EMBO/soda-nel huggingface dataset:
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 2

Figure 2. Distribution of PCNT and SYCP3 during interkinesis and the second meiotic division.

A-F Double immunolabelling of PCNT -PCNT- (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) interkinesis, (B) early prophase II, (C) late prophase II, (D) prometaphase II, (E) metaphase II and (F) telophase II. Chromatin has been stained with DAPI (blue). Scale bar in F represents 10 μm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 3

Figure 3. Distribution of CETN3 and SYCP3 during spermatogenesis.

A-L Double immunolabelling of CETN3 (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) leptotene, (B) zygotene, (C) pachytene, (D) metaphase I, (E) telophase I, (F) early interkinesis (F), late interkinesis (G), prophase II (H), metaphase II (I), telophase II (J), round spermatid (K), and elongated spermatid (L). Amplifications at 300% magnification for the centrosomic CETN3 signals are presented for each image. Chromatin has been stained with DAPI (blue). Scale bar in L represents 10 μm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 4

Figure 4: PLK1S137P distribution during male mouse meiosis.

A-J Double immunolabelling of PLK1S137P (green) and SYCP3 (red) on squashed WT mouse spermatocytes at (A) pachytene, (C) diplotene, (E) diakinesis, (G) metaphase I and (I) metaphase II. Chromatin has been stained with DAPI (blue). Fourth column shows the double Immunolabelling of PLK1-S137P (green) and γ-Tubulin (magenta) on squashed WT mouse spermatocytes at (B) pachytene, (D) diplotene, (F) diakinesis, (H) metaphase I and (J) metaphase II. Insets correspond to centrosomes at 200% magnification. White arrows indicate the location of centrosomes. Scale bar in J represents 10µm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 5

Figure 5. Triple immunolabelling of PCNT, CETN3 and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes.

A-L First column shows the triple immunolabelling of CETN3 (green), PCNT (red) and SYCP3 (grey) on squashed WT mouse spermatocytes. Second column shows the correspondent labellings for Plk1(∆/∆). Third column shows signals for BI2536-treated spermatocytes. Images are shown for (A, E, I) zygotene, (B, F, J) pachytene, (C, G, K) diplotene and (D, H, L) diakinesis. Amplifications at 300% magnification for all labellings are displayed for each image.

M-O Quantification of the percentage of cells without PCNT at centrosomes (PCNT-) in (M) zygotene, (N) pachytene and (O) diplotene in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data for BI2536 represent results for 100µM at 8h treatment on organotypic cultures of seminiferous tubules. Data are presented as mean ± SD for three biological replicates for each condition.

Data information: Scale bar in L represents 10µm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 6

Figure 6. Immunolabelling of γ-Tubulin and SYCP3 and CETN3 and γ-Tubulin.

A-C Immunolabelling of γ-Tubulin and SYCP3 during prophase I in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Images are show for pachytene, diplotene and diakinesis in Control (A), Plk1(∆/∆) (B) and BI2536-treated spermatocytes (C). First columns show the double immunolabelling of PCM -PCNT- (green) and SYCP3 (red), and second columns shows γ-Tubulin (γ-Tub, magenta) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Amplifications at 300% magnification for all labellings are displayed for each image. Data for BI2536 represent results for 8h at 100µM treatment on organotypic cultures of seminiferous tubules

(D) Immunolabelling of CETN3 and γ-Tubulin in control and Plk1(∆/∆). Double immunolabelling of CETN3 (red) and γ-Tubulin (green) in pachytene (a-b) and diakinesis (c-d). Chromatin has been stained with DAPI (blue).

(E) Quantification of the number of cells without γ-Tubulin at centrosomes (γ-Tub -) in pachytene and diplotene spermatocytes in control, Plk1(∆/∆) and BI2536-treated spermatocytes. Data represent average of two biological replicates per condition. Data are mean ± SD.

Data information: White arrows indicate the location of centrosomes. Scale bar in C-f and D-d represent 10µm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 7

Figure 7. Meiotic spindles I and II in control, Plk1(∆/∆), BI2536-treated and MLN8237-treated spermatocytes.

A-D Control: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) on control squashed spermatocytes for the first and the second meiotic divisions at metaphase I (A) and metaphase II (B). Double immunolabelling of PCNT (green) and α-Tubulin (red) on control squashed spermatocytes at metaphase I (C) and metaphase II (D). Chromatin has been stained with DAPI (pseudo coloured in grey).

E-H Plk1(∆/∆) mouse model: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) in monopolar spindle I (E) and monopolar spindle II (F). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (G) and monopolar spindle II (H). Chromatin has been stained with DAPI (pseudo coloured in grey).

I-L BI2536-treated spermatocytes: Double immunolabelling of SYCP3 (cyan) and α-Tubulin (red) at monopolar spindle I (I) and monopolar spindle II (J). Double immunolabelling of PCNT (green) and α-Tubulin (red) labelling in monopolar spindle I (K) and monopolar spindle II (L). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 100µM BI2536 for 8h treatment on organotypic cultures of seminiferous tubules.

M-P MLN8237-treated spermatocytes: Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (M) and monopolar spindle II (N). Double immunolabelling of PCNT (green) and α-Tubulin (red) in monopolar spindle I (O) and monopolar spindle II (P). Chromatin has been stained with DAPI (pseudo coloured in grey). Cells shown were treated for 10µM MLN8237 for 8h treatment on organotypic cultures of seminiferous tubules.

Q-R Quantitative analysis of the incidence of monopolar spindles during the first (Q) and second (R) meiotic divisions for each experimental condition. Percentage of monopolar spindles functional analysis are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Experiments were conducted in three different biological replicates. Bars and error bars indicate mean ± SD; (*) p<0.05, (**) p<0.01, (***) p<0.001, Student's t-test.

Data information: White arrows indicate the location of centrosomes. Scale bar in P represents 10 μm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 8

Figure 8. Analysis of AURKAP in control, Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes.

A-B AURKP distribution in control, Plk1(Δ/Δ), 100µM 8h BI2536-treated and 10µM 8h MLN8237-treated spermatocytes. (A) Double immunolabelling of AURKP (grey/green) and α-Tubulin (red) in control, Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Images are shown for bipolar spindles I and II for each condition. (B) Monopolar spindles I and II are shown for Plk1(Δ/Δ), BI2536-treated and MLN8237-treated spermatocytes. Chromatin has been stained with DAPI (blue)..

(C) Quantification of the percentage of bipolar spindles during the first and second meiotic divisions presenting (blue bars) or lacking (green bars) centrosomic AURKP signals for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD.

(D) Quantification of the percentage of monopolar spindles during the first and second meiotic division presenting (blue bars) or lacking (green bars) centrosomic AURKP signal for each experimental condition. Percentage of monopolar spindles are represented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules. Data represent the average of two biological replicates per condition. Data are mean ± SD.

(E) Quantitative analysis of AURKP signal at cell poles. Experiments were conducted for two biological replicates. Number of cells analysed: control (n=32), Plk1(Δ/Δ) (n=27), BI2536-treated spermatocytes (n= 27). Data are mean ± SD; (****) p<0.0001, Student's t-test.

Data information: White arrows indicate the location of centrosomes. Scale bars in A-h and B-f represent 10µm.
PLK1 REGULATES CENTROSOME MIGRATION AND SPINDLE DYNAMICS IN MALE MOUSE MEIOSIS (2021)
Figure 9

Figure 9: Distribution of CEP250, CETN3 and SYCP3 in control and Plk1(Δ/Δ) spermatocytes.

(A) Triple immunolabelling of CEP250 (green), CETN3 (magenta) and SYCP3 (grey) in (a-b) late diplotene and (c-d) metaphase I in control and Plk1(Δ/Δ) spermatocytes.

(B) Double immunolabelling of KIF11 (grey/green), and SYCP3 (red) in (a) control spindle, (b) Plk1(Δ/Δ) monopolar spindle, (c) BI2536 monopolar spindle and (d) MLN8237 monopolar spindle. Chromatin has been stained with DAPI (bue). Data is presented for 100µM BI2536 and 10µM MLN8237 for 8h treatments on organotypic cultures of seminiferous tubules.

Data information: White arrows in A indicate the location of centrosomes. Insets in A correspond to centrosomes at 300% magnification. Scale bar in A-d and B-d represents 10 μm.

Expanded View Figure Legends
Coenzyme Q deficiency causes impairment of the sulfide oxidation pathway (2016)
Figure 3

Figure 3. Sulfide respiration in fibroblasts

(A) Representative traces showing SQR-driven oxygen consumption in 3x106 control (C) and five patients fibroblasts (P1-5) in the absence (solid traces) or presence (dashed traces) of CoQ10 in the culture medium. See Materials and Methods for the role and concentration of the compounds used. (B) Relative rates of SQR-driven oxygen consumption are normalized to CIV respiration rates. Error bars represent SDs of two experiments. * Indicates a value of p< 0.05, ** indicates a value of p< 0.01 and *** indicates a value of p< 0.001.
Coenzyme Q deficiency causes impairment of the sulfide oxidation pathway (2016)
Figure 4

Figure 4. SQR, TST, ETHE1 and SUOX protein levels in fibroblasts

(A) Representative western blot showing the level of SQR, TST, ETHE1 and SUOX proteins in three control (C1-3) and four patients fibroblasts (P1-4). (B-E) Relative levels of proteins normalized to Vinculin. Error bars represent SDs of three experiments. * indicates a value of p< 0.05, ** indicates a value of p< 0.01.
Coenzyme Q deficiency causes impairment of the sulfide oxidation pathway (2016)
Figure 5

Figure 5. SQR, TST, ETHE1 and SUOX protein levels in fibroblasts after CoQ supplementation

(A) Representative western blots showing the level of SQR, TST, ETHE1 and SUOX proteins in two controls (C1, C2) and two patients fibroblasts (P3, P4). (B-E) Relative levels of proteins normalized to Vinculin. Error bars represent SDs of three experiments. ** indicates a value of p< 0.01.
Coenzyme Q deficiency causes impairment of the sulfide oxidation pathway (2016)
Figure 6

Figure 6. SQR, TST, ETHE1 and SUOX protein levels in 4-NB treated fibroblasts and ADCK3-depleted HeLa cells

(A) Representative western blots showing SQR, TST, ETHE1 and SUOX of two control fibroblasts treated with DMSO (-) or with DMSO+ 4-NB (+). (B) Quantification of proteins levels in fibroblasts treated with 4-NB relative to control. (C) Representative western blots showing ADCK3, SQR, TST, ETHE1 and SUOX in two control (EV) and six ADCK3-depleted clones (ADCK3 RNAi). (D) Quantification of proteins levels in ADCK3-depleted clones (ADCK3 RNAi) relative to controls (EV). Error bars represent SDs of at least three experiments with different cell lines. * indicates a value of p< 0.05.
Coenzyme Q deficiency causes impairment of the sulfide oxidation pathway (2016)
Figure 8

Figure 8. SQR, TST, ETHE1 and SUOX protein levels in the kidney of Pdss2kd/kd mouse

(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein in the kidney of three wild-type (WT) and three mutant (Mut) mice. (B-E) Relative levels of proteins normalized to Vinculin. Error bars represent SDs of 9 animals per group. *** indicates a value of p< 0.001.
Coenzyme Q deficiency causes impairment of the sulfide oxidation pathway (2016)
Figure 9

Figure 9. SQR, TST, ETHE1 and SUOX protein levels in brain of Pdss2kd/kd mice

(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein from the brain of 5 wild-type (WT) and five mutant (Mut) mice (B-E) Relative levels of proteins normalized to Vinculin. Error bars represent SDs of 5 animals per group.
Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity (2021)
Figure 3

Figure 3. PKD2 inactivation decreases lipids' absorption.

A. Western blot analysis of phosphorylated PKD (S916/S876) in small intestine of male C57BL/6 mice after overnight fasting, olive oil gavage (or PBS in controls) and dissection at different time-points.

B. Lipids tolerance test after oral gavage of olive oil (10µL per gram of body weight). n=6.

C. Lipids tolerance test 30 min after tyloxapol i.p. injection (250µg/g body weight). In this case, only 5µL of olive oil per gram of body weight were gavaged orally. n=6.

D. Western blot analysis and quantification of phosphorylated PKD2 (S876) and phosphorylated PKD (S916/S876) in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice. n=3.

E. Western blot analysis and quantification of phosphorylated PKD (S916/S876) in Caco2 cells expressing shscramble or shPkd2. n=3.

F. Representative light microscopy pictures of organoids derived from jejunum crypts of indicated male mice.

G. Percentage of released, uptaken and retained 14C-labeled fatty acids from Caco2 cells shscramble or shPkd2 (sequence 1) in a transwell system. n=6.

H. Percentage of released 14C-labeled fatty acids by Caco2 cells expressing shscramble* or shPkd2* (sequence 2) grown in a transwell system. n=6.

I. Transepithelial electric resistance of the Caco2 cells shscramble or shPkd2 (sequence 1). n=6.

Data information: For panels B and C, after acclimation, male mice after were treated as specified and triglycerides in circulation measured at the specified time-points. Data presented as average +/- SEM, * P≤0.05, ** P ≤0.01, *** P ≤0.001. Significances were assessed by using a two-tailed Student's t-test for independent groups.
Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity (2021)
Figure 4

Figure 4. Intestinal deletion of PKD2 reduces lipids absorption, protects from HFD-induced obesity and improves microbiota profile.

A. Western blot and quantification of phosphorylated PKD (S916/S876) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3.

B. Western blot and quantification of phosphorylated PKD (S744-748) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3.

C. Lipids tolerance test after oral gavage of olive oil to Pkd2flox/flox and Pkd2gutΔ/Δ male mice after 6 weeks in HFD. n=10.

D. Body weight gain of male mice of indicated genotypes in HFD. n=10.

E. Quantification of fat, free fluid and lean mass by nuclear magnetic resonance of mice in panel B. n=10.

F. Glucose tolerance test of male mice with indicated genotypes after 16 weeks in HFD. n=10.

G. Free-fatty acids and H. Triglycerides content in serum of male mice after 17 weeks in HFD. n=10.

I. Differences in bacterial alpha diversity as indicated by Shannon Index. n=7.

J. Changes in specific Operational taxonomic unit (OTU) in small intestinal samples. n=7.

Data information: Data presented as average +/- SEM. In panels I and J, central band indicates the median, the lower and upper part of the box indicate the 25 and 75 percentiles and the whiskers connect data points outside these quartiles. * P≤0.05, ** P ≤0.01, *** P ≤0.001. . Significances in panels A to H were assessed by using a two-tailed Student's t-test for independent groups. In panel G statistical testing was performed using Mann Whitney U test. In panel H statistical testing was performed using PERMANOVA with corrections for multiple testing using the Benjamini & Hochberg method.
Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity (2021)
Figure 5

Figure 5. PKD2 promotes chylomicron size.

A. Western blot (WB) analysis of specified proteins in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of the bands for each protein normalized to loading control and relative to Pkd2wt/wt. n=3.

B. WB of ApoliproteinA4 in jejunum of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of bands normalized to loading control and relative to Pkd2wt/wt. n=3.

C. WB of ApoliproteinA4 in jejunum of Pkd2flox/flox and Pkd2gutΔ/Δ male mice after one week of HFD. Quantification of bands normalized to loading control and relative to Pkd2flox/flox n=7.

D. WB analysis of specified apolipoproteins in 10µL of serum of overnight fasted, 2 hours refed male animals after 1 week in HFD. Quantification of bands relative to Pkd2wt/wt. n=4.

E. WB of 20µL of basal medium of Caco2 cells expressing shscramble or shPkd2 in transwell system after 12 hours stimulation with oleic acid. n=6.

F. Electron microscopy pictures of chylomicrons obtained by ultracentrifugation of plasma from Pkd2wt/wt and Pkd2ki/ki male mice as specified in methods.

G. Quantification of the diameter the chylomicrons shown in panel F. n=3.

H. WB with specified antibodies after an in-vitro kinase assay.

Data information: Data presented as average +/- SEM * P≤0.05, ** P ≤0.01, *** P ≤0.001. Significances were assessed by using a two-tailed Student's t-test for independent groups.
Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity (2021)
Figure 6

Figure 6. Inhibition of PKDs by small-molecule compound reduces the degree of diet-induced obesity and diabetes.

A. Basolateral release of 14C-labeled fatty acids in Caco2 cells grown in a transwell system and treated with CRT0066101 at the indicated concentrations for 24 hours. n=6.

B. Basolateral release of 14C-labeled fatty acids in Caco2 cells expressing shscramble or shPkd2 in a transwell system and treated with CRT0066101 for 24 hours. n=8.

C. Western blot analysis of phosphorylated PKD2 in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=3.

D. Energy content per gram of feces of male mice in HFD and receiving daily gavage of CRT0066101 or water as control. n=6 for control and n=5 for CRT0066101.

E. Lipids tolerance test after oral gavage of olive oil to male mice after 9 weeks in HFD and receiving daily CRT0066101 or control. n=6 for control and n=5 for CRT0066101.

F. WB of Apolipoprotein A4 (APOA4) in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. Quantification of bands normalized to GAPDH and relative to control (water) n=6.

G. Body weight of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=6 for control and n=5 for CRT0066101.

H. Glucose tolerance test in male C57BL/6 mice which received CRT0066101 or water (control). Measurements after 10 weeks of treatment and HFD feeding. n=6 for control and n=5 for CRT0066101.

I. Insulin tolerance test in male C57BL/6 mice which received CRT0066101 or water (control). Measurements after 11 weeks of treatment and HFD. n=6 for control and n=5 for CRT0066101.

Data information: Data presented as average +/- SEM, * P≤0.05, ** P ≤0.01. Significances in panels A and B were assessed by one way ANOVA followed by the post hoc tukey test. In panels C to I significances were assessed by using a two-tailed Student's t-test for independent groups.
Protein Kinase D2 drives chylomicron-mediated lipid transport in the intestine and promotes obesity (2021)
Figure 7

Figure 7. Reduction of PKD activity in intestine ameliorates pre-established obesity in mice and correlates with healthier blood profile in obese patients.

A. Body weight gain of male C57BL/6 mice that, after 7 weeks in HFD, started receiving treatment with CRT0066101 or water. HFD continued along with treatment. n=8.

B. Organ weight (expressed as percentage of total body weight) of different fat depots and liver of male mice in panel A.

C. Quantification of fat, free fluid and lean mass by nuclear magnetic resonance (NMR) of male mice in panel A.

D. Glucose tolerance test 8 weeks after starting the treatment of mice in panel A.

E. Insulin tolerance test 10 weeks after starting the treatment of mice in panel A.

F. Western blot analysis of PKD phosphorylation in proximal jejunum of morbidly obese female patients. Samples are loaded according to fasting triglyceride levels (lower to higher). n=7.

G. Scatter plot of PKD phosphorylation in jejunum (normalized to GAPDH) vs. triglycerides of morbidly obese female patients. n=7.

H. Scatter plot of PKD phosphorylation in jejunum (normalized to GAPDH) vs. percentage of glycated hemoglobin (HbA1c) of morbidly obese female patients. n=7.

I. Scatter plot of PKD phosphorylation in jejunum (normalized to GAPDH) vs. high density lipoprotein (HDL) of morbidly obese female patients. n=7.

Data information: Data presented as average +/- SEM, * P≤0.05, ** P ≤0.01, *** P ≤0.001. Significances were assessed by using a two-tailed Student's t-test for independent groups. In panels G-I, r2= square of the Pearson product-moment correlation coefficient. p determined for Pearson correlation coefficient for the given degrees of freedom.