15 + bulge and proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

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Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


Immunofluorescence images illustrating how palbociclib blocks the paclitaxel - induced accumulation of pH3 + ( H ) cells in the K15 + bulge and proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

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Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


( A - C ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS ( Earle ' s Balanced Salt Solution ) for 30 min . Total membranes ( mem ) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells . Reactions contained the indicated concentrations of PI3K inhibitor ( PI3KI ) 3 - methyladenine ( 3 - MA ) ( B ) or FYVE protein ( C ) . A diagram of the experimental scheme is shown in ( A ) . RPN1 , Ribophorin

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(A-C) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1


( D , E ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS in the absence or presence of 20 nM wortmannin ( Wtm ) or 10 mM 3 - methyladenine ( 3 - MA ) for 30 min . Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K × g , 25K × g and 100K × g pellet fractions followed by a lipidation assay as above ( E ) . A diagram is shown in ( D ) .

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(D, E) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D).


( F , G ) Atg5 KO MEFs were starved for 30 min . Membranes in the 25K × g and 100K × g pellets from a differential centrifugation were collected as described above . A similar lipidation assay was performed in the presence of indicated concentrations ( Conc in G ) of 3 - MA , wortmannin ( F ) and FYVE protein as well as a PI3P binding - deficient FYVE mutant protein ( C / S ) ( G ) . Quantification of lipidation activity is shown as the ratio of LC3 - II to LC3 - I ( II / I ) .

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(F, G) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


( B ) A small vesicle generation assay as above was performed with the indicated conditions . Slowly - sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction . 3 - MA , 5 mM ; Wortmannin , 20 nM ; S , starved Atg5 KO MEF cytosol ; N , untreated Atg5 KO MEF cytosol ; − / AP , in the absence of ATP regeneration and GTP and in the presence of apyrase ( AP ) .

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(B) A small vesicle generation assay as above was performed with the indicated conditions. Slowly-sedimenting vesicles were collected to determine activity in the LC3 lipidation reaction. 3-MA, 5 mM; Wortmannin, 20 nM; S, starved Atg5 KO MEF cytosol; N, untreated Atg5 KO MEF cytosol; −/AP, in the absence of ATP regeneration and GTP and in the presence of apyrase (AP).


( D ) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K , P and F1 - 4 fractions ( upper four panels : Small vesicle ) . SDS - PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction ( lower five panels : Fraction input ) . TFR , transferrin

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(D) A small vesicle generation assay followed by a lipidation reaction was performed with the 3K, P and F1-4 fractions (upper four panels: Small vesicle). SDS-PAGE and immunoblot with indicated antibodies were carried out to probe the organelle enrichment of each fraction (lower five panels: Fraction input). TFR, transferrin receptor


( E ) A small vesicle generation assay as shown in ( A ) was performed in the presence of indicated proteins or drugs . Slowly - sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST , H79G ( Sar1A ( H79G ) , 0 . 7 μM ) , wortmannin ( 20 nM ) , or BFA ( brefeldin A , 0 . 5 μg / ml ) .

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(E) A small vesicle generation assay as shown in (A) was performed in the presence of indicated proteins or drugs. Slowly-sedimenting vesicles were collected and incubated in a lipidation reaction in the presence of GST, H79G (Sar1A (H79G), 0.7 μM), wortmannin (20 nM), or BFA (brefeldin A, 0.5 μg/ml).


( F ) A small vesicle generation assay as shown in ( A ) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A ( H79G ) or 3 - MA followed by a lipidation reaction .

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(F) A small vesicle generation assay as shown in (A) was performed with cytosols from starved Atg5 KO MEF from control or Sec23A knockdown cells in the absence or presence of Sar1A (H79G) or 3-MA followed by a lipidation reaction.


( G ) Atg5 KO MEFs were starved for 30 min . Differential centrifugation as shown in Figure 1D was performed to collect the 25K × g and 100K × g pellet fractions . The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 ( H79G ) . Quantification of lipidation activity is shown as the ratio of LC3 - II to LC3 - I ( II / I ) .

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(G) Atg5 KO MEFs were starved for 30 min. Differential centrifugation as shown in Figure 1D was performed to collect the 25K ×g and 100K ×g pellet fractions. The LC3 lipidation was performed on these two fractions with indicated concentrations of SAR1 (H79G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).


( A ) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min . Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 ( F2 in Figure 2C , D ) of the OptiPrep gradient in the three - step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes .

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(A) Atg5 KO MEFs were either untreated or starved with or without 20 nM wortmannin for 30 min. Cells were harvested and ERGIC membranes were isolated by pooling fractions 3 and 4 (F2 in Figure 2C,D) of the OptiPrep gradient in the three-step fractionation approach followed by immunoblot to examine the amount of indicated markers on ERGIC membranes.


( B ) Atg5 KO MEFs were transfected with a plasmid encoding an HA - tagged SEC31A . After transfection ( 24 hr ) , the cells were treated as shown in ( A ) . Immunofluorescence was performed with anti - ERGIC53 and anti - HA antibodies and the cells were examined by confocal microscopy . Bar : 10 μM ( C ) Quantification of SEC31A overlapped with ERGIC53 shown in ( B ) using the Pearson ' s correlation coefficient ( PCC ) . Error bars represent standard deviations from ∼ 20 cells in three independent experiments . p values were calculated by T - test .

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(B) Atg5 KO MEFs were transfected with a plasmid encoding an HA-tagged SEC31A. After transfection (24 hr), the cells were treated as shown in (A). Immunofluorescence was performed with anti-ERGIC53 and anti-HA antibodies and the cells were examined by confocal microscopy. Bar: 10 μM (C) Quantification of SEC31A overlapped with ERGIC53 shown in (B) using the Pearson's correlation coefficient (PCC). Error bars represent standard deviations from ∼20 cells in three independent experiments. p values were calculated by T-test.


A - D ) Confocal image analysis of cryostat sections from adult CX3CR1 + / GFP ; Sfrp1 + / βgal ( A , B ) ; Sfrp1 + / βgal ( C ) and CX3CR1 + / GFP ; Sfrp1 - / - ( D ) and mouse brains three days after intra - cortical infusion of saline or LPS . Sections were immunostained for βgal ( magenta , green in C ) and Iba1 ( green in A ; red in C ) or Sfrp1 ( magenta ) and GFP ( green , B and D ) , and GFAP ( cyan in A ; B , D , red in C ) . Arrowheads indicate βgal / GFAP ( A ) and Sfrp1 / GFP co - localization ( B ) . No Sfrp1 protein is detected in the null mice independently of the treatment ( two bottom lines ) . Scale bar 25μm . E ) ELISA determination of Sfrp1 levels in brain extracts from 3 months - old wt and Sfrp1 - / - mice . WT mice were injected either with saline or LPS . Three days after injection the region around the injected side ( 10 mm3 cortical cube ) was isolated and SFRP1 content compared with that present in similar region of non - injected or Sfrp1 - / - mice used as negative control ( n = 5 mice for each group ) . Error bars represent Standard Error . Statistical significance : ns P > 0 . 5 * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni multiple comparisons test .

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A-D) Confocal image analysis of cryostat sections from adult CX3CR1+/GFP;Sfrp1+/βgal (A,B); Sfrp1+/βgal (C) and CX3CR1+/GFP;Sfrp1-/- (D) and mouse brains three days after intra-cortical infusion of saline or LPS. Sections were immunostained for βgal (magenta, green in C) and Iba1 (green in A; red in C) or Sfrp1 (magenta) and GFP (green, B and D), and GFAP (cyan in A;B, D, red in C). Arrowheads indicate βgal/GFAP (A) and Sfrp1/GFP co-localization (B). No Sfrp1 protein is detected in the null mice independently of the treatment (two bottom lines). Scale bar 25μm. E) ELISA determination of Sfrp1 levels in brain extracts from 3 months-old wt and Sfrp1-/- mice. WT mice were injected either with saline or LPS. Three days after injection the region around the injected side (10 mm3 cortical cube) was isolated and SFRP1 content compared with that present in similar region of non-injected or Sfrp1-/- mice used as negative control (n=5 mice for each group). Error bars represent Standard Error. Statistical significance: ns P>0.5 ****P<0.0001; One-way ANOVA followed by Bonferroni multiple comparisons test.


A ) Coronal cryostat sections of LV - IRES - Gfp or LV - Sfrp1 - IRES - Gfp infected brains 1 - month post - infusion , immunostained for SFRP1 ( magenta ) Iba1 ( green ) GFAP ( cyan ) , or TauP ( red ) . Lv , lateral ventricle . Scale bar 100μm . B ) The graph shows the normalised level of GFAP , Iba1 and CD45 immunoreactivity ( IR ) and the area occupied by TauP signal ( n = 24 acquisitions , white dots ; N = 3 mice , black dots , for each group ) , normalized to LV - IRES - Gfp infected brains . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * P < 0 . 01 ; * * * * P < 0 . 0001 by two - sided Student ' s t - test .

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A) Coronal cryostat sections of LV-IRES-Gfp or LV-Sfrp1-IRES-Gfp infected brains 1-month post-infusion, immunostained for SFRP1 (magenta) Iba1 (green) GFAP (cyan), or TauP (red). Lv, lateral ventricle. Scale bar 100μm. B) The graph shows the normalised level of GFAP, Iba1 and CD45 immunoreactivity (IR) and the area occupied by TauP signal (n=24 acquisitions, white dots; N=3 mice, black dots, for each group), normalized to LV-IRES-Gfp infected brains. Error bars represent Standard Error. Statistical significance: *P<0.05; **P<0.01; ****P<0.0001 by two-sided Student's t-test.


C , D ) Coronal sections from wt and Sfrp1 - / - mouse brains three days after infusion of saline or LPS , immunostained for GFAP ( cyan , C ) or CD45 ( magenta , D ) . The images are high power views of the somatosensory cortex ( lower power view in Fig EV1D ) . Scale bar 60μm . E , F ) The graphs show the normalised levels of immunoreactivity ( IR ) for GFAP ( E ) and CD45 ( F , P = 0 , 006 ) present in cortical sections ( n = 24 acquisitions white dots ; from N = 3 animals , black dots , per group ) from wt and Sfrp1 - / - animals infused with saline or LPS . Bars represent Standard Error . Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black . * * or # # P < 0 . 01 ; * * * or # # # P < 0 . 001 ; * * * * or # # # # P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test . * and # indicate significance between genotypes and treatments , respectively .

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C, D) Coronal sections from wt and Sfrp1-/- mouse brains three days after infusion of saline or LPS, immunostained for GFAP (cyan, C) or CD45 (magenta, D). The images are high power views of the somatosensory cortex (lower power view in Fig EV1D). Scale bar 60μm. E, F) The graphs show the normalised levels of immunoreactivity (IR) for GFAP (E) and CD45 (F, P=0,006) present in cortical sections (n=24 acquisitions white dots; from N=3 animals, black dots, per group) from wt and Sfrp1-/- animals infused with saline or LPS. Bars represent Standard Error. Statistical significance calculated per biological replicas is indicated in green and that based on number of acquisitions in black. ** or ## P<0.01; *** or ### P<0.001; **** or #### P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test. * and # indicate significance between genotypes and treatments, respectively.


C ) Wt and Sfrp1 - / - mice immunized with MOG and sacrificed 16 days post immunization . Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4 , Iba1 , GFAP and MBP . Images show the region dorsal fasciculus . White arrowheads point to the disruption of the pia surface in wt . Scale bar 100μm . D ) Quantification of CD4 + infiltrated lymphocytes , Iba1 normalised immunoreactivity , MBP normalised immunoreactivity and MBP immune - reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1 - / - mice 16 days after immunization ( n = 16 acquisitions , white dots ; from N = 4 animals , black dots , per genotype ) . Error bars represent Standard Error . Statistic refers to number of acquisitions .

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C) Wt and Sfrp1-/- mice immunized with MOG and sacrificed 16 days post immunization. Cryostat sections of the thoracic spinal cords were stained with antibodies against CD4, Iba1, GFAP and MBP. Images show the region dorsal fasciculus. White arrowheads point to the disruption of the pia surface in wt. Scale bar 100μm. D) Quantification of CD4+ infiltrated lymphocytes, Iba1 normalised immunoreactivity, MBP normalised immunoreactivity and MBP immune-reactive area in the dorsal fasciculus of spinal cord sections from wt and Sfrp1-/- mice 16 days after immunization (n=16 acquisitions, white dots; from N=4 animals, black dots, per genotype). Error bars represent Standard Error. Statistic refers to number of acquisitions.


D ) ELISA determination of SFRP1 levels present in the media of microglia ( n = 4 cultures per genotype ) , astrocytes ( n = 4 cultures per genotype ) or mixed astrocytes and microglial ( 2 : 1 , n = 7 cultures per genotype ) cultures derived from wt or Sfrp1 - / - pups exposed for 24h to saline or LPS ( 1μg / ml ) . Error bars represent Standard Error . Statistical significance : * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ; One - way ANOVA followed by Bonferroni Test .

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D) ELISA determination of SFRP1 levels present in the media of microglia (n=4 cultures per genotype), astrocytes (n=4 cultures per genotype) or mixed astrocytes and microglial (2:1, n=7 cultures per genotype) cultures derived from wt or Sfrp1-/- pups exposed for 24h to saline or LPS (1μg/ml). Error bars represent Standard Error. Statistical significance: *P<0.05; ***P<0.001; ****P<0.0001; One-way ANOVA followed by Bonferroni Test.


Secretory profile of cytokines present in the medium of microglia , astrocytes or mixed astrocytes and microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - and genotype ) . The content of IL1β , IL10 was determined by Multiplex ELISA . Values are represented in absolute concentration . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test .

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Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IL1β, IL10 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


F ) Secretory profile of cytokines present in the medium of microglia , astrocytes or mixed astrocytes and microglial ( 2 : 1 ) culture as above ( n = 5 - 6 cultures type - microglia , astrocytes or mixed cultures - and genotype ) . The content of IFNγ , TNFα , IL4 , IL6 was determined by Multiplex ELISA . Values are represented in absolute concentration . Error bars represent Standard Error . Statistical significance : ns P > 0 . 05 ; * P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 by two - way ANOVA followed by Bonferroni ' s multiple comparisons test .

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F) Secretory profile of cytokines present in the medium of microglia, astrocytes or mixed astrocytes and microglial (2:1) culture as above (n=5-6 cultures type -microglia, astrocytes or mixed cultures- and genotype). The content of IFNγ, TNFα, IL4, IL6 was determined by Multiplex ELISA. Values are represented in absolute concentration. Error bars represent Standard Error. Statistical significance: ns P>0.05; *P<0.05; ***P<0.001; ****P<0.0001 by two-way ANOVA followed by Bonferroni's multiple comparisons test.


B , C ) Volcano plots of differential gene expression from CX3CR1GFP / + ( B ) or CX3CR1GFP / + ; Sfrp1 - / - ( C ) microglial cell in response to LPS . Data are represented as Log2 Fold Change vs - Log10 adjusted p - value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method . Blue vertical lines represent an increase of 75 and 400 % in the expression levels respectively . Green horizontal lines represent a 0 . 05 or 0 . 01 adjusted p - value of statistical significance respectively . Genes with an expression change higher than 75 % and an adjusted p - value lower than 0 . 05 are highlighted in dark red ( + FC ) and dark blue ( - FC ) . Genes with an expression change higher than 400 % and an adjusted p - value lower than 0 . 01 are coloured in light red ( + FC ) and light blue ( - FC ) .

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B, C) Volcano plots of differential gene expression from CX3CR1GFP/+ (B) or CX3CR1GFP/+;Sfrp1-/- (C) microglial cell in response to LPS. Data are represented as Log2 Fold Change vs -Log10 adjusted p-value by the Wald test corrected for multiple comparisons by the Benjamini and Hochberg method. Blue vertical lines represent an increase of 75 and 400% in the expression levels respectively. Green horizontal lines represent a 0.05 or 0.01 adjusted p-value of statistical significance respectively. Genes with an expression change higher than 75% and an adjusted p-value lower than 0.05 are highlighted in dark red (+FC) and dark blue (-FC). Genes with an expression change higher than 400% and an adjusted p-value lower than 0.01 are coloured in light red (+FC) and light blue (-FC).


B ) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP / + and CX3CR1GFP / + ; Sfrp1 - / - mouse brains . Scale bar : 2 Kbp .

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B) Integrative Genomics Viewer transcription profile of represented genes in microglial cells isolated for saline and LPS treated CX3CR1GFP/+ and CX3CR1GFP/+;Sfrp1-/- mouse brains. Scale bar: 2 Kbp.


A . The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3 - GFP ( green ) . NSUN3 - GFP and staining with a Mitotracker ( red ) are shown separately and in an overlay with DAPI to indicate nuclei . The scale bar represents 5 m .

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A. The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3-GFP (green). NSUN3-GFP and staining with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.


B . To analyse submitochondrial localization of NSUN3 , human mitochondria were isolated and either left untreated , swollen in hypotonic buffer ( Mitoplasts ) or solubilized by sonication ( Sonic . ) before treatment with different amounts of Proteinase K ( PK ) where indicated , followed by SDS - PAGE and Western blotting using antibodies against human TIM44 , TIM23 , TOM70 or FLAG - tagged NSUN3 . Note that TIM44 localises to the matrix , while the N - terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface . The asterisk indicates a cross - reaction of the TOM70 antibody .

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B. To analyse submitochondrial localization of NSUN3, human mitochondria were isolated and either left untreated, swollen in hypotonic buffer (Mitoplasts) or solubilized by sonication (Sonic.) before treatment with different amounts of Proteinase K (PK) where indicated, followed by SDS-PAGE and Western blotting using antibodies against human TIM44, TIM23, TOM70 or FLAG-tagged NSUN3. Note that TIM44 localises to the matrix, while the N-terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface. The asterisk indicates a cross-reaction of the TOM70 antibody.


B . HEK293 cells expressing NSUN3 - HisPrcFLAG ( NSUN3 ) or the HisPrcFLAG - tag alone ( FLAG ) were either not crosslinked ( - ) , UV crosslinked ( UV ) or treated with 5 - azacytidine ( 5 - AzaC ) . The protein - RNA complexes were affinity purified and the bound RNA was trimmed , end - labelled with 32P phosphate and ligated to linkers . Protein - RNA complexes were separated by SDS - PAGE , transferred to nitrocellulose and exposed to an X - ray film .

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B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film.


G . 5 - AzaC crosslinking was performed and RNA associated with wildtype NSUN3 , the catalytically inactive NSUN3 mutant ( C265A ) or the FLAG tag alone was isolated as described in ( B ) . The RNA analysed by northern blot using probes against the mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on the left and eluates ( 50 % ) on the right .

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G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


A . In vitro methylation reactions were performed using recombinant His14 - MBP - NSUN3 ( NSUN3 ) or the catalytically inactive mutant His14 - MBP - NSUN3 - C265A ( C265A ) , 3H - labelled S - adenosyl methionine as a methyl group donor and in vitro transcribed mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . The RNA was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) to indicate inputs and exposed to an X - ray film to analyse methylation ( 3H - Me ) .

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A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me).


B . The distribution of Illumina sequence reads along the mt - tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV ( light grey ) or 5 - AzaC crosslinking ( dark grey ) is given as reads per million mapped reads . The position of the anticodon is indicated by a bar .

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B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar.


D . In vitro methylation assays were performed as described in ( A ) with His14 - MBP - NSUN3 and in vitro transcribed wildtype mt - tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in ( C ) . Two exposure times of X - ray films are shown : 16 hours ( short ) , 3 days ( long ) .

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D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).


E . In vitro methylation assay of in vitro transcribed mt - tRNAMet and chemically synthesized mt - tRNAMet containing an m5C modification at the wobble position . The experiment and analysis were performed as described in ( A ) .

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E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).


B . In vitro methylation assays using [ 3H - methyl ] - labelled S - adenosyl methionine , the in vitro transcripts of the mt - tRNAMet mutants described in ( A ) and recombinant His14 - MBP - NSUN3 . RNA was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) , dried and exposed to an X - ray film to detect methylated transcripts ( 3H - Me ) .

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B. In vitro methylation assays using [3H-methyl]-labelled S-adenosyl methionine, the in vitro transcripts of the mt-tRNAMet mutants described in (A) and recombinant His14-MBP-NSUN3. RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr), dried and exposed to an X-ray film to detect methylated transcripts (3H-Me).


C . In vitro methylation assay using chemically synthesised ASL . The experiment and analysis were performed as described in ( B ) .

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C. In vitro methylation assay using chemically synthesised ASL. The experiment and analysis were performed as described in (B).


A . The localization of ABH1 was analysed by immunofluorescence in HEK293 cells . ABH1 ( green ) localisation and mitochondria stained with a Mitotracker ( red ) are shown separately and in an overlay with DAPI to indicate nuclei . The scale bar represents 5 m .

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A. The localization of ABH1 was analysed by immunofluorescence in HEK293 cells. ABH1 (green) localisation and mitochondria stained with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.


B . HEK293 cells expressing ABH1 - HisPrcFLAG ( ABH1 ) or the HisPrcFLAG - tag alone ( FLAG ) were UV crosslinked ( UV ) and protein - RNA complexes were affinity purified . Co - precipitated RNA was isolated and analysis by northern blot using probes against the mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on the left and eluates ( 50 % ) on the right .

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

B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


A . In vitro transcribed mt - tRNAMet was methylated at C34 using recombinant NSUN3 and 3H - labelled S - adenosyl methionine as a methyl group donor . Radiolabelled mt - tRNAMet was re - extracted and then subjected to oxidation assays without protein ( - ) , with maltose binding protein ( MBP ) , with the dioxygenase FTO or using wildtype ( ABH1 ) or mutant ( R338A , D233A ) His14 - MBP - ABH1 . Besides ABH1 controls lacking a - ketoglutarate ( KG ) or Fe2 + ions , all samples contained a - ketoglutarate and Fe2 + ions . After oxidation , RNA was precipitated and tritium released upon oxidation of radiolabelled mt - tRNAMet was quantified in the supernatant and counts per minute ( CPM ) are shown for experiments performed in triplicate with error bars indicating ± SD ( upper panel ) . Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X - ray film to analyse the tritium retained ( 3H - Me ) .

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A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking a-ketoglutarate (KG) or Fe2+ ions, all samples contained a-ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me).


B . Synthetic anticodon stem loop ( ASL ) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in ( A ) using no protein ( - ) , MBP or wildtype His14 - MBP - ABH1 ( ABH1 ) . Experiments were performed in triplicate with error bars indicating ± SD .

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B. Synthetic anticodon stem loop (ASL) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in (A) using no protein (-), MBP or wildtype His14-MBP-ABH1 (ABH1). Experiments were performed in triplicate with error bars indicating ± SD.


C . Anion exchange HPLC analysis was performed on synthetic m5C containing ASL ( 20nt ) before and after oxidation by ABH1 . The small shift in retention time indicates formation of f5C modified RNA . The ABH1 oxidation product was then treated with NaBH4 to generate hm5C - modified RNA . All three samples were analysed by ESI - MS and the molecular weight ( m . w . ) is indicated on the HPLC trace . Only the f5C - containing RNA was labelled efficiently with 1 - ethyl - 2 , 3 , 3 - trimethylindoleninium - 5 - sulfonate ( TMI ) .

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C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI).


D . The different retention times of m5C , hm5C and f5C - modified ASL RNA were confirmed by co - injection of samples shown in ( C ) . HPLC was performed as in C .

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D. The different retention times of m5C, hm5C and f5C-modified ASL RNA were confirmed by co-injection of samples shown in (C). HPLC was performed as in C.


A . MTIF2 - dependent reading of initiation codons AUG , AUA or AUU in the P site of the ribosome by unmodified ( unmod . ) or C34 - modified [ 14C ] Met - tRNAMet . Binding was determined by nitrocellulose filtration and [ 14C ] Met - tRNAMet retrieved on the membrane was quantified by scintillation counting . Binding in the absence of ribosomes ( no 70S ) or mRNA ( no mRNA ) served as controls . Data from three independent experiments are presented with error bars indicating ± SEM . The statistical significance of the results was analysed by T - test and is indicated by the asterisks in the graph .

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A. MTIF2-dependent reading of initiation codons AUG, AUA or AUU in the P site of the ribosome by unmodified (unmod.) or C34-modified [14C]Met-tRNAMet. Binding was determined by nitrocellulose filtration and [14C]Met-tRNAMet retrieved on the membrane was quantified by scintillation counting. Binding in the absence of ribosomes (no 70S) or mRNA (no mRNA) served as controls. Data from three independent experiments are presented with error bars indicating ± SEM. The statistical significance of the results was analysed by T-test and is indicated by the asterisks in the graph.


B . TUFM - dependent recognition of A site codons during elongation . Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in ( A ) .

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B. TUFM-dependent recognition of A site codons during elongation. Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in (A).


A . HeLa cells were transfected with two different siRNAs against NSUN3 ( siNSUN3 _ 1 , siNSUN3 _ 2 ) , ABH1 ( siABH1 _ 1 , siABH1 _ 2 ) or with nontarget ( siNT ) siRNA and the knockdown efficiency was analysed by quantitative PCR . The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels . Data are presented as mean ± SD .

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A. HeLa cells were transfected with two different siRNAs against NSUN3 (siNSUN3_1, siNSUN3_2), ABH1 (siABH1_1, siABH1_2) or with nontarget (siNT) siRNA and the knockdown efficiency was analysed by quantitative PCR. The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels. Data are presented as mean ± SD.


B . Chemically synthesised f5C modified mt - tRNAMet and total RNA from wild - type ( WT ) cells or those transfected with siRNAs as in ( A ) were treated with TMI to specifically label f5C residues . Primer extension , using a radiolabelled antisense primer , was performed under limited dNTP conditions . Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager .

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B. Chemically synthesised f5C modified mt-tRNAMet and total RNA from wild-type (WT) cells or those transfected with siRNAs as in (A) were treated with TMI to specifically label f5C residues. Primer extension, using a radiolabelled antisense primer, was performed under limited dNTP conditions. Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager.


C . Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B . Stops corresponding to position C34 in mt - tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD .

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C. Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B. Stops corresponding to position C34 in mt-tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD.