EMBO/soda-nel huggingface dataset:
SHLD2 promotes class switch recombination by preventing 
inactivating deletions within the Ig locus (2020)
Figure 3

Figure 3. SHLD2-deficient B cells and B cells deficient in other NHEJ factors exhibit an Iglo population upon CSR induction.

A. WT and Shld2−/− B cells were purified from spleens and stimulated with LPS + IL4 and examined for IgM and IgG1 expression 3, 6, and 9 days post-stimulation by flow cytometry. Representative plots are shown for both WT and Shld2−/− B cells 6-days post stimulation. The graph plots show proportion of IgG1+ and Iglo cells, mean ± SD from 6 biological replicates; ** P ≤ 0.01, *** P ≤ 0.001, two-way ANOVA with post hoc Dunnett's test.

B. WT CH12 cells, as well as two each of 53BP1-, SHLD1-, SHLD2-, and SHLD3-deficient clones generated previously [21], as well as a LIG4-deficient CH12 clone were subjected to CSR induction with the CIT cocktail and measured for both IgM and IgA expression by flow cytometry. Representative flow plots for WT and 53bp1−/− CH12 cells are shown at day 3 post CIT stimulation. The graph plots show proportion of IgA+ and Iglo CH12 cells, mean ± SD from 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test. The letters below the x-axis represent the clone codes for the 53BP1, SHLD1, SHLD2, and SHLD3-deficient CH12 clones. One LIG4-deficient CH12 clone was also used.

C. The Xlf, Ku70, Ku80, Xrcc4, and Paxx genes were knocked out in CH12 cells by CRISPR, and two independent clones each were analyzed as in Figure 3B with mean ± 3 biological replicates; * P ≤ 0.05, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.
SHLD2 promotes class switch recombination by preventing 
inactivating deletions within the Ig locus (2020)
Figure 4

Figure 4. Reduced Ig expression in 53BP1, SHLD2, and SHLD3-deficient CH12 cells is permanent and dependent on CSR.

A. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 cells were stimulated with CIT and analyzed by flow cytometry for IgM and IgA expression. The Iglo population were reduced after 7 days in culture. Values are mean ± SD from 3 biological replicates; * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001, two-way ANOVA with post hoc Dunnett's test.

B. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort (see Figure EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post CIT treatment is shown.
SHLD2 promotes class switch recombination by preventing 
inactivating deletions within the Ig locus (2020)
Figure 5

Figure 5. Loss of Ig cell-surface expression in CH12 cells is accompanied by aberrant IgA transcripts.

A. mRNA expression analysis of IgM and IgA was carried out by RT-PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT-PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (bottom) as control. Appendix Figure S2 shows the expression of IgA and IgM by flow cytometry for each of these subclones.

B. mRNA expression analysis as in A of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones, and IgA+ subclones from Shld2-/-/- clone F3. The Iglo subclones derived from the Shld2-/-/- CH12 F2 clone are listed as H03-H40, while the Iglo subclones derived from the Shld2-/-/-CH12 F3 clone are listed as J02-J109.

C. Representative IgA cDNA sequence analysis from WT Iglo clones.
SHLD2 promotes class switch recombination by preventing 
inactivating deletions within the Ig locus (2020)
Figure 6

Figure 6. Loss of Ig expression in CH12 cells is accompanied by large deletions within the IgA constant region.

A. Schematic of the Ighm and Igha loci juxtaposed by their respective switch regions, along with primer binding sites for long-range PCR.

B. Long-range PCR of Iglo subclones from WT and two independent 53bp1-/- CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show long-range amplicons (top) and AID genomic DNA PCR control (bottom).

C. Long-range PCR as in B of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones (left 2 panels), and IgA+ subclones from Shld2-/-/- clone F3.

D. Sequence analysis of a representative subset of long range-PCR amplicons. Symbol annotations: (*) inverted sequence, (◊) rearranged fragment order, (†) amplified with the EμF primer rather than IμF, (‡) undamaged Cα coding sequence.

Expanded View Figure Legends
An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma (2022)
Figure 3

Fig 3. TgCRMPa and TgCRMPb are in complex with two additional membrane proteins, one of which is required for rhoptry exocytosis.

Mass spectrometric identification of proteins co-isolated with HA3-tagged TgCRMPa (left plot) and TgCRMPb (right plot). The volcano plots shown here were generated by plotting the ̶ log10 t-test P-value versus the t-test difference. The colored dots mark members of the CRMP complex. Baits are indicated by darker outlines.

Co-immunoprecipitation of TgCRMPa and TgCRMPb. Lysates from parasites co-expressing TgCRMPa-FLAG3 and TgCRMPb-HA3 were split and incubated with either anti-FLAG (left panels) or anti-HA beads (right panels). Eluates were subjected to SDS-PAGE and immunoblotted with anti-HA and anti-FLAG Abs. Untagged, TgCRMPa-FLAG3 and TgCRMPb-HA3 parasites were used as controls. TgCRMPs robustly associate with each other.

Protein domains of Tg247195 and Tg277910, co-purified with TgCRMPs. TSP: thrombospondin domain; H-lectin: lectin binding domain. Green: predicted signal peptides; blue: transmembrane domains.

Immunofluorescence images of untagged, Tg277910-HA3 and Tg277910_iKD intracellular tachyzoites. Parasites were stained with anti-HA and anti-ARO Abs to label Tg277910 and rhoptries, respectively. The nuclei (DNA) are stained with Hoechst. Tg277910 localization is similar to that of TgCRMPs, with an increased fluorescence in the iKD line (-ATc). Tg277910 signal almost completely disappeared upon 72h ATc-treatment. Single focal planes are shown.

Invasion of parental and Tg277910_iKD lines in absence of ATc, and upon 72h ATc treatment. Values are reported as mean ± SD (n=3 biological replicates, each with 3 technical replicates). The biological replicates are represented by different symbols.

Quantification of rhoptry secretion in Tg277910_iKD by SeCreEt system as described in Fig 2I. Tg277910-depleted parasites failed to deliver rhoptry content into the host cytosol. Values are reported as mean ± SD (n=3 biological replicates). The biological replicates are represented by different symbols.

Quantification of microneme secretion in control and Tg277910-depleted tachyzoites was measured as in Fig 2H. Blots were probed with anti-MIC2 (secretion of micronemes) and anti-GRA3 (constitutive secretion of dense granules). P: Parasites pellet. Sup: Supernatant from untreated parasites. Sup+Prop: Supernatant from parasites treated with propranolol. The results are representative of two independent experiments.

Data information: P values in E, F, were measured by two-tailed t-test.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 1

Figure 1

Characterization of single and double knock-out cell lines

A Schematic representation of AKAP450, PCNT and CDK5Rap2 proteins illustrating localization of the epitopes recognized by the antibodies and the sequences targeted by the siRNAs used in this study.

B Representative WBs of RPE-1 wild-type (WT) cells and the two selected KO clones for AKAP450 (akap KO), PCNT (pcnt KO) and CDK5Rap2 (c5rap2 KO) probed with the indicated antibodies.

C Confocal IF images of RPE-1 WT, single KO (akap KO, pcnt KO and c5rap2 KO) and double KO (pc-ak-2KO, left panels; pc-c5-2KO, right panels) cells stained with the indicated antibodies. The yellow dashed line indicates the nucleus contour (N) and the arrows point the centrosome (CTR). GA marks the Golgi Apparatus. Scale bars: 5 μm.

D Representative WB of RPE-1 WT cells and the two selected PCNT/AKAP450 (pc-ak-2KO) and PCNT/CDK5Rap2 (pc-c5-2KO) double knockout mutated clones probed with the indicated antibodies.

E Representative WB of RPE-1 WT cells and the indicated single or double KO cell lines probed with anti-AKAP450, PCNT and CDK5Rap2 antibodies (left). Quantification of signal intensity from three independent WB experiments is shown at right. # indicates KO proteins. The arrow points the dramatic decrease of CDK5Rap2 levels in pc-ak-2KO cell line.

Data information: GM130 was used as a loading control in B and D. Hsp70 was used as a loading control in E. For statistical analysis we used one-way ANOVA followed by Dunnett's multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001; n.s., non-significant.

Source data are available online for this figure.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 2

Figure 2

Analysis of centrosome- and GA-MT nucleation

A Confocal images of WT, akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO and pc-c5-2KO cells stained for EB1 (green) and the centrosomal protein CAP350 (red). High magnification single-channel images of selected areas are shown at right.

B Scatter plot showing quantification of EB1 comets from three independent experiments as that shown in A. A region of interest of 3 μm radius around the CAP350 signal was used to count the number of comets. Horizontal black lines represent the mean.

C MT regrowth experiments of WT cells mixed with either WT (as a control), akap KO, c5rap2 KO, pcnt KO, pc-ak-2KO or pc-c5-2KO cells. Samples were fixed 3 min after NZ washout and stained for EB1 (green) and the indicated protein in the red channel.

D Quantification of EB1 signal intensity around the centrosome from three experiments as that shown in C.

E WT and pc-ak-2KO cells were transfected with a combination of three siRNAs against CEP192, fixed and triple labelled for EB1, CEP192 and CAP350, as a centrosomal marker. Scramble siRNA was used as a control.

F EB1 comets around the centrosome were quantified as in A.

G Three min MT regrowth experiments in WT and pc-ak-2KO cells transfected with either scramble or CEP192 siRNAs.

H Quantification of the EB1 signal intensity around the centrosome as in D.

I MT regrowth experiments in WT and c5rap2 KO or pcnt KO cells. Cells were stained as indicated. Arrows point the centrosome.

J Quantification of EB1 signal intensity at Golgi membranes.

Data information: Data were collected from three independent experiments and normalized to WT mean. For statistical analysis in A-H we used one-way ANOVA followed by Dunnett's multiple comparisons test and for statistical analysis of J, unpaired two-tailed Student's t-test was employed. *** p<0.001 and **** p<0.0001. Scale bars, 5 μm.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 3

Figure 3

Distribution of γ-TuRC-binding factors containing complexes

A-B WB of AKAP450 (A) and PCNT (B) co-immunoprecipitation assays revealed with the indicated antibodies. Asterisks mark immunoglobulin heavy chains. Cntrl: non-specific Igs immunoprecipitates; IP: specific Igs immunoprecipitates; SP: supernatant.

C-G (Left) Centrosomes from WT, single and double KO cells, as indicated, were labelled for AKAP450 and PCNT (C-E), CDK5Rap2 (F) and γ-tubulin (G). Right panels show quantifications. A region of interest of 1,5 μm radius was drawn around the centrosome and fluorescence intensity was determined and normalized to WT mean.

H WT and pc-ak-2KO cells were transfected with either scramble or CEP192 siRNAs, fixed and stained for γ-tubulin. Quantification of the fluorescence intensity is show at right.

I IF images of WT and pcnt KO RPE-1 cells treated with NZ for 3h and triple stained for AKAP450 (green), CDK5Rap2 (red) and GMAP210 as a Golgi marker (blue). At right, quantification of fluorescence co-localization of either AKAP450 or CDK5Rap2 with the Golgi marker GMAP210 in WT and pcnt KO cells­.

J Scheme summing up the changes in AKAP450-based and PCNT-based complexes distribution in WT, akap KO and pcnt KO RPE-1 cells and their impact in γ-tubulin recruitment. GA, Golgi apparatus. CTR, centrosome.

Data information: Data were collected from three independent experiments except for E and I, which are based in two independent experiments, and normalized to WT mean. For statistical analysis (C-H) we used one-way ANOVA followed by Dunnett's multiple comparisons test and for statistical analysis of J, two-tailed Student's t-test was employed. ** p<0.01 and **** p<0.0001. Scale bars, 5 μm.

Source data are available online for this figure.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 4

Figure 4

The centrosome regulates MT nucleation activity of the GA

A Control and centrinone-treated cells were subjected to a 3-min MT regrowth assay, fixed and labelled for EB1 (green) and giantin (red). Insets show magnified images of boxed regions.

B Workflow of the method used for quantifying MT nucleation from the GA.

C Quantification of the total number of growing MTs per cell (left), the number of MTs nucleated per Golgi fragment (middle) and the number of MT-nucleating Golgi fragments (right) in both control and centrosome-less RPE-1 cells. Scatter plots show each individual data point. Horizontal black lines bars represent the mean.

D RPE-1 cells were treated wi­­th centrinone for either 7 days (left) or for 5 days and then allowed to recover for 48h in the absence of the drug prior to fixation (right). Representative images of a triple IF staining with EB1 (green), γ-tubulin (red) and giantin (blue) as a Golgi marker are shown. Insets show MT nucleation from Golgi elements (left) or from multiple centrioles (right) and are enlarged at right. Arrowheads indicate the position of Golgi elements.

E Quantification of the percentage of cells exhibiting Golgi-nucleating activity in centrinone-treated cells 48h after the washout of the drug. Cells were divided into two categories depending on the number of centrioles that were visualized by labelling for the centriole protein Cep63. At least 100 cells from two independent experiments were analyzed in each condition.

Data information: In C, data were collected from three independent experiments and normalized to WT mean. For statistical analysis we used unpaired two-tailed Student's t-test. *** p<0.001 and **** p<0.0001. Scale bars, 5 μm.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 5

Figure 5

Redistribution of γ-TuRC-binding factors to the GA in the absence of the centrosome

A Control (top) and centrinone-treated (bottom) RPE-1 cells double stained for either AKAP450, CDK5Rap2, PCNT or CEP192 (all shown in green) and giantin (red) as a Golgi marker. DNA was counterstained with DAPI and is shown in blue. Single channel (left, green) and merged images (right) are shown in each case. White arrows indicate the centrosome (CTR).

B IF images of control and centrinone-treated RPE-1 cells treated with NZ to induce GA fragmentation and double stained with either anti-AKAP450, anti-CDK5Rap2 or anti-PCNT (all shown in green) and anti-giantin antibodies (white). Enlarged views of the boxed areas are presented at the bottom with or without the signal of the Golgi marker. The yellow dashed line indicates the contour of the nucleus. Arrowheads point to accumulation of the respective proteins in Golgi membrane surfaces.

C Box-and-whisker plots showing quantification of association between the indicated protein and Golgi elements (see Materials and Methods for details). Top and bottom ends of the boxes represent 75th and 25th percentiles, and whiskers represent 90th and 10th percentiles. The median is depicted with a solid line. Individual Golgi elements from at least 14 cells were delineated (>500 elements/experiment) and the intensity of each protein spot associated with each Golgi element was measured. Data were collected from two independent experiments and normalized to WT mean.

D MT-regrowth experiment after NZ treatment in centrinone-treated cells. High magnification images at the bottom are representative of a triple staining with α-tubulin (green), GM130 (blue) and the indicated protein (red) and show MTs growing from the surface of the GA. Double labelling without α-tubulin are also shown (top) to allow better visualization of PCM-protein spots on Golgi elements acting as MT nucleation sites (arrowheads).

E Centrinone-treated pcnt KO, c5rap2 KO and akap KO cells, were incubated with NZ for 3h, fixed and double labelled for GM130 and either CDK5Rap2 or PCNT as indicated (n=3).

F MT regrowth assay in centrinone-treated WT, pcnt KO and akap KO RPE-1 cells stained with antibodies to EB1 and giantin. At right, quantification of EB1 intensity at the Golgi membranes in WT and pcnt KO cells treated with centrinone, as a measure of MT-nucleation from the GA. Data were collected from two independent experiments and normalized to WT mean.

Scale bars, 5 μm.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 6

Figure 6

Cytoplasmic MT nucleation in the absence of centrosome- and Golgi-mediated MT nucleation

A Representative MT network of a centrosome-less akap KO cell.

B MT-regrowth experiment after NZ treatment in centrinone-treated akap KO cells. A representative confocal image of a double labelling for EB1 (green) and giantin (blue) is shown. High magnification at right shows that growing MT asters are not associated to Golgi fragments.

C Summary table of proteins present (+) or excluded (-) from MT-nucleating cytoplasmic structures.

D Centrinone-treated akap KO cells were subjected to a MT regrowth assay and double stained with the indicated antibodies. High magnification images of the boxed areas are shown as merge (top) or individual green or red labelling (middle and bottom panels).

E Cells lacking AKAP450 were treated with centrinone in order to induce the formation of cytoplasmic aggregates and then transfected with siRNAs specific for CDK5Rap2 (top panels) or PCNT (bottom panels). After 3-min MT regrowth, cells were fixed and stained with the indicated antibodies.

F MT regrowth experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After a 3h NZ treatment, MTs were allowed to polymerize and, at the indicated time points after washout, cells were fixed and labelled for α-tubulin (green), PCNT (red) and CAP350 (blue).

G MT repolymerization experiments in centrinone-treated akap KO cells transfected with either scramble or PCNT siRNAs. After NZ treatment and removal, cell were incubated for 10 min on ice and then warmed for 3 min in culture medium containing either DMSO or 30 μM gatastatin. Cells were finally fixed and triple stained with EB1 (green), PCNT (red) and CAP350 (blue) antibodies. Enlarged views of the boxed areas are presented at right. Scale bars, 5 μm.
THE DUAL ROLE OF THE CENTROSOME IN ORGANIZING THE MICROTUBULE NETWORK IN INTERPHASE (2018)
Figure 7

Figure 7

The centrosome limits cytoskeleton MT density

A Confocal images showing MT network in WT, akap KO, c5rap2 KO, pcnt KO and pc-ak-2KO RPE-1 cells fixed and stained with antibodies against α-tubulin (left) or EB1 (right). The red and yellow lines indicate the contour of cells. A schematic representation of active MTOCs is shown in each case. GA, Golgi apparatus. CTR, centrosome.

B Same as in (A), but in centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO backgrounds. Cells containing extra centrosomes induced by centrinone washout were also quantified (bottom panels).

C-F Quantification of α-tubulin fluorescence intensity (as a measure of MT mass polymer, C), number of EB1 comets per cell (D), cell area (E) and MT density (MT mass polymer/cell area) (F) in control and centrinone-treated WT, akap KO, c5rap2 KO and pcnt KO cells. Non-centrinone treated double KO cells and centrinone-treated WT cells after washout were also included. Scatter plots show each individual data point and horizontal black lines bars represent the mean.

Data information: Scale bars, 10 μm. Data were collected from three independent experiments and normalized to the untreated WT control. Statistical analysis is shown in EV5.

Expandend View Figure Legends
PP2A Phosphatase Is Required for Dendrite Pruning via Actin Regulation in Drosophila (2020)
Figure 1

Fig. 1. PP2A is required for dendrite pruning.

A - F' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones.

G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses.

H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.
PP2A Phosphatase Is Required for Dendrite Pruning via Actin Regulation in Drosophila (2020)
Figure 2

Fig. 2. PP2A interacts genetically with the Sox14/Mical pathway.

A-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-)expressing lacZ. (B, G) C4da neurons (co-)overexpressing Sox14. (C, H) C4da neurons (co-)overexpressing Mical. (D, I) C4da neurons (co-)expressing Mical∆redox. (E, J) C4da neurons (co-)expressing Mical∆CH.

K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses.

L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.