Figure 7 ( A ) Representative single channel recordings from hippocampal neurons at 7 - 14 DIV with 500 nM BayK - 8644 , 1 µM ω - conotoxin GVIA , and 1 µM MVIIC in patch pipette upon depolarization from - 80 to - 30 , - 20 , - 10 , and 0 mV . The right panel depicts the single - channel current - voltage relationship . Mean amplitude of unitary currents for different membrane potential studied are - 2 . 07 pA ( - 30 mV ) , - 1 . 75 ( - 20 mV ) , - 1 . 53 ( - 10 mV ) and - 1 . 25 ( 0 mV ; n = 5 patches per test potential ) . Solid line represents best - fit of data using a linear equation ( R2 = 0 . 92 ) revealing a slope conductance for these channels of 27 ± 2 pS . ( B - D ) Representative single channel traces and summary plot upon depolarization from - 80 to 0 mV under control conditions and in the presence of ISO in the patch pipette . Cultures were pre - incubated for 15 min with 10 M 11R - PKI if indicated . The patch pipette contained either vehicle for control , nifedipine ( nif ; 1M ) , ISO ( 1M ) , or ISO plus nifedipine , which blocked all currents . The ISO - induced increase in NPo was prevented by 11R - PKI . ( E - H ) Representative single channel currents upon depolarization from - 80 to 0 mV and summary plot after pretreatment of whole cultures with ISO . Cultures were pre - incubated with vehicle ( H2O , mock wash , E ) or 1 M ISO for 5 min ( F , G ) and washed for 3 ( E , F ) or 10 min ( G ) before forming the cell - attached patch with ISO present in the patch pipette . The upregulation of NPo to ~ 0 . 4 ( cf . C , D ) occurred only if neurons were pretreated with vehicle instead of ISO ( E , H ; mock wash ) or if ISO wash out duration was 10 min ( G , H ) but not if wash out was only 3 min ( F , H ) .

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

Figure 7(A) Representative single channel recordings from hippocampal neurons at 7-14 DIV with 500 nM BayK-8644, 1 µM ω-conotoxin GVIA, and 1 µM MVIIC in patch pipette upon depolarization from -80 to -30, -20, -10, and 0 mV. The right panel depicts the single-channel current-voltage relationship. Mean amplitude of unitary currents for different membrane potential studied are -2.07 pA (-30 mV), -1.75 (-20 mV), -1.53 (-10 mV) and -1.25 (0 mV; n=5 patches per test potential). Solid line represents best-fit of data using a linear equation (R2=0.92) revealing a slope conductance for these channels of 27 ± 2 pS.(B-D) Representative single channel traces and summary plot upon depolarization from -80 to 0 mV under control conditions and in the presence of ISO in the patch pipette. Cultures were pre-incubated for 15 min with 10 M 11R-PKI if indicated. The patch pipette contained either vehicle for control, nifedipine (nif; 1M), ISO (1M), or ISO plus nifedipine, which blocked all currents. The ISO-induced increase in NPo was prevented by 11R-PKI.(E-H) Representative single channel currents upon depolarization from -80 to 0 mV and summary plot after pretreatment of whole cultures with ISO. Cultures were pre-incubated with vehicle (H2O, mock wash, E) or 1 M ISO for 5 min (F,G) and washed for 3 (E,F) or 10 min (G) before forming the cell-attached patch with ISO present in the patch pipette. The upregulation of NPo to ~0.4 (cf. C,D) occurred only if neurons were pretreated with vehicle instead of ISO (E,H; mock wash) or if ISO wash out duration was 10 min (G,H) but not if wash out was only 3 min (F,H).


Figure 8 ( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it membrane permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , and IP of β2AR ( A - C ) or simultaneously α11 . 2 and GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( A ) Myr - Pep 2 displaced α11 . 2 ( lane 5 vs . 3 , top of blot ) but not GluA1 ( middle , same blot ) from β2AR ( bottom , same blot ) ; the inverse was true for Myr - DSPL ( lane 1 vs . 3 ) . ( B , C ) For quantification , α11 . 2 and GluA1 immunosignals were normalized to β2AR signals ( * * p < 0 . 01 , * * * p < 0 . 001 , One Way ANOVA ) . ( A - F ) Forebrain slices were pre - incubated for 30 min with vehicle ( water ) or 10 μM Myr - Pep2 or Myr - DSPL . Like Myr - DSPL ( Joiner et al . , 2010 ) , Pep2 was myristoylated at its N terminus ( Myr - Pep2 ) to make it membrane permeant . Slices were then treated with ISO ( 10 M , 5 min ) or vehicle ( water ) before solubilization , ultracentrifugation , and IP of β2AR ( A - C ) or simultaneously α11 . 2 and GluA1 with a combination of corresponding antibodies within same samples ( D - F ) . ( D ) Myr - Pep2 blunted ISO - induced phosphorylation of α11 . 2 S1700 ( lane 6 vs . 4 , top of blot ) but not GluA1 S845 ( middle , same blot ) ; the inverse was true for Myr - DSPL ( lane 2 vs . 4 ) . ( E , F ) For quantification , pS1700 and pS845 immunosignals were normalized to α11 . 2 and GluA1 signals , respectively ( * * * p < 0 . 001 , One Way ANOVA ) . ( G ) Representative cell - attached recordings from hippocampal neurons as in Figure 7 . In interleafed experiments cultures were pre - incubated for 30 min with 10 M Myr - Pep2 or scrambled Myr - Pep2 ( Myr - Pep2scr ) . The patch pipette contained either vehicle ( H2O ; control ) or 1 µM ISO . The ISO induced increase in NPo was prevented by Myr - Pep2 but not Myr - Pep2scr . Arrows indicate the 0 - current level ( i . e . closed channel ) . ( H ) Summary plot for ( G ) . For statistical analysis , the NPo value was determined for each recording and pooled under each condition for comparison ( n = 10 - 12 patches ; * p < 0 . 05 , One Way ANOVA with Tukey post - hoc test ) .

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

Figure 8(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(A) Myr-Pep 2 displaced α11.2 (lane 5 vs. 3, top of blot) but not GluA1 (middle, same blot) from β2AR (bottom, same blot); the inverse was true for Myr-DSPL (lane 1 vs. 3).(B,C) For quantification, α11.2 and GluA1 immunosignals were normalized to β2AR signals (**p<0.01, ***p<0.001, One Way ANOVA).(A-F) Forebrain slices were pre-incubated for 30 min with vehicle (water) or 10 μM Myr-Pep2 or Myr-DSPL. Like Myr-DSPL (Joiner et al., 2010), Pep2 was myristoylated at its N terminus (Myr-Pep2) to make it membrane permeant. Slices were then treated with ISO (10 M, 5 min) or vehicle (water) before solubilization, ultracentrifugation, and IP of β2AR (A-C) or simultaneously α11.2 and GluA1 with a combination of corresponding antibodies within same samples (D-F).(D) Myr-Pep2 blunted ISO-induced phosphorylation of α11.2 S1700 (lane 6 vs. 4, top of blot) but not GluA1 S845 (middle, same blot); the inverse was true for Myr-DSPL (lane 2 vs. 4).(E,F) For quantification, pS1700 and pS845 immunosignals were normalized to α11.2 and GluA1 signals, respectively (***p<0.001, One Way ANOVA).(G) Representative cell-attached recordings from hippocampal neurons as in Figure 7. In interleafed experiments cultures were pre-incubated for 30 min with 10 M Myr-Pep2 or scrambled Myr-Pep2 (Myr-Pep2scr). The patch pipette contained either vehicle (H2O; control) or 1 µM ISO. The ISO induced increase in NPo was prevented by Myr-Pep2 but not Myr-Pep2scr. Arrows indicate the 0-current level (i.e. closed channel).(H) Summary plot for (G). For statistical analysis, the NPo value was determined for each recording and pooled under each condition for comparison (n=10-12 patches; *p<0.05, One Way ANOVA with Tukey post-hoc test).


Figure 9Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz / 3 min ( A - C ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO . Inserts show sample traces immediately before ( left ) and ~ 30 min after ( right ) tetani . ( A - C ) Litter - matched WT but not conditional Cav1 . 2 KO mice showed PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) . ( D - F ) WT as well as Cav1 . 2 KO mice showed NMDAR - dependent 100 Hz LTP . Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz / 3 min ( A - C , G - I ) or 100Hz / 1sec tetanus ( D - F ) . Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr - Pep2 or Myr - Pep2scr . Inserts show sample traces immediately before ( left ) and ~ 30 min after ( right ) tetani . ( G - I ) Myr - Pep2 but not Myr - Pep2scr blocked PTT - LTP ( * * p < 0 . 001 , one - way ANOVA ) .

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

Figure 9Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(A-C) Litter-matched WT but not conditional Cav1.2 KO mice showed PTT-LTP (**p < 0.001, one-way ANOVA).(D-F) WT as well as Cav1.2 KO mice showed NMDAR-dependent 100 Hz LTP.Graphs depict fEPSP initial slopes recorded from hippocampal CA1 before and after either a 5Hz/3 min (A-C, G-I) or 100Hz/1sec tetanus (D-F). Arrowheads mark onset of tetani and bars perfusion with 1 µM ISO and 10 µM of Myr-Pep2 or Myr-Pep2scr. Inserts show sample traces immediately before (left) and ~30 min after (right) tetani.(G-I) Myr-Pep2 but not Myr-Pep2scr blocked PTT-LTP (**p < 0.001, one-way ANOVA).


Figure 1 ( A - C ) Distribution of spindle pole factors in centrosomal and acentrosomal spindles . DMSO - treated control mitotic spindles ( 2 - centrosomes ) and centrinone - treated acentrosomal spindles ( 0 - centrosome ) of HeLa cells . ( A ) Red , gray , green , and blue represent NuMA , α - tubulin , Cep152 , and DNA , respectively . Z - projections of eight sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Red , gray , and blue represent NuMA , Katanin p60 , and DNA , respectively . Z - projections of five sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Red , gray , and blue represent p150glued , ASPM , and DNA , respectively . Z - projections of 10 ( 2 - centrosomes ) or five ( 0 - centrosome ) sections , every 0 . 3 μm . Scale bar , 5 μm . ( D ) The structure of NuMA and microtubules in centrinone - treated acentrosomal spindles of HeLa cells . Two - way arrows indicate the bipolarity of elongated NuMA structures . Gray , red , green and blue represent α - tubulin , NuMA , Cep152 and DNA , respectively . Scale bar , 5 μm . ( E ) The localization of p150glued in centrinone - treated acentrosomal spindles of HeLa cells . Red , gray , green and blue represent NuMA , p150glued , Cep152 and DNA , respectively . Scale bar , 5 μm . ( F ) NuMA distribution in 3D . Images of NuMA ( Red ) and chromosomes ( blue ) were reconstructed in 3D and surface - rendered . ( G ) Representative images of NuMA ( arrowheads ) in centrosomal and acentrosomal cells before and after photobleaching . DMSO - or centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were observed . Time after photobleaching ( sec ) is indicated . Scale bar , 5 μm . ( H ) Normalized fluorescence intensities of NuMA - mClover at centrosomal poles ( control , 2 - centrosomes ) or at acentrosomal spindle pole ( 0 - centrosome ) during the FRAP time course ( N = 20 cells for each condition ) . The first three time points are before photobleaching . Error bars show standard deviation ( SD ) . ( I ) Mobile fractions from the normalized recovery curves of NuMA - mClover at centrosomal spindle poles ( control , 2 - centrosomes ) or at acentrosomal spindle poles ( 0 - centrosome ) . Mobile fractions ( roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting . Line and error bars represent the median and interquartile range ( N = 20 cells for each condition ) . The Mann - Whitney U test ( two - tailed ) was used to obtain a P value .

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

Figure 1(A-C) Distribution of spindle pole factors in centrosomal and acentrosomal spindles. DMSO-treated control mitotic spindles (2-centrosomes) and centrinone-treated acentrosomal spindles (0-centrosome) of HeLa cells. (A) Red, gray, green, and blue represent NuMA, α-tubulin, Cep152, and DNA, respectively. Z-projections of eight sections, every 0.3 μm. Scale bar, 5 μm. (B) Red, gray, and blue represent NuMA, Katanin p60, and DNA, respectively. Z-projections of five sections, every 0.3 μm. Scale bar, 5 μm. (C) Red, gray, and blue represent p150glued, ASPM, and DNA, respectively. Z-projections of 10 (2-centrosomes) or five (0-centrosome) sections, every 0.3 μm. Scale bar, 5 μm.(D) The structure of NuMA and microtubules in centrinone-treated acentrosomal spindles of HeLa cells. Two-way arrows indicate the bipolarity of elongated NuMA structures. Gray, red, green and blue represent α-tubulin, NuMA, Cep152 and DNA, respectively. Scale bar, 5 μm.(E) The localization of p150glued in centrinone-treated acentrosomal spindles of HeLa cells. Red, gray, green and blue represent NuMA, p150glued, Cep152 and DNA, respectively. Scale bar, 5 μm.(F) NuMA distribution in 3D. Images of NuMA (Red) and chromosomes (blue) were reconstructed in 3D and surface-rendered.(G) Representative images of NuMA (arrowheads) in centrosomal and acentrosomal cells before and after photobleaching. DMSO- or centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed. Time after photobleaching (sec) is indicated. Scale bar, 5 μm.(H) Normalized fluorescence intensities of NuMA-mClover at centrosomal poles (control, 2-centrosomes) or at acentrosomal spindle pole (0-centrosome) during the FRAP time course (N=20 cells for each condition). The first three time points are before photobleaching. Error bars show standard deviation (SD).(I) Mobile fractions from the normalized recovery curves of NuMA-mClover at centrosomal spindle poles (control, 2-centrosomes) or at acentrosomal spindle poles (0-centrosome). Mobile fractions (roughly corresponds to normalized fluorescence intensities at the last time point were determined by single exponential fitting. Line and error bars represent the median and interquartile range (N=20 cells for each condition). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


Figure 2 ( A ) Time - lapse observation of the establishment of bipolarity in acentrosomal cells . HeLa cells expressing EGFP - centrin1 and mCherry - NuMA were observed with a 63 × objective . Magenta and green represent mCherry - NuMA and EGFP - centrin1 , respectively . Arrowheads indicate the assembling NuMA after NEBD . Two - way arrows indicate the bipolarity . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( B ) Time - lapse observation of the dynamics of NuMA and chromosomes . Centrinone - treated HeLa cells expressing mCover - NuMA were observed with a 63 × objective . Magenta and green represent SiR - DNA and mClover - NuMA , respectively . Arrowheads and two - way arrows indicate the assembling NuMA after NEBD and bipolarity , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( C ) The timing of bipolarity establishment in the acentrosomal pole , chromosome alignment , and anaphase onset Each plot shows the cumulative percentage of each event at individual time points ( N = 49 cells from two independent experiments ) . ( D ) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells . Two - way arrow indicates the bipolarity of elongated NuMA structures . Red , gray , green , and blue represent NuMA , CENP - C , GT335 , and DNA , respectively . Scale bar , 5 μm . ( E , F ) Time - lapse observation of the structure of NuMA and microtubules upon centrosome removal . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were observed with a 60 × objective . Red and green represent NuMA and SiR - tubulin , respectively . Z - projections of 17 sections , every 1 μm . Scale bar , 5 μm . Time zero corresponds to NEBD . ( F ) An example of NuMA formed several asters ( arrowheads ) at the time of NEBD .

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

Figure 2(A) Time-lapse observation of the establishment of bipolarity in acentrosomal cells. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads indicate the assembling NuMA after NEBD. Two-way arrows indicate the bipolarity. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.(B) Time-lapse observation of the dynamics of NuMA and chromosomes. Centrinone-treated HeLa cells expressing mCover-NuMA were observed with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads and two-way arrows indicate the assembling NuMA after NEBD and bipolarity, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD.(C) The timing of bipolarity establishment in the acentrosomal pole, chromosome alignment, and anaphase onset Each plot shows the cumulative percentage of each event at individual time points (N=49 cells from two independent experiments).(D) The distribution of kinetochores during acentrosomal spindle pole separation was confirmed in fixed cells. Two-way arrow indicates the bipolarity of elongated NuMA structures. Red, gray, green, and blue represent NuMA, CENP-C, GT335, and DNA, respectively. Scale bar, 5 μm.(E, F) Time-lapse observation of the structure of NuMA and microtubules upon centrosome removal. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were observed with a 60× objective. Red and green represent NuMA and SiR-tubulin, respectively. Z-projections of 17 sections, every 1 μm. Scale bar, 5 μm. Time zero corresponds to NEBD. (F) An example of NuMA formed several asters (arrowheads) at the time of NEBD.


Figure 3 ( A ) NuMA structure upon microtubule depolymerization . Nocodazole - treated HCT116 CMV - OsTIR1 CEP152 - mClover - mAID cells were treated with 100 ng / ml nocodazole . Gray , red , green and blue represent α - tubulin , NuMA , CEP152 ( mClover ) and DNA , respectively . Scale bar , 5 μm . ( B ) Immunostaining of DHC1 and NuMA in centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover cells . Gray , green , red and blue represent DHC1 ( mClover ) , GT335 , NuMA , and DNA , respectively . Scale bar , 5 μm . ( D ) NuMA structure upon DHC1 depletion . Centrinone - treated HCT116 TetOsTIR1 DHC1 - 3X - mAID - mClover cells were treated with 1 μg / ml doxycycline ( Dox ) and 500 μM indole - 3 - acetic acid ( IAA ) . Arrowhead indicates the spindle pole . Green , red , gray and blue represent DHC1 ( mClover ) , NuMA , GT335 and DNA , respectively . Scale bar , 5 μm . ( E ) Frequency of acentrosomal pole patterns upon DHC1 depletion in ( D ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 24 spindles in each experiment ) . ( ( F ) The distribution of NuMA upon DHC1 depletion . Arrowheads indicate the presence of NuMA on microtubules . Green , red , and blue represent α - tubulin , NuMA , and DNA , respectively . Scale bars , 5 μm . ( G ) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA - mAID - mClover - FLAG with either mCherry - NuMA WT or 5A - 3 . Arrowheads indicate the assembled NuMA structure . Green , red , gray and blue represent endogenous NuMA ( mClover ) , expressed NuMA ( mCherry ) , GT335 and DNA , respectively . Z - projections of 21 sections , every 1 μm . Scale bar , 5 μm . ( H ) The number of NuMA structure in ( G ) . Values are mean percentages ± SD . from four independent experiments ( N ≧ 15 spindles in each experiment ) . One - way ANOVA with Tukey ' s multiple comparisons test was used to obtain a P value .

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

Figure 3(A) NuMA structure upon microtubule depolymerization. Nocodazole-treated HCT116 CMV-OsTIR1 CEP152-mClover-mAID cells were treated with 100 ng/ml nocodazole. Gray, red, green and blue represent α-tubulin, NuMA, CEP152 (mClover) and DNA, respectively. Scale bar, 5 μm.(B) Immunostaining of DHC1 and NuMA in centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells. Gray, green, red and blue represent DHC1 (mClover), GT335, NuMA, and DNA, respectively. Scale bar, 5 μm.(D) NuMA structure upon DHC1 depletion. Centrinone-treated HCT116 TetOsTIR1 DHC1-3X-mAID-mClover cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM indole-3-acetic acid (IAA). Arrowhead indicates the spindle pole. Green, red, gray and blue represent DHC1 (mClover), NuMA, GT335 and DNA, respectively. Scale bar, 5 μm. (E) Frequency of acentrosomal pole patterns upon DHC1 depletion in (D). Values are mean percentages ± SD. from three independent experiments (N≧24 spindles in each experiment). ( (F) The distribution of NuMA upon DHC1 depletion. Arrowheads indicate the presence of NuMA on microtubules. Green, red, and blue represent α-tubulin, NuMA, and DNA, respectively. Scale bars, 5 μm.(G) Structure of acentrosomal spindle poles upon replacement of endogenous NuMA-mAID-mClover-FLAG with either mCherry-NuMA WT or 5A-3. Arrowheads indicate the assembled NuMA structure. Green, red, gray and blue represent endogenous NuMA (mClover), expressed NuMA (mCherry), GT335 and DNA, respectively. Z-projections of 21 sections, every 1 μm. Scale bar, 5 μm. (H) The number of NuMA structure in (G). Values are mean percentages ± SD. from four independent experiments (N≧15 spindles in each experiment). One-way ANOVA with Tukey's multiple comparisons test was used to obtain a P value.


Figure 4 ( A , B ) Structure of the spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with 1 μg / ml doxycycline ( Dox ) and 500 μM IAA . Two - way arrow indicates the bipolarity . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , GT335 , and DNA , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( C ) Frequency of spindle structure patterns upon NuMA depletion in ( A , B ) . Values are mean percentages ± SD . from three independent experiments ( N = 30 spindles in each experiment ) . ( ( D ) The localization of Eg5 in centrinone - treated acentrosomal HeLa cells . Red , gray , green and blue represent NuMA , Eg5 , GT335 and DNA , respectively . Scale bar , 5 μm . ( E ) The localization of Eg5 in the spindle upon NuMA depletion . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with Dox and 500 μM IAA . Gray , green , red , and blue represent Eg5 , NuMA ( mClover ) , CP110 , and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm . ( F ) Structure of the spindle upon Eg5 inhibition . Centrinone - treated HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells were treated with 30 μM monastrol . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , GT335 , and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm . ( G ) Frequency of spindle structure patterns upon Eg5 inhibition in ( F ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 23 spindles in each experiment ) .

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

Figure 4(A, B) Structure of the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 1 μg/ml doxycycline (Dox) and 500 μM IAA. Two-way arrow indicates the bipolarity. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (C) Frequency of spindle structure patterns upon NuMA depletion in (A, B). Values are mean percentages ± SD. from three independent experiments (N=30 spindles in each experiment). ( (D) The localization of Eg5 in centrinone-treated acentrosomal HeLa cells. Red, gray, green and blue represent NuMA, Eg5, GT335 and DNA, respectively. Scale bar, 5 μm.(E) The localization of Eg5 in the spindle upon NuMA depletion. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with Dox and 500 μM IAA. Gray, green, red, and blue represent Eg5, NuMA (mClover), CP110, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.(F) Structure of the spindle upon Eg5 inhibition. Centrinone-treated HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells were treated with 30 μM monastrol. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), GT335, and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm. (G) Frequency of spindle structure patterns upon Eg5 inhibition in (F). Values are mean percentages ± SD. from three independent experiments (N≧23 spindles in each experiment).


Figure 5 ( A ) Time - lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5 . HeLa cells expressing EGFP - centrin1 and mCherry - NuMA were observed in the presence of 50 μM of monastrol with a 63 × objective . Magenta and green represent mCherry - NuMA and EGFP - centrin1 , respectively . Arrowheads and two - way arrows indicate the monopolar and bipolar states , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( B ) The time of bipolarity establishment and being stuck in the monopolar - like state of acentrosomal spindle poles in ( A ) . Each plot shows the cumulative percentage of each event at each time point ( N = total 75 cells from two independent experiments ) . ( ( C ) Time - lapse observation of the dynamics of mClover - NuMA and chromosomes . HeLa cells expressing mClover - NuMA were observed in the presence of SiR - DNA with a 63 × objective . Magenta and green represent SiR - DNA and mClover - NuMA , respectively . Arrowheads indicate initial bipolarity in the acentrosomal spindle pole ( 8 min ) , dispersed chromosomes ( 328 min ) , and the monopolar - like state of the acentrosomal spindle pole ( 332 min ) , respectively . Z - projections of 20 sections , every 1 . 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( D ) The time of bipolarity establishment and being stuck in the monopolar - like state of acentrosomal spindle poles in ( C ) . Each plot shows the cumulative percentage of each event at each time point ( N = total 35 cells from three independent experiments ) .

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Figure 5(A) Time-lapse observation of the establishment of bipolarity within the acentrosomal spindle pole upon inhibition of Eg5. HeLa cells expressing EGFP-centrin1 and mCherry-NuMA were observed in the presence of 50 μM of monastrol with a 63× objective. Magenta and green represent mCherry-NuMA and EGFP-centrin1, respectively. Arrowheads and two-way arrows indicate the monopolar and bipolar states, respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (B) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (A). Each plot shows the cumulative percentage of each event at each time point (N=total 75 cells from two independent experiments). ( (C) Time-lapse observation of the dynamics of mClover-NuMA and chromosomes. HeLa cells expressing mClover-NuMA were observed in the presence of SiR-DNA with a 63× objective. Magenta and green represent SiR-DNA and mClover-NuMA, respectively. Arrowheads indicate initial bipolarity in the acentrosomal spindle pole (8 min), dispersed chromosomes (328 min), and the monopolar-like state of the acentrosomal spindle pole (332 min), respectively. Z-projections of 20 sections, every 1.2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (D) The time of bipolarity establishment and being stuck in the monopolar-like state of acentrosomal spindle poles in (C). Each plot shows the cumulative percentage of each event at each time point (N=total 35 cells from three independent experiments).


Figure 6 ( A ) Structure of the spindle upon NuMA and Eg5 inhibition . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml doxycycline ( Dox ) , 500 μM IAA and monastrol . Green , red , and blue represent NuMA ( mClover ) , α - tubulin , and DNA , respectively . Z - projections of 10 sections , every 0 . 3 μm . Scale bar , 5 μm . ( B ) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in ( A ) . Values are mean percentages ± SD . from three independent experiments ( N ≧ 22 spindles in each experiment ) . ( ( C ) The localization of Eg5 in the spindle upon NuMA depletion . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml doxycycline Dox and 500 μM IAA . Gray , green , red , and blue represent α - tubulin , NuMA ( mClover ) , Eg5 and DNA , respectively . Z - projections of 5 sections , every 0 . 3 μm . Scale bar , 5 μm . ( D ) Time - lapse observation of the establishment of spindle bipolarity upon NuMA depletion . HCT116 TetOsTIR1 NuMA - mAID - mClover - FLAG cells with 2 - centrosomes were treated with 1 μg / ml Dox and 500 μM IAA and observed with a 40 × objective . Red and green represent SiR - tubulin and NuMA , respectively . Z - projections of 14 sections , every 2 μm . Scale bar , 10 μm . Time zero corresponds to NEBD . ( E ) The time required for the initial establishment of spindle bipolarity in ( D ) . Line and error bars represent the mean and SD . ( N ≧ 60 cells from two independent experiments ) . The Mann - Whitney U test ( two - tailed ) was used to obtain a P value .

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Figure 6(A) Structure of the spindle upon NuMA and Eg5 inhibition. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline (Dox), 500 μM IAA and monastrol. Green, red, and blue represent NuMA (mClover), α-tubulin, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (B) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in (A). Values are mean percentages ± SD. from three independent experiments (N≧22 spindles in each experiment). ( (C) The localization of Eg5 in the spindle upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline Dox and 500 μM IAA. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), Eg5 and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm.(D) Time-lapse observation of the establishment of spindle bipolarity upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml Dox and 500 μM IAA and observed with a 40× objective. Red and green represent SiR-tubulin and NuMA, respectively. Z-projections of 14 sections, every 2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (E) The time required for the initial establishment of spindle bipolarity in (D). Line and error bars represent the mean and SD. (N≧60 cells from two independent experiments). The Mann-Whitney U test (two-tailed) was used to obtain a P value.


Figure 1A - C . CHO cells expressing PD - L1 alone ( A ) or co - expressing CD80 ( B ) or CD86 ( C ) were stained with CD80 Ig , PD - 1 Ig , CTLA - 4 Ig or anti - PD - L1 Ab clones MIH1 , MIH3 , 29E . 2A3 ) at 1µg / ml for 30 min at 37 ° C . Representative FACS plots show staining plotted against total PD - L1 mCherry levels . E . Western blot analysis of an immunoprecipitation by anti - mCherry antibody ( PDL1 ) from indicated DG - 75 cell lysates , with and without the BS3 crosslinker , and probed for mCherry ( PD - L1 ) or GFP ( CD80 ) .

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Figure 1A-C. CHO cells expressing PD-L1 alone (A) or co-expressing CD80 (B) or CD86 (C) were stained with CD80 Ig, PD-1 Ig, CTLA-4 Ig or anti-PD-L1 Ab clones MIH1, MIH3, 29E.2A3) at 1µg/ml for 30 min at 37°C. Representative FACS plots show staining plotted against total PD-L1 mCherry levels.E. Western blot analysis of an immunoprecipitation by anti-mCherry antibody (PDL1) from indicated DG-75 cell lysates, with and without the BS3 crosslinker, and probed for mCherry (PD-L1) or GFP (CD80).


Figure 2A . Transendocytosis assays were carried out overnight using CHO donor cells ( CD80GFP alone or CD80GFP with PD - L1mCherry co - expression ) incubated with CTLA - 4 recipient cells at a ratio of 1 : 1 . Recipient cells either lacked CTLA - 4 ( No CTLA - 4 ) or expressed CTLA - 4 . FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA - 4 + recipients . C - F . Transendocytosis assays were performed at a ratio of 2 donor : 1 CTLA - 4 recipient for the times indicated . ( C ) , Representative FACS plots showing CD80GFP levels and ( D ) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA - 4 control ( mean ± SEM , 3 independent experiments , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons test ) . ( E ) Representative FACS plots showing PD - L1mCherry level at time points indicated . ( F ) Full kinetic analysis of PD - L1mCherry level on donor cells ( mean ± SEM , 3 independent experiments ) .

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Figure 2A. Transendocytosis assays were carried out overnight using CHO donor cells (CD80GFP alone or CD80GFP with PD-L1mCherry co-expression) incubated with CTLA-4 recipient cells at a ratio of 1:1. Recipient cells either lacked CTLA-4 (No CTLA-4) or expressed CTLA-4. FACS plots show CD80GFP ligand loss from donor cells highlighted in blue quadrants in the presence of CTLA-4+ recipients.C-F. Transendocytosis assays were performed at a ratio of 2 donor:1 CTLA-4 recipient for the times indicated. (C), Representative FACS plots showing CD80GFP levels and (D) full kinetic analysis of CD80 or CD86 downregulation on donor cells quantified as a percentage relative to no CTLA-4 control (mean ± SEM, 3 independent experiments, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test). (E) Representative FACS plots showing PD-L1mCherry level at time points indicated. (F) Full kinetic analysis of PD-L1mCherry level on donor cells (mean ± SEM, 3 independent experiments).


Figure 3D . Levels of CD86GFP and PD - L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA - 4 WT or Del36 plotted relative to no CTLA - 4 control ( mean ± SEM , 6 independent experiments ) . * * * * P ≤ 0 . 0001 , RM one - way ANOVA .

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Figure 3D. Levels of CD86GFP and PD-L1mCherry remaining on donor cells over a 24h transendocytosis period with CTLA-4 WT or Del36 plotted relative to no CTLA-4 control (mean ± SEM, 6 independent experiments). ****P≤0.0001, RM one-way ANOVA.


Figure 4A - B . APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was used to detect PD - L1 in DG - 75 cells co - expressing CD80GFP ( A ) or CD86GFP ( B ) . Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA - 4 , CTLA - 4 WT or CTLA - 4 Del36 ( at a ratio of 1 : 1 ) for the indicated durations . ( A ) shows representative FACS plots of CD80GFP vs . PD - 1 Ig at the time points indicated , with full kinetic data plotted below ( mean ± SEM , 3 independent experiments , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 : two - way ANOVA with Tukey ' s multiple comparisons test ) . ( B ) As for ( A ) except using CD86 - PD - L1 expressing cells .

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Figure 4A-B. APC-labelled PD-1 Ig (0.75µg/ml) was used to detect PD-L1 in DG-75 cells co-expressing CD80GFP (A) or CD86GFP (B). Staining was carried out following transendocytosis using Jurkat cells expressing no CTLA-4, CTLA-4 WT or CTLA-4 Del36 (at a ratio of 1:1) for the indicated durations. (A) shows representative FACS plots of CD80GFP vs. PD-1 Ig at the time points indicated, with full kinetic data plotted below (mean ± SEM, 3 independent experiments, ***P≤0.001, ****P≤0.0001: two-way ANOVA with Tukey's multiple comparisons test). (B) As for (A) except using CD86-PD-L1 expressing cells.


Figure 5A . Staining of free PD - L1 with APC - conjugated PD - 1 Ig in DG - 75 cells expressing only PD - L1mCherry , using cell lines with increasing ratios of CD80GFP : PD - L1mCherry compared to CD80GFP only . FACS plots are representative of 4 independent experiments .

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Figure 5A. Staining of free PD-L1 with APC-conjugated PD-1 Ig in DG-75 cells expressing only PD-L1mCherry, using cell lines with increasing ratios of CD80GFP: PD-L1mCherry compared to CD80GFP only. FACS plots are representative of 4 independent experiments.


Figure 6A . DG - 75 B cells expressing CD80GFP or CD86GFP ( green ) and PD - L1mCherry ( red ) were incubated with Jurkat cells ( nuclei shown in grey ) expressing CTLA - 4 WT or mutant CTLA - 4 Del36 for the indicated durations and analysed by confocal microscopy . 2 hours prior to assay endpoint , APC - labelled PD - 1 Ig ( 0 . 75µg / ml ) was added . CD80 / CD86 and PD - L1 co - localisation at the immune synapse is shown in yellow with PD - 1 Ig binding shown in purple . Scale bar , 10µm .

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Figure 6A. DG-75 B cells expressing CD80GFP or CD86GFP (green) and PD-L1mCherry (red) were incubated with Jurkat cells (nuclei shown in grey) expressing CTLA-4 WT or mutant CTLA-4 Del36 for the indicated durations and analysed by confocal microscopy. 2 hours prior to assay endpoint, APC-labelled PD-1 Ig (0.75µg/ml) was added. CD80/CD86 and PD-L1 co-localisation at the immune synapse is shown in yellow with PD-1 Ig binding shown in purple. Scale bar, 10µm.


Figure 7B . Flow cytometry plots of CD80GFP levels on DG - 75 B - cells ( gated on CTFR + cells ) at 24h with graphical representation of the MFI in each condition ( RH graph ) . The Jreg cells used , and the anti - PD - 1 status is indicated above the plot . C . Flow cytometry plots of CD28 levels indicating contact between the CD80 + APC and the PD - 1 + CD28 + responder Jurkat T - cells , following CD80 transendocytosis . Graphical representation of the CD28 MFI in each condition is shown on the right . D . Flow cytometry of CD69 levels on responder Jurkat T - cells and graphical representation of CD69 MFI in each condition . E . Flow cytometry of CD69 levels on CD28KO ( PD - 1 + ) responder Jurkat T - cells and graphical representation of the CD69 + cells MFI in each condition . Cartoons on the left of B - E illustrate the cell type analysed . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : two - way ANOVA with Tukey ' s multiple comparisons test .

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Figure 7B. Flow cytometry plots of CD80GFP levels on DG-75 B-cells (gated on CTFR+ cells) at 24h with graphical representation of the MFI in each condition (RH graph). The Jreg cells used, and the anti-PD-1 status is indicated above the plot. C. Flow cytometry plots of CD28 levels indicating contact between the CD80+ APC and the PD-1+ CD28+ responder Jurkat T-cells, following CD80 transendocytosis. Graphical representation of the CD28 MFI in each condition is shown on the right. D. Flow cytometry of CD69 levels on responder Jurkat T-cells and graphical representation of CD69 MFI in each condition. E. Flow cytometry of CD69 levels on CD28KO (PD-1+) responder Jurkat T-cells and graphical representation of the CD69+ cells MFI in each condition. Cartoons on the left of B-E illustrate the cell type analysed. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: two-way ANOVA with Tukey's multiple comparisons test.


Figure 8B . Titration of Abatacept staining ( left hand panel ) and EC50 values of Abatacept ( right hand panel ) for CD80 only or PD - L1 / CD80 co - expressing DG - 75 . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : paired t - test ( B ) D . Comparison of PD - L1 detection using PD - 1 - Ig or anti - PD - L1 antibodies ( MIH3 or 29E . 2A3 clone ) with and without prior incubation with 10µg / ml of abatacept , based on data from ( C ) . E . Graphs showing PD - L1 detection under conditions used in ( C ) following different Abatacept incubation periods ( 30mins , 3h or 6h ) . Data information : Data is representative of 3 independent experiments showing mean ± SEM . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , ns - not significant : RM one - way ANOVA ( D ) .

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Figure 8B. Titration of Abatacept staining (left hand panel) and EC50 values of Abatacept (right hand panel) for CD80 only or PD-L1/CD80 co-expressing DG-75. Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: paired t-test (B)D. Comparison of PD-L1 detection using PD-1-Ig or anti-PD-L1 antibodies (MIH3 or 29E.2A3 clone) with and without prior incubation with 10µg/ml of abatacept, based on data from (C). E. Graphs showing PD-L1 detection under conditions used in (C) following different Abatacept incubation periods (30mins, 3h or 6h). Data information: Data is representative of 3 independent experiments showing mean ± SEM. *P≤0.05, **P≤0.01, ns - not significant: RM one-way ANOVA (D).


Figure 1D Western blot of Sec63 , mCherry and Pgk1 from WT cells ( SSY1404 ) , cells harboring the estradiol - inducible system ( SSY1405 ) or ∆ opi1 cells ( SSY1607 ) , all of which expressed Sec63 - mNeon and Rtn1 - mCherry . Cells were untreated or treated with 800 nM estradiol for 6 h . Pgk1 served as a loading control . E Quantification of ER size in estradiol - treated cells harboring the inducible system ( SSY1405 ) , untreated ∆ opi1 cells ( SSY1607 ) and WT cells ( SSY1404 ) treated with 8 mM DTT for 1 h . Bars represent mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with 0 nM estradiol or ∆ opi1 cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 ; n . s . , not significant .

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Figure 1D Western blot of Sec63, mCherry and Pgk1 from WT cells (SSY1404), cells harboring the estradiol-inducible system (SSY1405) or ∆opi1 cells (SSY1607), all of which expressed Sec63-mNeon and Rtn1-mCherry. Cells were untreated or treated with 800 nM estradiol for 6 h. Pgk1 served as a loading control.E Quantification of ER size in estradiol-treated cells harboring the inducible system (SSY1405), untreated ∆opi1 cells (SSY1607) and WT cells (SSY1404) treated with 8 mM DTT for 1 h. Bars represent mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with 0 nM estradiol or ∆opi1 cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01; n.s., not significant.


Figure 2A Sec63 - mNeon images of cells of the indicated genotypes harboring the inducible system . Cells were treated with 800 nM estradiol for 6 h . Two examples of each phenotypic class are shown . C Sec63 - mNeon and Rtn1 - mCherry images of ∆ lnp1 and ∆ sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h .

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Figure 2A Sec63-mNeon images of cells of the indicated genotypes harboring the inducible system. Cells were treated with 800 nM estradiol for 6 h. Two examples of each phenotypic class are shown.C Sec63-mNeon and Rtn1-mCherry images of ∆lnp1 and ∆sey1 cells harboring the inducible system and treated with 800 nM estradiol for 6 h.


Figure 3C Quantification of peripheral ER structures in WT and ∆ ice2 cells harboring the inducible system ( SSY1405 , 1603 ) and treated with 800 nM estradiol for the times indicated . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant . E Quantification of peripheral ER structures in untreated WT , Δice2 , Δopi1 and Δice2 Δopi1 cells ( SSY1404 , 2356 , 2595 , 2811 ) . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

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Figure 3C Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


Figure 4C , D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63 - mNeon and Rtn1 - mCherry ( SSY1405 , 1603 ) that were untreated ( C ) or treated with 8 mM DTT for 1 h ( D ) .

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Figure 4C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).


Figure 5B Growth assays of untreated WT , Δice2 , Δnem1 , Δnem1 Δice2 , Δspo7 , Δspo7 Δice2 , Δpah1 and Δpah1 Δice2 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 , 2807 , 2808 ) . Numbers represent areas under the curves and serve as growth indices . Mean + s . e . m . , n = 3 biological replicates . Data for WT and ∆ ice2 cells are the same in the left and middle panels . C , D Lipidomic analysis of WT , Δice2 , Δnem1 , Δice2 Δnem1 , Δspo7 and Δice2 Δspo7 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 . Data for WT and Δice2 cells are the same as in both panels . E Sec63 - mNeon images of untreated WT , Δnem1 , Δnem1Δice2 , Δspo7 and Δspo7 Δice2 cells ( SSY1404 , 2482 , 2484 , 2481 , 2483 ) .

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Figure 5B Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.C, D Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).


Figure 6D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1 - HA from ∆ nem1 mutants ( SSY3065 ) and microsomes from cells of the indicated genotypes ( SSY3053 , 3074 , 3075 , 3095 ) . Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase ( PPase ) are shown for reference . E Western blot of HA , Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ ice2 cells expressing Nem1 - HA ( SSY3140 , 3141 ) . Nem1 is undetectable in cell lysates due to its low abundance .

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Figure 6D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.


Figure 7Western blots of FLAG , HA and Dpm1 from cell lysates or anti - FLAG immunoprecipitates of WT or ∆ nem1 cells containing Spo7 - FLAG , Nem1 - FLAG or Ice2 - HA as indicated ( SSY122 , 2421 , 3183 , 3184 , 3195 , 3196 , 3197 ) . The asterisk marks the position of the light chain from the antibody used for immunoprecipitation . The diamond marks a protein that is non - specifically precipitated by the anti - FLAG antibody . IP , immunoprecipitate . E Images of cells with endogenously tagged Ice2 - mScarlet and Spo7 - mNeon or Nem1 - mNeon ( SSY3244 , 3245 ) . Arrows indicate foci containg both Ice2 and either Spo7 or Nem1 . F Quantification of Spo7 and Nem1 foci in WT and ∆ ice2 cells ( SSY2916 , 3238 , 2917 , 3239 ) . Mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with the respective WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 .

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Figure 7Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, Nem1-FLAG or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197). The asterisk marks the position of the light chain from the antibody used for immunoprecipitation. The diamond marks a protein that is non-specifically precipitated by the anti-FLAG antibody. IP, immunoprecipitate.E Images of cells with endogenously tagged Ice2-mScarlet and Spo7-mNeon or Nem1-mNeon (SSY3244, 3245). Arrows indicate foci containg both Ice2 and either Spo7 or Nem1. F Quantification of Spo7 and Nem1 foci in WT and ∆ice2 cells (SSY2916, 3238, 2917, 3239). Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the respective WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


Figure 8A Lipidomic analysis of WT and ∆ ice2 cells in which PAH1 was replaced with PAH1 - HA or pah1 ( 7A ) - HA as indicated ( SSY2841 , SSY2842 , SSY2970 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 .

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Figure 8A Lipidomic analysis of WT and ∆ice2 cells in which PAH1 was replaced with PAH1-HA or pah1(7A)-HA as indicated (SSY2841, SSY2842, SSY2970). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


Figure 9A Sec63 - mNeon images of mid and cortical sections of untreated WT and ∆ opi1 cells , overexpressing ICE2 where indicated ( SSY1404 , 2588 , 2595 , 2596 ) . D Growth assays on solid media of WT , ∆ hac1 , Δice2 , and Δhac1 Δice2 cells ( SSY1404 , 2356 , 2805 , 2806 ) in the absence or presence of 0 . 2 µg / ml tunicamycin . For each series , cells were diluted fivefold from one step to the next .

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Figure 9A Sec63-mNeon images of mid and cortical sections of untreated WT and ∆opi1 cells, overexpressing ICE2 where indicated (SSY1404, 2588, 2595, 2596).D Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.


Figure 1A . Example of littermate males of the indicated genotypes 26 days post partum . Additional examples are provided in Appendix Fig S2 . B . Weights of mice of the indicated genotypes from 18 to 120 days post partum . Boxplots indicate 5 - 95 percentile . A minimum of 8 animals is plotted at each age . Asterisks denote statistical significance ( NS , not significant , * P value < 0 . 05 , * * P value < 0 . 01 and * * * P value < 0 . 001 ) determined by the unpaired two - way Wilcoxon rank - sum test . C . Example of dissected brains ( upper panel ) and coronal sections ( left and right panels ) stained with H & amp ; amp ; E of the indicated genotypes . Note the severe hydrocephaly and ventricular dilation of the lateral ventricle ( LV ) and third ventricle ( TV ) in a Gemc1 - / - mouse ( bottom right panel ) compared to a Gemc1 + / + littermate animal ( bottom left panel ) . Periventricular malacia ( asterisks ) and hippocampal atrophy ( arrow ) is also evident in the Gemc1 - / - sample . Scale bars = 500 µm . D . Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers ( n ) . The study was terminated at 16 months and mice were examined for histology . 97 % ( 31 / 32 ) of the Gemc1 - / - mice examined to date exhibited hydrocephaly ( additional histological examples in Appendix Fig S2 ) . E . RT - QPCR analysis of Gemc1 expression in murine tissues from Gemc1 + / + mice using a Taqman probe . Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted . F . RT - QPCR analysis of Gemc1 in trachea , oviduct and ovary tissue from Gemc1 + / + and Gemc1 - / - mice shows that the knockout reduces Gemc1 mRNA to undetectable levels . Mean and standard deviation of duplicate samples from 2 female mice are plotted . Additional examples from other Gemc1 - / - tissues are shown in Appendix Fig S1 .

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Figure 1A. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2.B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test.C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&amp;E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm.D. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2).E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted.F. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.


Figure 2 . Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age . B . Testes from Gemc1 - / - mice have strongly reduced cellularity compared to Gemc1 + / + or Gemc1 + / - animals , as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber ( n = 3 ) . Mean and standard deviation are indicated . C . Histological analysis of tubules from 6 - week - old Gemc1 - / - mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids . Scale bars = 100 µm ( left panels ) and 50 µm ( right panels ) . D . Sperm counts from the cauda epididymis of wild type and Gemc1 - / - mice revealed no sperm in mice lacking GEMC1 ( n = 3 ) . Mean and standard deviation are indicated . E . Examples of sperm morphology from spermatocyte spreads from 2 month old mice . Scale bars = 10 µm . F and G . Histological evaluation of the caput epididymis in sagittal ( F ) and coronal ( G ) orientation from 2 and 4 month old animals , respectively . Note the absence of luminal spermatozoa in the Gemc1 - / - mice compared to wild type . Scale bars = 100 µm ( F , left panels ) and 50 µm ( F , right panels and G ) .

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Figure 2. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age.B. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated.C. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels).D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm.F and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).


Figure 3A . Examples of ovaries from Gemc1 + / + ( top panels ) and Gemc1 - / - mice ( bottom panels ) at 6 weeks of age . Ovaries were smaller in Gemc1 - / - mice and contained primarily degenerated antral follicles . A histologically normal antral follicle is indicated in the Gemc1 + / + ovary ( top right , black arrow ) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1 - / - ovaries ( bottom right , red arrows ) . Scale bars = 200 µm ( left panels ) ) and 100 µm ( right top panels and right bottom panel respectively ) . B . Morphological abnormalities in the oviduct of 3 - week - old Gemc1 - / - mice ( right panels ) compared to Gemc1 + / + ( left panels ) . A clear lack of cilia ( bottom panels ) in Gemc1 - / - oviducts is observed by H & amp ; amp ; E staining . Scale bars = 50 µm ( top panels ) . C . Staining of frozen sections of oviduct with antibodies for Ac - tubulin ( cilia ) and γ - tubulin ( basal bodies ) reveals normal cilia organization in the wild type oviduct ( left ) while basal bodies and cilia are not detectable in Gemc1 - / - oviducts ( right ) . D . FOXJ1 and E . RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1 + / + oviducts and absent in Gemc1 - / - mice . Scale bars = 50 µm ( C - E ) .

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Figure 3A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&amp;E staining. Scale bars = 50 µm (top panels).C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).


Figure 4A . H & amp ; amp ; E staining of the nasal mucosa of E17 . 5 embryos of the indicated genotype . Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1 + / + embryos compared to the attenuated , flat columnar to squamous epithelium of Gemc1 - / - embryos . Scale bars = 20 µm . B . PAS staining ( purple ) of the nasal mucosa of E18 . 5 embryos of the indicated genotype . Normal PAS positive goblet cells ( inset , red arrows ) are interspersed between MCCs ( inset , black arrows ) with their characteristic goblet or cup - like shape in Gemc1 + / + embryos . The Gemc1 - / - mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell ( inset , red arrows ) and lacks typical goblet cells . Scale bars = 20 µm . C . Transmission electron micrographs of cross - sections of adult trachea from Gemc1 + / + animals revealed the presence of secretory cells ( SC ) and multiciliated cells ( MCC ) . The apical membrane of MCCs accumulated basal bodies ( BB ) nucleating multiple cilia ( C ) and microvilli ( MV ) . In the Gemc1 - / - airway epithelium , no MCCs are identifiable and only secretory - like cells with MV at the apical surface are detectable . Scale bars = 5µm . Higher magnifications of the boxed regions are shown below , scale bars = 0 . 5µm . D . Lack of Ac - tubulin ( top panel ) and RFX3 ( second panel from top ) staining in the trachea of 1 month old mice . E . Cilia ( Ac - tubulin staining ) are not detectable in the ependymal cells of 2 month old Gemc1 - / - mice ( right panel ) . Choroid plexus ( CP ) and lateral ventricles ( LV ) are indicated . Scale bars = 20

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Figure 4A. H&amp;E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1+/+ embryos compared to the attenuated, flat columnar to squamous epithelium of Gemc1-/- embryos. Scale bars = 20 µm.B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm.C. Transmission electron micrographs of cross-sections of adult trachea from Gemc1+/+ animals revealed the presence of secretory cells (SC) and multiciliated cells (MCC). The apical membrane of MCCs accumulated basal bodies (BB) nucleating multiple cilia (C) and microvilli (MV). In the Gemc1-/- airway epithelium, no MCCs are identifiable and only secretory-like cells with MV at the apical surface are detectable. Scale bars = 5µm. Higher magnifications of the boxed regions are shown below, scale bars = 0.5µm.D. Lack of Ac-tubulin (top panel) and RFX3 (second panel from top) staining in the trachea of 1 month old mice.E. Cilia (Ac-tubulin staining) are not detectable in the ependymal cells of 2 month old Gemc1-/- mice (right panel). Choroid plexus (CP) and lateral ventricles (LV) are indicated. Scale bars = 20 µm


Figure 5E and F . RT - QPCR analysis of individual genes related to MCC formation in the oviduct ( E ) and trachea ( F ) of Gemc1 - / - mice compared to wild type littermates , using 18S rRNA as a reference ( n = 2 ) . Genes examined generally fall into 3 classes : centriole duplication ( Cep152 , Plk4 , Cetn2 ) , Deuterosome mediated centriole expansion ( Deup1 , Ccdc78 , Ccno ) and transcription of genes required for MCC differentiation ( FoxJ1 , Myb , Mcidas ) .

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Figure 5E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).


Figure 6A . GEMC1 interacts with E2F4 and DP1 . Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel . FLAGimmunoprecipiation ( IP ) of GEMC1co - IPs DP1 ( α - HA , lane 5 ) and E2F4 ( α - HA , lane 6 ) . The interaction is enhanced upon co - expression of HA tagged E2F4 and DP1 ( α - HA , lane 7 ) . B . GEMC1 interacts with E2F5 but not E2F1 . Co - IP of E2F5 ( α - HA , lane 3 ) and E2F5 - DP1 ( α - HA , lane 6 ) but not E2F1 or E2F1 - DP1 ( α - HA , lanes 2 and 5 ) . In the FLAG - IP panels , a short ( top ) and long ( second from top ) exposure of the Western are shown . C . Identification of the domain required for E2F4 interaction . GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP . Wild type GEMC1 ( lane 5 ) or GEMC1 lacking the CC domain ( lane 6 ) pulled down E2F4 - DP1 . A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4 - DP1 ( lanes 7 , 8 ) . D . Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients ( G313D ) impairs the interaction with E2F4 - DP1 ( compare land 5 and 6 ) . E . GEMC1 interacts with Multicilin through its CC domain . Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc - tagged Multicilin ( anti - Myc blot , lanes 4 and 6 ) while a CC domain mutant does not ( anti - Myc blot , lane 5 ) .

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Figure 6A. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAGimmunoprecipiation (IP) of GEMC1co-IPs DP1 (α-HA, lane 5) and E2F4 (α- HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7).B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown.C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8).D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6).E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5).


Figure 7A . Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression ( RT - QPCR ) in HEK293T cells . Results for MCIDAS are graphed on a smaller scale in the right panel : V = vector and G = GEMC1 . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar induction of FOXJ1 was observed in U2OS and T98G ( Fig EV3 ) . B . Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 ( RT - QPCR ) . A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar results were also observed in U2OS ( Fig EV3 ) . C . Activation of FOXJ1 by GEMC1 is inhibited by the co - expression of Geminin ( compare 3rd and 4th lanes ) . Example Western blot is shown below . Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells . D . Co - expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression ( measured by RT - QPCR ) as Multicilin alone in HEK293T cells . The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed . E and F . The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin . HEK293T cells were transfected with a control siRNA ( siC ) or 2 different siRNAs against MCIDAS ( si1 and si2 ) prior to being transfected with GFP or GFP - GEMC1 expression vectors . Relative MCIDAS ( E ) and FOXJ1 ( F ) expression levels were assessed by RT - QPCR . The mean and standard deviation of a representative experiment performed in triplicate is graphed . An additional experiment is presented in Fig EV3 .

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Figure 7A. Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression (RT-QPCR) in HEK293T cells. Results for MCIDAS are graphed on a smaller scale in the right panel: V = vector and G = GEMC1. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar induction of FOXJ1 was observed in U2OS and T98G (Fig EV3).B. Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 (RT-QPCR). A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar results were also observed in U2OS (Fig EV3).C. Activation of FOXJ1 by GEMC1 is inhibited by the co-expression of Geminin (compare 3rd and 4th lanes). Example Western blot is shown below. Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells.D. Co-expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression (measured by RT-QPCR) as Multicilin alone in HEK293T cells. The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed.E and F. The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin. HEK293T cells were transfected with a control siRNA (siC) or 2 different siRNAs against MCIDAS (si1 and si2) prior to being transfected with GFP or GFP-GEMC1 expression vectors. Relative MCIDAS (E) and FOXJ1 (F) expression levels were assessed by RT-QPCR. The mean and standard deviation of a representative experiment performed in triplicate is graphed. An additional experiment is presented in Fig EV3.


Figure 1 A ) Transcriptome comparison of hemocytes from stage 16 embryos ( E16 ) and wandering 3rd instar larvae ( WL ) . The x - axis is the average genes expression levels ( n = 3 ) and the y - axis the Log2 Fold Change WL / E16 . P - values are indicated with the color code . Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in hemocytes Scatter plots as in ( A ) highlighting in black subsets of known genes associated with the GO terms extracellular matrix Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in phagocytosis F - G ) Phagocytosis assay on E16 ( F ) and WL hemocytes ( G ) srp ( hemo ) - moesin - RFP . The beads ( in green ) are phagocytosed by the hemocytes ( in red ) . The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure . Full stacks are displayed and the scale bars represent 20 µm .

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Figure 1 A) Transcriptome comparison of hemocytes from stage 16 embryos (E16) and wandering 3rd instar larvae (WL). The x-axis is the average genes expression levels (n=3) and the y-axis the Log2 Fold Change WL/E16. P-values are indicated with the color code.  Scatter plots as in (A) highlighting in black subsets of known genes expressed in hemocytes  Scatter plots as in (A) highlighting in black subsets of known genes associated with the GO terms extracellular matrix  Scatter plots as in (A) highlighting in black subsets of known genes expressed in phagocytosis  F-G) Phagocytosis assay on E16 (F) and WL hemocytes (G) srp(hemo)-moesin-RFP. The beads (in green) are phagocytosed by the hemocytes (in red). The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure. Full stacks are displayed and the scale bars represent 20 µm.


Figure 2 A ) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL ( NI dataset ) . D ) Top 5 markers of each cluster . The expression levels is represented by the gradient of purple levels ) and the percentage of cells with the size of the dots .

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Figure 2 A) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL (NI dataset). D) Top 5 markers of each cluster. The expression levels is represented by the gradient of purple levels) and the percentage of cells with the size of the dots.


Figure 3 A ) Identification of the position ( circulating / resident ) of the clusters within the larva by qPCR . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment . Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment ( n = 5 , mean + / - SD are represented on the graph ) . The p - values are estimated by bilateral paired student test B ) Circulating and resident hemocytes ( top and bottom panels , respectively ) from Mimic - Lsp1beta - MI05460 WL , which express Lsp1beta tagged with GFP . The immunolabelling was done with anti - GFP ( in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI and GFP , the right panels show the GFP alone . The scale bars represent 10 µm . C ) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC - Lsp1beta - MI05460 larvae . The OregonR value indicates the background level . The number of cells included in the analysis is displayed on the x - axis label and were quantified from two independent preparation , p - values were estimated after variance analysis with bilateral student test for equal variance . D ) Resident hemocytes located around the oenocytes or along the muscles ( top and bottom panels , respectively ) from WL srp ( hemo ) - moesin - RFP , that express moesin tagged with RFP in hemocytes . The immunolabelling was done with anti - RFP ( in red ) , anti - Pendulin ( Pen , in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , Pen and RFP , the middle panels RFP alone and the right panels Pen alone . Arrow heads in the top panels indicate cells co - expressing RFP and Pen . The scale bars represent 50 µm .

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

Figure 3 A) Identification of the position (circulating/resident) of the clusters within the larva by qPCR. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment. Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment (n=5, mean +/- SD are represented on the graph). The p-values are estimated by bilateral paired student test  B) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC-Lsp1beta-MI05460 larvae. The OregonR value indicates the background level. The number of cells included in the analysis is displayed on the x-axis label and were quantified from two independent preparation, p-values were estimated after variance analysis with bilateral student test for equal variance. D) Resident hemocytes located around the oenocytes or along the muscles (top and bottom panels, respectively) from WL srp(hemo)-moesin-RFP, that express moesin tagged with RFP in hemocytes. The immunolabelling was done with anti-RFP (in red), anti-Pendulin (Pen, in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, Pen and RFP, the middle panels RFP alone and the right panels Pen alone. Arrow heads in the top panels indicate cells co-expressing RFP and Pen. The scale bars represent 50 µm.


Figure 4A ) Heatmap representing the z - score for the top 5 regulons of each cluster determined with SCENIC . The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset . The z - score is indicated with a gradient from magenta ( z - score < 0 , the regulon is repressed ) to yellow ( z - score > 0 , the regulon is active ) .

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

Figure 4A) Heatmap representing the z-score for the top 5 regulons of each cluster determined with SCENIC. The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset. The z-score is indicated with a gradient from magenta (z-score < 0, the regulon is repressed) to yellow (z-score > 0, the regulon is active).


Figure 5A ) UMAP projection representing the 16 clusters of cells identified in the WI sample . Note the presence of two additional clusters , LMLM - 1 and LM - 2 , compared to the NI sample . The two clusters correspond to the lamellocytes . B ) Estimation of the number of cells of each cluster per larva in normal condition ( NI , orange ) and after wasp infestation ( WI , blue ) . For each cluster , the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets . D ) Dot plot representing the expression levels ( gradient of red levels ) and the percentage of cells ( size of the dots ) that express the top 5 markers of each cluster in the WI dataset .

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

Figure 5A) UMAP projection representing the 16 clusters of cells identified in the WI sample. Note the presence of two additional clusters, LMLM-1 and LM-2, compared to the NI sample. The two clusters correspond to the lamellocytes. B) Estimation of the number of cells of each cluster per larva in normal condition (NI, orange) and after wasp infestation (WI, blue). For each cluster, the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets. D) Dot plot representing the expression levels (gradient of red levels) and the percentage of cells (size of the dots) that express the top 5 markers of each cluster in the WI dataset.