EMBO/soda-nel huggingface dataset:
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 6

Figure 6. Rescue of abnormal migration by HDAC6 inhibition

(A) Rescue of a delayed cortical migration in CAMDI-KO mice by Tubastatin A. Immunohistochemical analysis of Cux1 expression in the somatosensory cortex at P2 after intraperitoneal injections of Tubastatin A into pregnant CAMDI-KO mice from E12.5–17.5. Scale bar, 100 µm.

(B) Quantification of the number of Cux1-positive neurons. Note the abnormal distribution of neurons in deep cortical layers of CAMDI-KO mice. n = 3 mice/genotype (WT (vehicle) = 3,628 cells, KO (vehicle) = 2,816 cells, WT (Tubastatin A) = 1,847 cells, KO (Tubastatin A) = 2,364 cells). *, p<0.05, **, p<0.01, Two-way ANOVA followed by Tukey HSD test. Data are presented as mean ± SEM.

(C) Enhanced α-tubulin acetylation at P0 by Tubastatin A. Ac-tubulin level was assessed by immunoblot analysis.

(D) Quantification of the α-tubulin acetylation. n=3 independent experiments. *, p<0.05, Two-way ANOVA followed by Scheffe’s post-hoc test. Data are presented as mean ± SEM.
Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor (2016)
Figure 7

Figure 7. Rescue of psychiatric behaviors by HDAC6 inhibition

(A) Representative traces from control (WT) and CAMDI-KO (KO) mice in the open field test at P21. Sample traces of locomotor activity were shown.

(B) Distance traveled for 10 min. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 43)=6.25, main effect of drug F(1, 43)=2.14, interaction F(1, 43)=12.68). Data are presented as mean ± SEM.

(C) Repetitive jumping behavior. n = 13 for WT (vehicle) mice, n = 27 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 18 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 59)=5.04, main effect of drug F(1, 59)=10.13, interaction F(1, 59)=4.98). Data are presented as mean ± SEM.

(D) Grooming time in small open field test. n = 13 for WT (vehicle) mice, n = 13 for KO (vehicle) mice, n = 5 for WT (Tubastatin A) mice, n = 16 for KO (Tubastatin A) mice. **, p<0.01, ***, p<0.001. Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 43)=11.18, main effect of drug F(1, 43)=7.23, interaction F(1, 43)=9.34). Data are presented as mean ± SEM.

(E) Distance traveled for 15 min. n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. **, p<0.01, Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 28)=1.73, main effect of drug F(1, 28)=3.96, interaction F(1, 28)=11.80). Data are presented as mean ± SEM.

(F) Light-dark test. Time in latency to the light chamber (latency). n = 10 for adult WT (Vehicle) mice, n = 5 for KO (Vehicle) mice, n = 6 for WT (Tubastatin A) mice, n = 11 for KO (Tubastatin A) mice. *, p<0.05, Two-way ANOVA followed by Scheffe’s post-hoc test (main effect of genotype F(1, 28)=2.34, main effect of drug F(1, 28)=3.82, interaction F(1, 28)=10.64). Data are presented as mean ± SEM.
Overlapping Roles for PLK1 and Aurora A During Meiotic Centrosome Biogenesis in Mouse Spermatocytes (2021)
Figure 1

Figure 1. Centriole duplication and centrosome separation patterns during meiosis in mouse spermatocytes.

Tubule squash preparations were performed on wild type juvenile C57BL/6J mice undergoing the first wave of spermatogenesis.

A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.

B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage. Error bars show mean ± SEM. P values obtained from two-tailed Mann Whitney t-test. **P<0.01.

C. Representative images of unseparated (< 1 μm apart), initial separation (1-5 μm apart), intermediate separation (5-11 μm apart), and pole separation (>11 μm apart) of centrosomes. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.

D. Centrosome separation was scored in n = 30 spermatocytes at each prophase substage using the four centrosome separation classifications shown in C. P value obtained from a Kruskal-Wallis one-way ANOVA. **P<0.01.

E. Spermatocytes undergoing meiosis I through meiosis II were immunolabeled against PCNT (purple), CETN3 (green), and SYCP3 (red), and stained with DAPI (blue). Zoomed images of centrosomes below each panel. Scale bars = 5 μm.

F. Model for centriole duplication and centrosome separation patterns throughout the meiotic prophase and division stages. During the leptonema to zygonema transition the first round of centriole duplication occurs. As cells progress through meiotic prophase I, centrosomes mature and acquire distal appendages. During diakinesis centriole separation occurs, allowing the formation of a bipolar spindle during the first meiotic division. During interkinesis centrioles duplicate a second time and separate to form a bipolar spindle at meiosis II.
Overlapping Roles for PLK1 and Aurora A During Meiotic Centrosome Biogenesis in Mouse Spermatocytes (2021)
Figure 2

Figure 2. Conditional deletion of Plk1 or Aurka causes meiosis I arrest in mouse spermatocytes.

A. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. Scale bars = 50 μm.

B. Percent testis to body weight ratios from control (n = 8), Plk1 cHet (n = 4), and Plk1 cKO (n = 7) adult mice.

C. TUNEL staining of paraffin embedded testis sections of adult control, Plk1 cHet, and Plk1 cKO mice. Scale bars = 50 μm.

D. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Plk1 cHet, and Plk1 cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.

E. Hematoxylin and eosin staining of 5-micron thick testis sections of adult control, Aurka cHet, and Aurka cKO mice. Stars indicate examples of spermatocytes with condensed chromosomes. Scale bars = 50 μm.

F. Percent testis to body weight ratios from control (n = 5), Aurka cHet (n = 3), and Aurka cKO (n = 5) adult mice.

G. TUNEL staining of paraffin embedded testis sections of adult control, Aurka cHet, and Aurka cKO mice. Scale bars = 50 μm.

H. The number of TUNEL positive cells per seminiferous tubule cross-section was quantified in control Aurka cHet, and Aurka cKO mice. Two technical replicates were performed per genotype, and 100 seminiferous tubule cross sections were analyzed per replicate.

I. Representative images of spermatocytes containing monopolar, bipolar, and multipolar spindles immunolabeled against alpha-tubulin (red), centromere (green), and stained with DAPI. Scale bars = 10 µm.

J. Spindle pole number per primary spermatocyte was quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO. n = 3 experimental replicates with > 40 metaphase I spermatocytes analyzed per genotype in each experiment.

Data information: For all graphs (B, D, F, H, J) - Error bars show mean ± SEM. P values obtained from two-tailed Student's t-test. n.s. (not significant) and, ***P<0.001.
Overlapping Roles for PLK1 and Aurora A During Meiotic Centrosome Biogenesis in Mouse Spermatocytes (2021)
Figure 3

Figure 3. Centriole duplication and centrosome separation patterns in PLK1 and AURKA mutant spermatocytes.

A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.

B. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage. Error bars show mean ± SEM.

C. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.

D. Quantification of the number of CETN3 foci per spermatocyte. n = 30 for each meiotic prophase substage. Error bars show mean ± SEM. P values obtained from two-tailed Mann Whitney t-test. **P<0.01.

E. Centrosome (PCNT) separation was scored per spermatocyte. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications defined in Fig 1C. Error bars show mean ± SEM. P values obtained from two-tailed Student's t-test comparing the percent of unseparated centrosomes in each mouse mutant to control. n.s. (not significant), ***P<0.001 .
Overlapping Roles for PLK1 and Aurora A During Meiotic Centrosome Biogenesis in Mouse Spermatocytes (2021)
Figure 4

Figure 4. Centriole and centrosome maturation defects are observed in Plk1 cKO spermatocytes.

A. Spermatocytes were immunolabeled against CETN3 (green), CEP164 (red), SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom and top left insets. Scale bars = 5 μm.

B. The percent of centrioles containing CEP164 distal appendages was quantified from three biological replicates (n = 30 total cells) in control, Aurka cKO, Plk1 cHet, and Plk1 cKO spermatocytes at the pachytene-stage of meiotic prophase and metaphase I.

C. Quantification of gamma-tubulin positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 137, 169, 198, and 198, respectively.

D. Spermatocytes were immunolabeled against gamma-tubulin (green), alpha-tubulin (red), and stained with DAPI (blue). Plk1 and Aurka cKO spermatocytes arrest at a prometaphase stage. Scale bars = 5 μm.

E and F. Western blots of gamma-tubulin from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (E). Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (F). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.

Data information: For graphs (B, C) - Error bars show mean ± SEM. P values obtained from two-tailed Student's t-test. n.s. (not significant), *P<0.05, and ***P<0.001. For the western blot shown in E, the same samples were used for blotting in Fig EV3C. For the western blot shown in F, the same samples were used for blotting in Fig 5C and Fig EV3E. Therefore, the TCE images are the same for these blots. Full western blot images and replicates are presented in Figure Source Data for Figure 4.
Overlapping Roles for PLK1 and Aurora A During Meiotic Centrosome Biogenesis in Mouse Spermatocytes (2021)
Figure 5

Figure 5. The localization of AURKA and PLK1 substrates is perturbed in Aurka cKO and Plk1 cKO spermatocytes.

A. Spermatocytes were immunolabeled against TACC3 (green), SYCP3 (red), CETN3 (magenta), and stained with DAPI (blue). Zoom images of the centrosomes are presented to the right. Scale bars = 5 μm.

B. Quantification of TACC3 positive centrosomes per metaphase I spermatocyte. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 97, 103, 96, and 105, respectively.

C, D. Western blots of TACC3 from protein extracts obtained from 19dpp and 22dpp control, Aurka cKO, and Plk1 cKO spermatocytes (D). Protein extracts from control and Plk1 cHet spermatocytes at 16, 19 and 22 dpp (C). Total protein stained with 2,2,2-Trichloroethanol (TCE) display protein loading.

E, F. Spermatocytes were immunolabeled against beta-catenin (green), SYCP3 (red), PCNT (magenta), and stained with DAPI (blue). Middle columns emphasize beta-catenin signal in diplonema and diakinesis spermatocytes. Scale bars = 5 μm.

G. Quantification of beta-catenin positive centrosomes per spermatocyte at diakinesis. Immunolabeling was performed on three biological replicates, with ≥20 spermatocytes quantified per replicate. The total number of cells quantified for control, Aurka cKO, Plk1 cHet, and Plk1 cKO mice were 89, 106, 122, and 93, respectively.

Data information: For graphs (B, G) - Error bars show mean ± SEM. P values obtained from two-tailed Student's t-test. n.s. (not significant) and, ***P<0.001. For the western blot shown in C, the same samples were used for blotting in Fig 4F and Fig EV3E. Therefore, the TCE images are the same. Full western blot images and replicates are presented in Figure Source Data for Figure 5.
Overlapping Roles for PLK1 and Aurora A During Meiotic Centrosome Biogenesis in Mouse Spermatocytes (2021)
Figure 6

Figure 6. Centrosome separation defects are compounded in spermatocytes lacking both PLK1 and AURKA.

A. Spermatocytes were immunolabeled against PCNT (red), CETN3 (green), and SYCP3 (blue), and stained with DAPI (upper right inset). Zoomed images of the centrosome are shown in the bottom left inset. Scale bars = 5 μm.

B. Quantification of the number of CETN3 foci per spermatocyte (n = 30) at each meiotic prophase substage.

C. Centrosome (PCNT) separation was assessed in Aurka; Plk1 dKO spermatocytes. Three biological replicates were analyzed with > 30 metaphase I spermatocytes assessed per replicate using the four centrosome separation classifications defined in Fig 1C.

D. Aurora A- and PLK1-mediated regulation of meiotic centrosome biogenesis during spermatogenesis. After spermatocytes enter meiotic prophase and complete the first round of centriole duplication, presumably stimulated by PLK4, the centrioles must mature in order to effectively act as MTOCs during the first meiotic division. PLK1 and AURKA regulate this maturation process by ensuring the recruitment of various maturation factors including TACC3, CEP164, and γ-tubulin (γ-TUB). PLK1 and AURKA are also required for the efficient degradation of β-catenin (β-CAT), a necessary step to ensure centrosome separation prior to bipolar spindle establishment at metaphase I. The mechanism by which the first and second rounds of centriole duplication are restricted to early meiotic prophase I and interkinesis, respectively, is not understood and warrants further investigation. However, we find that high levels of PLK1 are necessary to avoid a premature second round of centriole duplication, which insinuates that PLK1 counteracts the centriole duplication function of PLK4.

Data information: For graphs (B and C) - Error bars show mean ± SEM. P values obtained from two-tailed Student's t-test. n.s. (not significant) and , ***P<0.001.

Expanded View Figure Legends:
A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis (2021)
Figure 2

Figure 2. The R145W mutation in Sema7a markedly reduces the levels of canalicular membrane bile acid (BA) efflux transporters, Bsep and Mrp2 in hepatocytes.

(A-C) The relative levels of (A) canalicular membrane (BA) transporter Bsep, Mrp2, Mdr1b, Mdr2, Abcg5 and Abcg8 mRNA transcripts, (B) basolateral membrane BA transporter Mrp3, Mrp4, Ostβ, Ntcp and Oatp1a1 mRNA transcripts, and (C) BA synthetic enzyme Cyp7a1, Cyp7b1, Cyp8b1, Cyp27a1, and Cyp2c70 mRNA transcripts. Wild type, wild type mice, n=6; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=7. Data are shown as means ± SD. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.

(D) Western blot analysis of the relative levels of Bsep, Mrp2, Mdr2, Mrp3, Mrp4, Ostα/β, Ntcp, Cyp7a1 and Cyp8b1 protein expression in mouse livers. Contrary to the mRNA alterations, the levels of canalicular membrane Bsep and Mrp2 proteins were dramatically reduced in homozygous mouse livers, suggesting that the decreased these proteins were might be mediated by post-translational regulation in the livers of Sema7aR145W mutant mice. Wild type, wild type mice, n=4; Heterozygote, Sema7aR145W heterozygous mice, n=5; Homozygote, Sema7aR145W homozygous mice, n=5. *P < 0.05 versus the WT mice, #P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test and the Mann-Whitney U test.

(E, F) Multiplex IF analyses of Bsep and Mrp2 expression in the livers (E) and primary hepatocytes in sandwich cultures (F) from WT and homozygous mice. Scale bar, 50 µm.

(G) Multiplex IF analyses revealed that transfection with the SEMA7A_WT or SEMA7A_R148W construct decreased the levels of canalicular membrane Bsep and Mrp2 expression in mouse primary hepatocytes in collagen sandwich cultures in a dose-dependent manner (0, 0.2, 1.0 μg per well of 6-well plate; 1.5 ml culture medium per well). Scale bar, 50 µm. Together, the Sema7aR145W mutation can reduce hepatic Bsep and Mrp2 protein expression and lead to intrahepatic BA accumulation and cholestasis (Appendix Table S16). Subsequently, cholestasis triggered an adaptive response in the liver by down-regulating the expression of BA synthetic enzymes of Cyp7a1 and Cyp8b1 and up-regulating the expression of BA efflux transporters Mrp3, Mrp4 and Ostα/β.
A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis (2021)
Figure 3

Figure 3. Administration of UDCA and GSH improved liver function in the child patient with this new type of PFIC.

Treatment with UDCA (13 mg/kg/d) and GSH (40 mg/kg/d) for 2 weeks restored normal liver function in the child. However, when the treatment was ceased the levels of serum ALT, AST and TBA returned to abnormally high levels in the patient. Interestingly, treatment with half of the dose of UDCA and GSH also maintained the normal levels of serum ALT and AST, and reduced serum TBA in the child patient.

Expanded View figure legends:
Sequential Treatment with 5-aza-2’deoxycitidine and Deacetylase Inhibitors Reactivates HIV-1 (2015)
Figure 1

Figure 1: The DNA methylation inhibitor 5-AzadC induces HIV-1 expression in latently-infected T cells.

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A); metabolic activity was assessed by a WST-1 assay (B); viral protein expression was analyzed by FACS (C); and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D). The selected dose was indicated by an arrow.

E J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400nM) or TNFα (10ng/ml) as positive control. At 24 h, 48 h or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR.

Data information: For D and E, results were normalized using β-actin gene primers and are presented as histograms indicating the fold inductions compared to mock-treated condition for each time period. For A-E, means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (D, E) and 100% (B).
Sequential Treatment with 5-aza-2’deoxycitidine and Deacetylase Inhibitors Reactivates HIV-1 (2015)
Figure 4

Figure 4: Sequential 5-AzadC + HDACI treatments synergistically activate HIV-1 gene expression and production in latently-infected cells.

A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B), FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D), initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).
Sequential Treatment with 5-aza-2’deoxycitidine and Deacetylase Inhibitors Reactivates HIV-1 (2015)
Figure 9

Figure 9: 5-AzadC + Panobinostat and 5-AzadC + Romidepsin treatments do not induce global T-cell activation.

A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69- CD25- CD4+ T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+ T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.
Sequential Treatment with 5-aza-2’deoxycitidine and Deacetylase Inhibitors Reactivates HIV-1 (2015)
Figure 10

Figure 10: 5-AzadC + Panobinostat and 5-AzadC + Romidepsin treatments induce a significant decrease in the cell surface CD4 receptor expression.

One night after cell purification, HLA DR- CD69- CD25- CD4+ T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+ T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.
Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2 (2020)
Figure 2

Figure 2. Distinct binding of ubiquitin and ISG15 enables separation of PLpro function.

A) Detail of the S1 ubiquitin binding site of SARS2 PLpro, bound to ubiquitin (left) and ISG15 (right), highlighting differential interactions of ubiquitin with the Fingers subdomain, and of ISG15 with the Thumb subdomain of PLpro. Labelled residues were mutated, see Fig EV3.

B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods.

C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.