EMBO/soda-nel huggingface dataset:
Plasmodium sporozoite disintegration during skin passage limits 
malaria parasite transmission (2022)
Figure 6

Figure 6 - Concavin is essential for efficient transmission by mosquitoes

A. Growth curve of blood stage parasites in C57BL/6 mice infected by the bite of 10 mosquitoes (left) or 10.000 sporozoites intra venously (right) at two different times post mosquito infection as indicated. Shown is the mean ± SEM. Mice infected with Concavin(-) parasites by bite represent 2 independent biological replicates with 3 mice each. Wild type bite back and Concavin(-) intra venous injections represent one biological replicate using 3 or 4 mice respectively.

B. Sum table of infected mice from A with pre-patency period (time to detect a blood stage infection) and parasitemia on day 6. Note that in concavin(-) by bite infected mice not all develop a blood stage infection.

C. Liver cell invasion assay of concavin-gfp and concavin(-) parasites. Parasites are positively stained for CSP in case they remain extracellular. Both, normal and deformed parasites were detected intracellularly. Graph shows quantification of CSP positive (red, hence extracellular) and negative (grey, hence intracellular) sporozoites. Small graph shows the percentage of deformed and normally shaped intracellular concavin(-) sporozoites. Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed. Shown is the mean ± SEM. P-values are calculated using the Mann Whitney test. Scale bar: 5 µm.

D. Liver-stage development of concavin(-) parasites compared to wildtype, both expressing cytoplasmic GFP (white). Parasite size was measured 24h and 48h post infection. Data points represent individual parasites from 4 independent biological replicates. Shown is the mean ± SEM. P-values calculated using the Mann Whitney test. Scale bar: 5 µm.

E. Sporozoite ejection of immobilized concavin(-) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes. * indicates individual sporozoites in the ejected saliva. Scale bar: 10 µm.

F, G. Only normally shaped concavin(-) sporozoites released by salivary glands move on helical paths (arrows) through polyacrylamide gels that mimic the skin (F). Quantification of two individual experiments Scale bar 10 µm. (G): only normal shaped sporozoites were able to migrate through the gel.
Plasmodium sporozoite disintegration during skin passage limits 
malaria parasite transmission (2022)
Figure 7

Figure 7 - Migrating concavin(-) sporozoites lose their cellular integrity

A. Percentage of mosquitoes depositing WT or concavin(-) sporozoites in the skin of a mouse during a bite (left) and sporozoites deposited during a mosquito bite (right). P-values are calculated using the Fishers exact test (left) and the Mann Whitney test (right). Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT. For each mosquito infection 2-3 mice were imaged, for a total of 3-5 ears.

B. Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito-bite transmission. Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10, 20 and 30 minutes of recording, numbers analysed as well as sporozoite speed. Pooled data from 4-5 WT or 5-8 concavin (-) bite sites per time point. Scale bar: 50 µm. P-values are calculated using the Mann Whitney test.

C. Motile WT or concavin(-) sporozoites entering blood vessels during the first hour following micro-injection. Number above bars shows numbers of sporozoites observed. Pooled data from four WT or five concavin(-) experiments. P-values are calculated using the Fishers exact test.

D, E (D) Deformation and (E) disintegration of concavin(-) sporozoites migrating in the skin. Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection (left) of 13-min movies. Arrowheads indicate constrictions of the parasites. Graphs below time-lapse show that deformation and disintegration are preceded by a decrease in speed. Color of dots correspond to the time-points displayed in the time-lapse images. Scale bars: 10 µm.

F. Percentage of deformation and disintegration events observed in 54 WT and 57 concavin(-) sporozoites that were tracked for at least 10 min. Pooled data from 5 WT or 10 concavin(-) bite sites.

G. Consequence of deformation and disintegration from the 22 sporozoites in F include immobilization and parasite death.

EXPANDED VIEW FIGURE LEGENDS
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf1

(a) COS-7 cells expressing EGFP-tagged wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin exon 1 (green) for 48 h stained for endogenous mTOR (red) show mTOR in mutant aggregates. (b) Double-labeling immunohistochemistry of huntingtin (EM48; green) and mTOR (red) in brain tissue of mice expressing mutant huntingtin. (c) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease or unaffected controls using antibody to mTOR. (d) Western blot of lysates from stable inducible PC12 cells 48 h after expression of wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin showing mTOR in the stacking gel (mTOR insoluble) in cells expressing mutant huntingtin. (e) High-molecular-mass aggregates of mTOR in both PC12 (i) and COS-7 (ii) cells expressing mutant (Gln74; Q74) huntingtin. Blots of cell lysates from PC12 cells were probed with antibodies to phosphorylated mTOR (Ser2448; p-mTOR; iii) and to GFP (iv); both showed insoluble material in the stack. (f) Cell lysates from stable inducible PC12 cells and transiently transfected COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin for 48 h were immunoprecipitated using antibody to GFP and immunoblotted with antibody to mTOR. Uninduced PC12 cells with mutant huntingtin (UI) and untransfected COS-7 cells (UT) were controls. (g) Brain lysates from mice expressing mutant huntingtin (Tg) or control (Cont) littermates were immunoprecipitated with antibody to mTOR and immunodetected with an antibody recognizing the polyglutamine tract (IC2). Htt, huntingtin.
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf2

(a) Reduced levels of soluble mTOR (as a function of tubulin) in stable inducible PC12 cells expressing mutant (Gln74; Q74) huntingtin compared with those expressing wild-type (Gln23; Q23) huntingtin (four independent experiments; P 0.0001; unpaired t-test). (b) Reduced levels of soluble mTOR in brain lysates from mice expressing mutant huntingtin (M1, M2; n = 9) compared with age-matched wild-type littermates (C1, C2; n = 9; P 0.001, unpaired t-test). (c) COS-7 cells transiently transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) labeled for endogenous 4E-BP1 (red). (d) Immunohistochemistry of brain tissue from individuals with grade III Huntington disease showed 4E-BP1 in intranuclear inclusions. No 4E-BP1-stained inclusions were seen in controls (data not shown). (e) Western blot of cell lysates from stable inducible PC12 cells transfected with wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin shows that cells expressing mutant huntingtin had 31% (± 4.3% s.e.) less phosphorylated S6K1 (top) relative to total S6K1 (bottom) than cells expressing wild-type huntingtin. (f) Similarly, cells expressing mutant (Gln74; Q74) huntingtin had 30% (± 4.5% s.d.) less phosphorylated 4E-BP1 (top) relative to total 4E-BP1 (bottom) than cells expressing wild-type (Gln23; Q23) huntingtin. Expression of wild-type and mutant huntingtin was induced for 48 h (e,f). (g) Cells expressing mutant (Gln74; Q74) huntingtin also had lower levels of phosphorylated forms of transiently transfected FLAG-tagged 4E-BP1 than cells expressing wild-type (Gln23; Q23) huntingtin. The multiple bands in 4E-BP1 (f,g) are due to its different phosphorylated species.
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf3

(a) COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin (green) were labeled for S6 phosphorylated at Ser235 and Ser236 (red). Percentages of cells expressing wild-type or mutant huntingtin that did (+agg) or did not (−agg) contain aggregates were scored for negative staining for phosphorylated S6 (p-S6). (b) Brain sections from control (WT) and mice expressing mutant huntingtin (HD) were stained for phosphorylated S6 (red) and with antibody to huntingtin (EM48; green). (c,d) Impaired mTOR-dependent TOP translation in cells expressing mutant huntingtin. (c) PC12 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin were transfected with luciferase constructs containing 5′ untranslated sequence with (+) or without (−) a TOP sequence along with β-gal. Each bar represents the difference between the luciferase activities in the presence versus the absence of rapamycin. Data shown are from one representative experiment (of three) done in sextuplicate (error bars represent s.e.; raw data are given in Supplementary Fig. 4 online). (d) COS-7 cells were cotransfected with wild-type or mutant huntingtin, a nontypical TOP or control luciferase vector and β-gal. The experiment was done and analyzed as in c (raw data are given in Supplementary Fig. 4 online) (e–g) Relevance of mTOR sequestration to other polyglutamine expansion diseases (e) COS-7 cells expressing Xpress-tagged ataxin-1 with 92 polyglutamine repeats (Q92) for 48 h were doubly labeled with antibodies to Xpress (green) and to mTOR (red). (f) mTOR immunohistochemistry in brains of individuals with dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia types 2, 3 and 7 (SCA2, SCA3 and SCA7). (g) Staining for phosphorylated S6 (red) in COS-7 cells transiently transfected with mutant (ataxin Q92; green) or wild-type (ataxin Q2) ataxin-1 constructs. Results were significant and similar to those in a. Arrows in a, b, e and g show cells containing aggregates.
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf4

(a) LC3-II levels, which closely correlate with autophagic activity, were higher in mutant lines (expressing Gln74 huntingtin; Q74; lanes 3 and 4) than in wild-type lines (expressing Gln23 huntingtin; Q23; lanes 1 and 2). Actin was used as a loading control. Data are shown for two independent clonal lines expressing mutant or wild-type huntingtin. Quantification of the band intensity is shown in the right panel. (b) Immunofluorescence analysis of COS-7 cells expressing wild-type (Gln23; Q23) or mutant (Gln74; Q74) huntingtin with antibody to LC3 showed more autophagic vacuoles in cells expressing mutant huntingtin that had aggregates (arrow). COS-7 cells without the primary antibody (negative) and COS-7 cells without any treatment (untreated) stained for LC3 are also shown. The numbers of EGFP-positive cells expressing wild-type or mutant huntingtin with (+agg) and without (−agg) aggregates that had higher numbers of autophagic vacuoles (>15–20 vesicles per cell) is shown in the graph. COS-7 cells grown in starvation medium (starvation) or treated with rapamycin (Rap) for 4 h to induce autophagy had more autophagic vacuoles (arrows).
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf5

(a) Activation of mTOR activity with rheb increases aggregation and toxicity of mutant (Gln74; Q74) huntingtin. COS-7 cells were cotransfected with a mammalian expression vector encoding rheb (or empty vector control) and mutant huntingtin for 48 h at a 3:1 mass ratio (rheb or empty vector to huntingtin) to ensure that almost all cells expressing huntingtin also expressed rheb. The percentages of EGFP-positive cells with aggregates or apoptotic nuclear morphology (as described previously) were markedly higher when rheb was overexpressed. Control cells express mutant huntingtin (green) without rheb. The middle panels show images captured at the same comparatively low gain to distinguish aggregates from soluble EGFP. The left panels show the same fields at a similar higher gain to indicate the total number of EGFP-positive cells (this shows that the effect seen in the middle panels is not due to loss of cells). Nuclear staining with DAPI (blue) is shown in the right panels. ***P 0.0001. Error bars represent s.e. for a representative triplicate experiment. (b,c) Treatment with rapamycin rescues huntingtin-induced degeneration in flies 2 d after eclosion. Photographs of ommatidia of flies expressing mutant (Gln120; Q120) huntingtin treated with DMSO or rapamycin from the larval stage into adulthood. More rhabdomeres are visible in the ommatidia of flies treated with rapamycin. This effect is significant (P 0.0001; Mann-Whitney U test). At 2 d after eclosion, there is no reduction in the number of visible rhabdomeres in wild-type untreated flies (data not shown).
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf7

(a) Weights of mice expressing mutant huntingtin (Tg) and nontransgenic (NT) littermates treated with CCI-779 or with a placebo. (b) Brain weights of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.023), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.90) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.22) of age. (c) Ratio of brain weight to body weight of mice expressing mutant huntingtin treated with CCI-779 (black bars) or with a placebo (white bars) and nontransgenic untreated littermates (gray bars) at 8 weeks (CCI-779, n = 3; placebo, n = 3; nontransgenic, n = 7; CCI-779 versus placebo, P = 0.06), 12 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 5; CCI-779 versus placebo, P = 0.38) and 16 weeks (CCI-779, n = 2; placebo, n = 3; nontransgenic, n = 2; CCI-779 versus placebo, P = 0.56) of age.
Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease (2004)
figf8

(a) CCI-779 reduces aggregate load in mice expressing mutant huntingtin. Peroxidase immunohistochemistry of brains of mice expressing mutant huntingtin at 16 weeks of age. Mice treated with CCI-779 had fewer aggregates per unit area in the striatum than mice treated with a placebo. Overall cell staining in the tissues of mice treated with CCI-779 was weaker than in those treated with a placebo and in nontransgenic untreated littermates (data not shown). Aggregates, when visible, were smaller in mice treated with CCI-779 than in those treated with a placebo, as apparent in the higher magnification images of single cells with aggregate (bottom panels). Six coronal sections per mouse were analyzed; these corresponded to the bregma ± 1 mm. We counted aggregates in five different high-power fields (60× objective) per slide on each side of the brain, sampling fields from the dorsal to the ventral part of the striatum just underneath the external capsule. The striata of mice treated with CCI-779 had 70% fewer aggregates than the striata of mice treated with a placebo (P = 0.0001, two-tailed t-test). (b) CCI-779 blocks mTOR signaling in vivo. Immunohistochemistry of mice with Huntington disease at 16 weeks of age with antibody to phosphorylated S6, in the cortical area and striatum. Levels of phosphorylated S6 were lower in mice treated with CCI-779 than in those treated with a placebo.
FACT maintains nucleosomes during transcription and stem cell viability in adult mice (2022)
Figure 1

Figure 1. Effect of Ssrp1 KO in adult mice.

A, B. Normalized weight (A) and survival (B) of Ssrp1fl/fl; CreERT2+/+ mice of different ages (weeks) after the start of tamoxifen (TMX) treatment. A. Mean weight (%) ± SD. P < 0.05 for all tamoxifen treated groups versus vehicle (ANOVA). B. Kaplan-Meier survival curves. Log-rank test p = 0.032 for the oldest male age group versus all other male groups; p = 0.013 for all females versus all males; for all other comparisons p > 0.05. n = 6-10 mice.

C. Normalized blood cell counts in Ssrp1fl/fl; CreERT2+/+ mice treated with vehicle or tamoxifen on day 7 after start of treatment. Mean ± SD, * indicates p < 0.05 (unpaired t-test). n = 3 mice.

D. Photographs of vehicle- or tamoxifen-treated Ssrp1fl/fl; CreERT2+/+ mice seven days after start of treatment.

E. Gross pathology examination of Ssrp1fl/fl; CreERT2+/+ mouse euthanized due to 20% weight loss after treatment with tamoxifen. Vehicle-treated mouse is shown for comparison. Green and blue ovals indicate subcutaneous and inguinal fat deposits, respectively.

F. MRI image of Ssrp1fl/fl; CreERT2+/+ mouse before and after tamoxifen treatment.
FACT maintains nucleosomes during transcription and stem cell viability in adult mice (2022)
Figure 3

Figure 3. FACT depletion in mice leads to reduced proliferation and cells death in several organs.

A, B. Effect of tamoxifen on replication in the spleen (A) and intestine (B) of Ssrp1fl/fl; CreERT2+/+ mice. EdU was given to mice three days after the end of tamoxifen treatment (1 hour before tissue collection). Organ sections were stained for total DNA (Hoechst), EdU, and SSRP1. Scale bars - 100 μM.

C. Quantitation of EdU staining. Mean number of EdU positive cells per field of view +/- SD. n > 5 fields of view per mouse. 3 mice were analyzed.

D. Quantitation of cleaved caspase-3 staining. Mean number of positive cells per fields of view +/- SD, n > 5 fields of view per mouse Two male and female mice were analyzed.

Data information: P-values for C and D were calculated using the unpaired t-test.
FACT maintains nucleosomes during transcription and stem cell viability in adult mice (2022)
Figure 5

Figure 5. Effect of FACT depletion on cell composition of the intestine from Ssrp1fl/fl; CreERT2+/+ mice.

A. UMAP plots with unbiasedly identified clusters shown in different colors.

B. UMAP plots are colored according to cell type identified by transcriptional signatures using data from Gao et al., 2018() as a reference. LI - large intestine, SI - small intestine.

C. Distribution of intestinal cell types in samples from vehicle- and tamoxifen-treated mice. Pie charts are colored accordingly to panel B.

D. UMAP plots with color-coded intestinal stem cells identified by transcriptional signatures of intestinal stem cells from Haber et al., 2017() or by expression of Lgr5 or Gkn3 genes.

E. UMAP plots showing the distribution of cells with transcriptional signatures of proximal and distal enterocytes from Haber et al., 2017() and cells expressing the Dclk1 gene.

F. GSEA of gene markers of clusters 2 and 3 (lost upon Ssrp1 KO, blue) versus clusters 8 and 9 (emerged upon Ssrp1 KO, red).

Data information: Pooled data from two mice on day 1 after the end of treatment.
FACT maintains nucleosomes during transcription and stem cell viability in adult mice (2022)
Figure 6

Figure 6. FACT depletion affects the viability of stem cells but not differentiated cells.

A, B. Effect of 4-OHT administration on colony formation by bone marrow cells of different genotypes. A. Photographs of colonies from bone marrow cells kept in culture for seven days in the presence or absence of 4-OHT. B. Quantitation of bone marrow colony assay. Mean colony number per plate ± SD, n = 3 plates per mouse. P values < 0.05 are shown (unpaired t-test). 2 mice of each genotype were analyzed.

C, D. Growth of intestinal organoids in the presence or absence of 4-OHT. C. Photographs of small intestine organoids on different days after plating. D. Number of organoids in the presence and absence of 4-OHT. Mean fold change between number of organoids in control and 4-OHT treated wells ± SD, n = 4 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from one mouse of each genotype in two independent experiments.

E. Caspase activity in lysates of control and 4-OHT treated MSCs of different genotypes. Mean fold change ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from 2-3 mice of each genotype.

F, G. Effect of 4-OHT administration on intestinal organoids of different genotypes kept in the presence (undifferentiated) or absence (differentiated) of R-spondin-1. F. The viability of organoids assessed by the resorufin assay. Mean ± SD, n = 3 wells per mouse. P < 0.05 are shown (unpaired t-test). Organoids were isolated from two mice of each genotype. G. IF images of intestinal organoids from Ssrp1fl/fl; CreERT2+/+ mice kept in the presence or absence of R-Spondin-1. The organoids were stained with antibodies to LGR5 and SSRP1 and counterstained with Hoechst (DNA).

H-J. Effect of 4-OHT administration on MSCs and their differentiated counterparts. H. Methylene blue viability assay for undifferentiated MSCs or MSCs differentiated into osteocytes or adipocytes for 21 days and then treated with vehicle or 4-OHT. Mean ± SD, n = 3 plates per mouse. P < 0.05 are shown (unpaired t-test). MSCs were isolated from two mice of each genotype. I. Staining of MSCs, osteocytes, and adipocytes with Alizarine Red (osteocytes) or Oil Red (adipocytes). J. Western blotting of protein extracts from MSCs, adipocytes, and osteocytes from mice of different genotypes treated with vehicle or 4-OHT.
FACT maintains nucleosomes during transcription and stem cell viability in adult mice (2022)
Figure 7

Figure 7. Comparison of transcription between Ssrp1fl/fl; CreERT2+/+ MSCs treated with vehicle or 4-OHT.

A. EU incorporation measured by flow cytometry. Histograms of EU fluorescent signals from two biological replicates for each condition (except no EU).

B. SSRP1 staining of the cells in A.

C. Volcano plot of changes in transcript counts in 4-OHT-treated cells versus control. Red dots indicate transcripts with fold-change > 2 and FDR < 0.05. n = 2 biological replicates.

B. MA plot showing the difference between the changes in transcript counts in 4-OHT-treated MSCs vs. control and the average expression of the same transcripts between conditions (baseMean). n = 2 cell cultures.

E, F. Dot plots with the results of GO enrichment for genes upregulated (C) and downregulated (D) upon FACT depletion.