EMBO/soda-nel huggingface dataset:
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes (2021)
Figure 3

Figure 3. AP2-O3 represses transcription of the male-associated genes in the female gametocyte.

Number of differentially expressed genes in the purified female gametocyte determined by RNA-seq. Detailed information is available in the Figure EV2.

Percentage of female and male genes in down- and up-regulated genes.

Gene ontology enrichment analysis of the upregulated genes indicates male-specific or preferential biological processes.

Transcript expression heatmap by RNA-seq of 26 genes involving DNA replication and DNA repair between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains.

Quantitative RT-PCR analysis of 26 genes in D. mean±SEM from three independent experiments. Two-tailed unpaired Student's t-test applied.

Linear correlation of gene expression among the 26 genes (DNA replication and DNA repair) detected via RNA-seq and quantitative RT-PCR.
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes (2021)
Figure 4

Figure 4. Protein expression of upregulated male genes in AP2-O3 null female gametocytes

Four male genes in DNA replication and DNA repair pathways, including dpod2, dpod1, rpa1, and mcm7.

Diagram of CRISPR/cas9-mediated C-terminally tagging of these four genes with gfp in the female reporter strain ccp2::mCherry, generating four DTS (double tagged strain: DTS1-DTS4). Next, the endogenous ap2-o3 gene was deleted in these four DTS, generating four DTS;Δap2-o3 mutants.

Representative fluorescence microscopy images of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 (ccp2::mCherry;dpod2::gfp) and DTS1;Δap2-o3 strains. mCherry is specifically expressed in female gametocytes. Scale bars = 5 μm.

Flow cytometry detection of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 and DTS1;Δap2-o3 strains. The female and male gametocyte populations are circled by solid and dashed line respectively.

E,F Similar analysis (as in C, D) in DTS2 (ccp2::mCherry;dpod1::gfp) and DTS2;Δap2-o3 strains. Scale bars = 5 μm.

G,H Similar analysis (as in C, D) in DTS3 (ccp2::mCherry;rpa1::gfp) and DTS3;Δap2-o3 strains. Scale bars = 5 μm.

I,J Similar analysis (as in C, D) in DTS4 (ccp2::mCherry;mcm7::gfp) and DTS4;Δap2-o3 strains. Scale bars = 5 μm.
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes (2021)
Figure 5

Figure 5. AP2-O3 binds upstream promoter of specific male genes

The number of overlapped peaks (1186) and genes (1085) in two ChIP-seq experiments. Statistical differences were determined using Fisher exact test.

Correlation of read density among the 1186 peaks between two ChIP-seq experiments. Pearson correlation is indicated.

The enriched DNA motif is identified by the motif-discovery algorithm MEME in AP2-O3 binding sites.

EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.

The number (319) of overlapping genes between AP2-O3-bound genes (1085) in ChIP-seq and upregulated genes (1141) in RNA-seq. Statistical differences were determined using Fisher exact test.

Gene ontology enrichment analysis of the 319 genes indicates male-specific or preferential biological processes. Hypergeometric test was applied.

Mapped views showing the RNA-seq and ChIP-seq results of four male genes in DNA replication and DNA repair pathways.

ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes in G. The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes (2021)
Figure 6

Figure 6. Decreased expression of highly expressed female genes in AP2-O3 null female gametocytes

Transcriptome of female gametocyte by RNA-seq between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains. The genes were ranked by transcripts level (FPKM average of three sample replicates) in the heatmap. In spite of global upregulation of vast genes with low or medium transcript levels (left panel), the highly expressed genes (FPKM>1500, right panel) are downregulated in KO over WT. The FPKM values in each row (each gene) were normalized by Z-score normalization.

Gene percentage of downregulation and upregulation in populations of the highly expressed genes (n=111, FPKM>1500) and the others (n=4589, FPKM<1500) between KO and WT.

Line chart of 50 representative highly expressed female genes in A. The dot is the mean value of FPKM in three repeats.

qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes in A. The numbers indicate the gene ID. mean±SEM from three to five independent experiments. Two-tailed unpaired Student's t-test applied.

Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes (2021)
Figure 7

Figure 7. Downregulation of AP2-O3 its expression after fertilization is required for ookinete development

AP2-O3 protein expression from female gametocyte, female gamete, zygote, to retort stage of the ap2-o3::6HA strain. The activated gametocytes were collected at 30, 60, and 120 min and co-stained with HA and P28 antibodies. Pie charts indicate the percentage of AP2-O3+ and AP2-O3- cells. "n" is the number of cells analyzed at each time point. Scale bars = 5 μm.

Western blot of AP2-O3 protein expression in parasites from A.

Diagram of CRISPR/cas9-mediated promoter swap in the ap2-o3::6HA. The promoter (467 bp) of ap2-o3 gene was replaced with the ccp2 gene promoter (1202 bp), generating the Pccp2 mutant to drive gene expression post-fertilization.

Western blot of AP2-O3 protein expression in non-activated (NAG) and activated gametocytes (AG) of ap2-o3::6HA and Pccp2 strains.

IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains. Scale bars = 5 μm.

Gametocyte formation in mouse.

In vitro exflagellation rate of male gametocytes.

Female gamete fertilization to zygote analysis by co-staining of P28 and GAP45.

Mature ookinete formation in vitro.

Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.

Data information: In F-I, mean±SEM from three infected mice or three experiments. Two-tailed unpaired Student's t-test applied in G-I, Mann-Whitney test applied in J.

Expanded view figure legends
GRASP55 regulates intra-Golgi localization of glycan enzymes to control glycosphingolipid synthesis (2021)
Figure 2

Figure 2. GRASP55 regulates SL biosynthesis:

(A) HeLa cells were treated with control or indicated siRNA (pool of 4 or 2 as indicated in methods) for 72 hours and SL biosynthesis measured by [3H] - sphingosine pulse-chase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls. Data represented are mean ± SD of 3 independent experiments **p <0.01 (Student's t test).

(B, C) Effect of GRASP55 depletion on SL biosynthesis monitored by [3H] - sphingosine pulse-chase assay in GRASP55 KO cells or cells treated with GRASP55 siRNA or following expression of GRASP55-GFP in GRASP55 depleted cells. GSL levels are expressed as fold changes respect to control. Data represented are mean ± SD of 3 independent experiments *p <0.05, **p <0.01, ***p <0.001 (Student's t test). (C) The levels of GM and Gb were quantified and represented as GM/Gb ratio. Data represented are mean ± SD of 3 independent experiments *p <0.05, ***p <0.001 (Student's t test).

(D) Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow cytometry analysis. Mean fluorescence intensity was measured and represented. Data represented are mean ± SD of 3 independent experiments *p <0.05, ***p <0.001 (Student's t test). Scale bar: 10 μm

(E, F) SL levels as assessed by LC/MS or MALDI-MS (Gb3) in control and GRASP55 KO (#2) cells. Data represented are mean ± SD of 3 independent experiments *p <0.05, ***p <0.001 (Student's t test).
GRASP55 regulates intra-Golgi localization of glycan enzymes to control glycosphingolipid synthesis (2021)
Figure 3

Figure 3. GRASP55 regulates the intra-Golgi localization of GSL biosynthetic enzymes:

(A, B) Control and GRASP55 KO clones were transfected with HA-tagged GSL biosynthetic enzymes, treated with nocodazole (33 µM) for 3 hours, and processed for immunofluorescence with anti-HA (green), anti-GM130 (red), and anti-TGN46 (blue) antibodies. The relative position of HA-tagged enzymes with respect to GM130 and TGN46 was measured by line scanning and expressed as normalized positions of the peak intensity with the start of GM130 peak indicated as Cis and the end of TGN46 peak indicated as trans in the graph. The double headed arrows show the distance between the peak localization of GM130 and the enzymes. Scale bar 1μm.

(C) Control and GRASP55 KO cells were transfected for 16 hours with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labelling.

(D, E) Distribution of indicated enzymes across the Golgi stack was quantified and represented as fraction of Gold particles in each cisternae for GCS-HA (D) and LCS- HA (E) (n indicated in the graph; data are Mean ± SEM).
GRASP55 regulates intra-Golgi localization of glycan enzymes to control glycosphingolipid synthesis (2021)
Figure 5

Figure 5. GRASP55 compartmentalizes the enzymes by preventing their entry into retrograde carriers:

(A-C) Control and GRASP55 KO (#2) cells were transfected for 16 hours with the indicated HA-tagged enzymes and processed for cryo-immunolabeling with anti-HA antibody (10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles indicate GM130 labelling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as normalized linear density (n indicated in the graph; data are Mean ± SEM).

(D) COPI vesicles were reconstituted using the two-stage incubation system (detailed in the Methods section). After the second-stage incubation, samples were centrifuged to obtain the pellet fraction that contains Golgi membranes and the supernatant fraction that contains reconstituted COPI vesicles. Both fractions were immunoblotted for LCS, GCS and transferrin receptor (Tfr) to show their relative distributions on Golgi membranes and in COPI vesicles.

(E) The COPI vesicle reconstitution system was performed as described above (in D), and then the fraction of LCS and GCS in COPI vesicles versus their total distribution (on Golgi membranes and in COPI vesicles) was calculated. The mean and standard error from 3 independent experiments are shown, * p < 0.05, Student's t-test.

(F) HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 hours and were processed for cryoimmunolabeling with anti-HA antibody (10-nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead indicates the clathrin-coated vesicle that marks the TGN area and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles. (n indicated in the graph; data are Mean ± SEM).

(G) Model represents GRASP55-mediated compartmentalization of GCS and LCS. A cyclical and balanced activity of GRASP55 and GOLPH3 compartmentalizes LCS/GCS to the trans-Golgi. The anterograde transport of enzymes (forward direction arrow; cis to trans direction) counterbalances their retrograde transport (reverse direction arrow; trans to cis direction) resulting in the compartmentalization of these enzymes.
GRASP55 regulates intra-Golgi localization of glycan enzymes to control glycosphingolipid synthesis (2021)
Figure 6

Figure 6. Change in intra-Golgi localization of GSL biosynthetic enzymes changes GSL output:

(A, B) Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5μg/ml) for 30 min and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase). Total GSL levels were quantified and expressed as fold changes with respect to control (A). The GM/Gb ratio was calculated and represented (B). Data are mean ± SD of 2 independent experiments.

(C) HeLa cells were transfected with indicated GCS constructs for 16 hours and SL output monitored by [3H] - sphingosine pulse-chase assay (8 h chase) in the presence or absence of BFA (5μg/ml). Total GSL levels were quantified and expressed as fold changes respect to control. NT refers to non-transfected cells. Data are mean ± SD of 2 independent experiments. *p <0.05, **p <0.01, (Student's t test).

(D) HeLa cells KO for LCS were transfected with indicated LCS RUSH constructs for 16 hours, and expressed enzymes were retained in the ER (-biotin) or placed in the Golgi by addition of biotin (40 μM) (+biotin) and SL output monitored by [3H] - sphingosine pulse-chase assay. The ratio of GM/Gb is represented. Data are mean ± SD of 2 independent experiments.

(E) Schematic representation of how the change in localization of GCS in GRASP55 KO with respect to control (pink dashed box vs blue dashed box) from trans- to cis-Golgi results in preferential access to ceramide and thus an increased production of GSLs.

(F) CERT was silenced using siRNAs for 72 hours in control and GRASP55 KO (#2) clone before subjecting to [3H] -sphingosine pulse chase assay. GSLs were quantified and represented as fold change with respect to control. Values are mean ± SD (n=3). *p <0.05, (Student's t test).
SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination. (2022)
Figure 1

Figure 1. FBXW7 loss-of-function bypasses requirement for autocrine Wnt signaling in Wnt-addicted PDAC cells.

A HPAF-II cells contain an autocrine Wnt signaling loop, where Wnt secreted by cells is required for the cells to grow. Treatment with the Porcupine (PORCN) inhibitor LGK974 (which inhibits Wnt secretion) leads to cell cycle arrest.

B Clonogenic growth assay in control HPAF-II cells or in cells treated with 150nM LGK974 for 10 days; representative of three independent replicates.

C Schematic representation of the positive selection screen conducted to identify genes involved in LGK974 sensitivity.

D Volcano plot with results of the positive selection screen identifying FBXW7 along with AXIN1, CSNK1A1, and APC as genes overcoming LGK974-induced cell cycle arrest when knocked out.

E Schematic representation of FBXW7-/- and APC-/- cell line generation in HPAF-II using LGK974 treatment to enrich edited cells.

F Clonogenic growth assay of indicated HPAF-II cells left untreated or in the presence of 150nM LGK974 for 10 days; representative of three independent replicates. Quantification of n=3 biological replicates, one-way ANOVA, mean ± SEM.

G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF-II cells from indicated genotype, treated with vehicle or 100nM LGK974 for 48h; representative of three biological replicates.
SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination. (2022)
Figure 3

Figure 3. CCNL1 protein stability is mediated by SCFFBXW7.

A Immunoblot of CCNL1 and FBXW7 expression in HPAF-II wild-type and FBXW7-/- cells, representative blot of 3 independent replicates.

B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF-II wild-type and FBXW7-/- cells , representative blot of 3 independent replicates.

C Quantification of cycloheximide chase in B, mean ± SEM of three independent replicates. Two-way ANOVA at T8.

D Immunoblot of CCNL1 expression in HPAF-II wild-type and dN-Cul1 expressing cells, representative blot of 3 independent replicates.

E Sequence alignment of well-characterized CPD degron motifs, and identification of potential FBXW7 phosphodegron motif in a conserved region of CCNL1.

F Fluorescence polarization assay of CCNL1 and cyclin E peptides binding FBXW7-Skp1 complex, three independent replicates, mean ± SEM.

G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild-type or degron-mutated CCNL1, representative blot of 3 independent replicates.

H Quantification of cycloheximide chase in G, mean ± SEM of three independent replicates. Two-way ANOVA at T8.
SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination. (2022)
Figure 4

Figure 4. FBXW7 is involved in the ubiquitination of CCNL1

A Immunoblot of immunoprecipitation of FLAG-FBXW7 overexpressed in HEK293T cells, detecting endogenous CCNL1. Representative of three independent replicates.

B Immunoblot of immunoprecipitation of FLAG-CCNL1 overexpressed in HEK293T cells, detecting endogenous FBXW7. Representative of three independent replicates.

C Immunoblot of cellular ubiquitination assay demonstrating the requirement of the 325-TPALS-329 degron for ubiquitination of CCNL1. Representative of three independent replicates.

D Immunoblot of cellular ubiquitination assay demonstrating the requirement of FBXW7 for ubiquitination of CCNL1. Representative of three independent replicates.

E Immunoblot of cellular ubiquitination assay showing re-expression of FBXW7 in knockout cells leads to increased CCNL1 ubiquitination. Representative of three independent replicates.
SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination. (2022)
Figure 5

Figure 5. CCNL1 regulates mitotic timing.

A Cell cycle profile of nocodazole treated and released HPAF-II wild-type and FBXW7-/- cells measured by flow cytometry, n=3 independent replicates, mean± SEM.

B Immunoblot of HPAF-II wild-type and FBXW7-/- released from overnight nocodazole treatment at indicated time points, representative of 3 independent replicates

C Representative cell cycle profile of HPAF-II wild-type, FBXW7-/-, and CCNL1OE cells stained with propidium iodide, with quantification of G2-M phase. Three independent replicates, mean ± SEM, one-way ANOVA.

D Representative images of GFP-tubulin labeled wild-type, FBXW7-/-, and CCNL1OE HPAF-II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol (150μM for 18 hours). Arrows indicate tracked cell, 20μm scale bars

E Quantification of cytokinetic timing, n=20, 25, 26 (wild-type, FBXW7-/, CCNL1OE ) pooled from three independent experiments, one-way ANOVA, mean ± SEM.

F Immunoblot of wild-type, FBXW7-/- and CCNL1OE HPAF-II cell lines demonstrating varying levels of CCNL1.

G Cell cycle profiles of wild-type, FBXW7-/- and APC-/- HPAF-II cells following treatment with 500nM LGK974 for 24 hours. Representative images of 3 independent replicates, mean ± SEM, 2-way ANOVA, *p=0.044, ****p<0.001.
SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination. (2022)
Figure 6

Figure 6. Cells harboring FBXW7-LOF mutations or CCNL1 overexpression are highly sensitive to CDK11 inhibitor, OTS964.

A Clonogenic growth assays for HPAF-II wild-type and FBXW7-/- polyclonal, and FBXW7-/- clones in presence of various OTS964 doses for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and plotted mean ± SEM.

B Clonogenic growth assays for HPAF-II wild-type and CCNL1OE polyclonal, and CCNL1OE clones in presence of various doses of OTS964 for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and plotted mean ± SEM.

C Immunoblot for the indicated proteins from lysates extracted from the individual FBXW7-/- and CCNL1OE clones.

D Cell cycle distribution plots with and without 24h OTS964 treatment, representative of 4 independent replicates. Normalized to untreated, mean ± SEM, one-way ANOVA.
SCF-FBXW7 regulates G2-M progression through control of CCNL1 ubiquitination. (2022)
Figure 7

Figure 7. Cervical cancer cell lines exhibit differential sensitivity to OTS964 depending on FBXW7 mutational status.

A Oncoprint from cBioPortal demonstrates mutual exclusivity between FBXW7 and CCNL1 alterations

B Immunoblot of lysates from cervical cell lines demonstrating C33A cells (FBXW7R465H) express high levels of CCNL1, representative of three independent replicates.

C Clonogenic growth assay of C33A, Caski, and SiHa cells in presence of various doses of OTS964 for 14 days. Representative images of 3 independent replicates, quantified by crystal violet absorbance at A595 and plotted mean ± SEM.

D Cell cycle distribution plots with and without 24h OTS964 treatment, representative of 3 independent replicates. Normalized to untreated, mean ± SEM, one-way ANOVA.

E Model of proposed mechanism.

Expanded View Figure Legends
Oxidized LDL-dependent pathway as new pathogenic trigger in Arrhythmogenic Cardiomyopathy. (2021)
Figure 1

Figure 1. ACM patients show high plasma oxLDL, cardiac lipid peroxidation and oxLDL receptor levels.

A) oxLDL plasma concentration in ACM patients and HC (n=36; Mann-Whitney test).

B) oxLDL plasma concentration in mutated ACM (n=7) and NON ACM relatives, carriers of the same causative mutation (n=9; Mann-Whitney test).

C) oxLDL plasma concentration in ACM patients carriers of a PKP2 mutation and ACM patients carriers of other desmosomal or non desmosomal mutations, or gene elusive (n=10 vs. n=26; One-Way ANOVA).

D) Representative images of MDA immunostaining (green) on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue).

E) Representative images of CD36 immunostaining on ACM and HC ventricular tissue sections and relative quantification (n=4 biological replicates; Two-tailed Student's t-test). Nuclei are counterstained with Hoechst33342 (blue).

Data information: mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001.