EMBO/SourceData huggingface dataset:
Figure 2A. Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum-starved control and MASTL knockout MDA-MB-231 cells using the isgMASTL system. The plot shows the quantification of phospho-AKT T308 after 30 min of insulin stimulation. Plots displays mean data + SEM from 5 independent experiments. *P<0.05, Student's t-test.B. TSC2 was knocked down in control or MASTL-null MDA-MB-231 cells using specific (+) or Scrambled (−) shRNAs. Cells were starved for glucose for 1 h. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.C. MDA-MB-231 control (−) or MASTL null (+) cells were treated with 100 nM rapamycin or vehicle as control. Cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min; rapamycin was added 15 min before glucose stimulation. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.
Figure 3A. Immunofluorescence for GLUT4 (magenta) in MDA-MB-231 cells in conditions of serum starvation and insulin stimulation. DAPI (DNA) is in cyan and a cell mask is in yellow. Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane (PM). Representative magnifications are also shown. Scale bars, 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM. Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA. Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies.
Figure 4E. GLUT4 staining (magenta) in skeletal muscle sections. Vinculin (membrane) and DAPI (DNA) staining is shown in yellow and cyan, respectively. Scale bars, 50 µm. Mice were fasted overnight for 16 h, and re-fed for 2 h before sample collection. Quantification of the mean intensity [relative units x103; n=3 Mastl(+/+) and 3 Mastl(∆/∆)] is shown. Data points reflect the mean + SEM; *P<0.05; ***P <0.001; unpaired Student's t-test.G. Immunoblot with the indicated antibodies in liver tissues from Mastl(+/+) (n=6) and Mastl(Δ/Δ) (n=7) mice. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of phospho-AKT T308 (normalized to total AKT level). n=6 Mastl(+/+) and 7 Mastl(Δ/Δ) mice. Charts reflect the mean + SEM; *P<0.05; unpaired Student's t-test.
Figure 5A. Co-depletion of ENSA and ARPP19 proteins in the MDA-MB-231 cell line using specific siRNAs or control siRNAs against luciferase (Luc). 72 h after transfection cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min. Total cell extracts were collected and analyzed for the indicated antibodies.B. isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG- and HA-tagged versions of ENSA and ARPP19 phospho-mimetic mutants (S67D and S62D) or GFP as a control. Cells were treated with Dox to induce MASTL CRISPR/Cas9-dependent knock-out, and 72 h later cells were treated as specified in A. The plot displays mean + SEM from 3 independent experiments. Significance determined by Student's t-test comparing ENSA/ARPP19 overexpression versus control cells (ns, not significant; *P<0.05).C. Control (Dox −) and MASTL knockout (Dox +) cells were treated with 5 µM fostriecin, 50 nM okadaic acid (OA) or vehicle (Veh) 10 min before insulin stimulation for 30 min. Total lysates were analyzed for the indicated antibodies and P-T308-AKT signal was quantified. β-Actin was used as a loading control. The plot represents mean + SEM from 3 independent experiments, except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown. *P<0.05; Student's t-test.
Figure 6A. MDA-MB-231 cells were co-transduced with specific siRNAs against the PPP2R2A (B55α) and PPP2R2D (B55δ) transcripts (B55αδ) or against luciferase (Luc) as a control. Asynchronous cells were collected and blotted for the indicated antibodies. The histograms represent the quantifications of the specified phospho-residues. Plots show mean + SEM from 3 independent experiments. *P<0.05, Student's t-test.B. Immunodetection of the indicated antigens in control (−) and MASTL knockout (+) cells starved from glucose and re-stimulated with glucose for 15 min.C. siRNA-mediated genetic depletion of B55alpha and delta subunits (B55αδ) in control (Dox −) and MASTL-null (Dox +) cells. The phosphorylation status of the different proteins was scored in conditions of glucose re-addition. n=3 independent experiments. The plot shows mean + SEM from 3 independent experiments. *P<0.05, 1-way ANOVA.D. Immunoblot with the indicated antibodies in skeletal muscle (gastrocnemius) extracts. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1, and phospho-S6K1 T389 (normalized to total S6K1 level; n=6 Mastl(+/+) and 7 Mastl(∆/∆)). vinculin were used as a loading control. Data are mean + SEM; *P<0.05, unpaired Student's t-test.
Figure 7A. MDA-MB-231 cells were starved for 1 h and re-stimulated with glucose for 15 min and T194 phosphorylation was tested with a specific antibody. The plot shows the quantification of phospho-MASTL T194 (n=2 independent experiments). Data are mean + SEM from 3 independent experiments. ns, not significant; Student's t-test.B. Cas9-expressing control (−) cells or cells with CRIPSR/Cas9-mediated TSC2 knockout (sgTSC2) were treated with rapamycin (Rap), Torin1 (Tor), or vehicle as control (V). MASTL-knockdown cells (shMASTL) were used as an additional control of the specificity of the antibody. Inhibitors were added 15 min before glucose re-stimulation for additional 15 min and total cell lysates were harvested for immunoblot. The plot shows the quantification of phospho-MASTL T194 as mean from 2 independent experiments.C. In vitro kinase assay using purified GST-human MASTL as a substrate, and recombinant mTORC1 and S6K1 kinases. Red arrowheads show the phosphorylation of MASTL in the presence of mTORC1 or S6K1. Note the appearance of other phosporylation signals in mTORC1 and S6K1 reactions, independently of the presence of MASTL, which correspond to autophosphorylation of these kinases. Imperial staining shows the amount of GST-MASTL protein used as a substrate in each reaction.F. In vitro kinase activity of Mastl mutant isoforms. HEK-293T cells were transiently transfected with the indicated GFP fusions of mouse Mastl mutants. Anti-GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 (white circles) or ENSA (filled circles) as a substrate. The graph shows the relative catalytic activity of the indicated mutants. Kinase activity of GFP-mMastl wild-type was set as 1 (n=3 independent experiments). Data are mean + SEM from 3 independent experiments. ***P <0.001; 1-way ANOVA.G. isgMASTL MDA-MB-231 cells were transduced with the indicated Mastl mutants or GFP alone as a control, and treated with doxycycline (Dox) to induce Mastl deletion. Cells were collected after glucose stimulation, and total lysates blotted for the indicated antibodies.
Figure 1B sgRNA frequencies (log2 of fold change) in PD-L1low cells unstimulated (left) or IFN-γ stimulated (right). Enriched sgRNAs (dashed line) are highlighted; hit genes (black, red) and controls (green, yellow or purple) are shown and labeled with target gene names.C sgRNA frequencies (log2 of fold change) in viability screen. sgRNAs targeting controls are highlighted; essential (orange), non-essential (blue). Genes attributed to PD-L1 modulation are shown in different colors (red, yellow and purple).
Figure 2A PD-L1 cell surface presentation (percentage ± SD) in RKO mock control (black) or monoclonal MLLT6 knockout cells (grey) (Student's t-test; **p < 0.01; n=3 biological replicates; degrees of freedom (df)=2). B MLLT6 BAC mediated rescue in U2OS cells (left) showing percentage ± SD of PD-L1 cell surface presentation in mock control (black), MLLT6 polyclonal knockout (light grey) and MLLT6 knockout + BAC cells (dark grey) (Student's t-test; *p < 0.05; n=3 biological replicates; df=2). Representative flow cytometry plots (right) of PD-L1 cell surface presentation after staining with anti-PD-L1 antibody (top right, bottom left, bottom right) and isotype control (top left). Percentages of cells in the PD-L1+ gate as indicated.  C Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in monoclonal RKO MLLT6 knockout, mock control or MLLT6 knockout + BAC cells visualized by immunoblotting. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.  D PD-L1 transcript levels in U2OS mock (black) and polyclonal MLLT6 knockout lines (grey) treated with and without IFN-γ normalized to expression in mock U2OS cells without IFN-γ treatment (ANOVA; **p < 0.01; n=4 biological replicates; df=12).  E Representative flow cytometry plots of PD-L1 cell surface presentation in the presence and absence of IFN-γ in mock, polyclonal MLLT6 knockout and PD-L1 knockout cells. Percentages of cells in the PD-L1+ gate after staining with anti-PD-L1 antibody or isotype control as indicated.  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.
Figure 3 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).  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 ).
Figure 4A 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.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.
Figure 5A 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.
Figure 1C. 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.
Figure 2A. 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).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).
Figure 3A-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.
Figure 4A. 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.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.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.
Figure 5B. 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.
Figure 7A. 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).C. Summary graphs of the diameter and total size of the vGluT1-positive presynaptic area of the calyx of Held.
Figure 1100 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.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.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.
Figure 2The 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.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.E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.
Figure 3A) 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.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.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.
Figure 4Ai) 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.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.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.