EMBO/soda-nel huggingface dataset:
SOX9 maintains human fetal lung tip progenitors by enhancing WNT and RTK signalling (2022)
Figure 2

Figure 2. SOX9 regulates human fetal lung progenitor cell self-renewal.

(A) Human fetal lung tip organoids faithfully express in vivo tip progenitor markers, SOX2 and SOX9. Upper panel: SOX2/SOX9 dual expression in human fetal lungs. Lower panel: SOX2/SOX9 in human fetal lung tip organoids. SOX9 green, SOX2 red, E-Cadherin white.

(B) qRT-PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript levels using inducible CRISPRi after 5 days of Dox and TMP treatment. 3 different organoid lines and 2 different SOX9 gRNAs were used. Error bars: mean ± SEM. Two-sided Student's t-test with equal variance ***p < 0.001.

(C) SOX9 was knocked down at the protein level using the inducible CRISPRi system after 4 days of Dox/TMP treatment.

(D) SOX9 knockdown organoids at Passage #4 (P4).

(E) Summary of the serial passaging assay results for SOX9 knockdowns. 3 independent organoid lines each were transduced with 2 different gRNAs against SOX9, making 6 different organoid lines altogether. For serial passaging assay, organoids were maintained in Dox and TMP and broken into pieces every 3-4 days.

(F) Schematic of RNA-Seq to identify SOX9 downstream targets. 2 independent inducible CRISPRi parental organoid lines were transduced with 2 different non-targeting control gRNAs, two different SOX9 gRNAs and two different SOX2 gRNAs respectively. Organoids were supplemented with Dox/TMP for 5 days before harvesting for RNA-Seq.

(G) Venn Diagram showing the overlapping number of differentially expressed (DE) genes in SOX9 knockdown organoids from different parental lines.

(H) All SOX9 positively and negatively regulated genes were used to score against an scRNA-Seq dataset from 9-11pcw human fetal lung epithelium (left panel, He et al., 2022). SOX9 negatively-regulated genes were primarily enriched in secretory lineage populations (middle panel). SOX9 positively-regulated genes were primarily enriched in tip progenitor cells (right panel).

Data information: Scale bars denote 50 μm (A, C) and 100 μm (D).
SOX9 maintains human fetal lung tip progenitors by enhancing WNT and RTK signalling (2022)
Figure 3

Figure 3. Targeted DamID (TaDa) identified SOX9 direct regulating targets.

(A) Schematic of SOX9 TaDa lentiviral construct design. SOX9 is fused with E.coli DNA adenine methylase (Dam). The fusion protein binds to SOX9 binding targets and methylates adenine in the sequence GATC. Methylated GATC is specifically recognised and cleaved by DpnI restriction enzyme. TaDa is designed to produce an extremely low level of Dam-Sox9 fusion protein: an mNeonGreen open reading frame (ORF1) was placed in front of Dam-SOX9 fusion (ORF2). The two ORFs were separated by two stop codons and a frame-shift C (represented by 2 black diamonds), such that the Dam-SOX9 fusion protein is translated at very low levels after rare translational re-initiation events.

(B) Venn Diagram showing the overlap between DE genes identified in the SOX9 knockdown RNA-Seq experiment and genes annotated from SOX9 TaDa peaks.

(C) Heatmap showing expression level of all 171 SOX9 directly regulated genes across non-targeting control and SOX9 knockdown organoid lines.

(D) CD44 protein expression in SOX9+ tip progenitor cells. SOX9 green, SOX2 red, CD44 white.

(E) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor cells. SOX9 red, LGR5 cyan.

(F) Tip progenitor cells are of high WNT signalling activity. NOTUM yellow, SOX9 red.

(G) Design of constitutively activated β-catenin over-expression lentiviral construct.

(H) qRT-PCR showing rescue of SOX9 transcription after constitutively activated β-catenin overexpression in organoids cultured without WNT activators. N = 3 different organoid lines. Error bars: mean ± SEM.

(I) WB showing SOX9 protein was rescued after constitutively activated β-catenin overexpression in organoids cultured without WNT activators.

Data information: Scale bars = 50 μm in all panels.
SOX9 maintains human fetal lung tip progenitors by enhancing WNT and RTK signalling (2022)
Figure 4

Figure 4. SOX9 directly regulates ETV4 and ETV5 expression thereby enhancing FGF signalling.

(A,B) HCR images showing ETV5 (A) and ETV4 (B) expression in SOX9 expressing tip progenitors. ETV4/5 red, SOX9 cyan.

(C) qRT-PCR showing ETV4 and ETV5 dual knockdown in tip organoids. N =3 different organoid lines and 2 different ETV4 and ETV5 gRNA combinations were used. Error bars: mean ± SEM. Two tailed paired t-test: **p < 0.01; ***p < 0.001.

(D) Summary of the serial passaging assay results for ETV4; ETV5 dual knockdowns. 3 independent organoid lines each were transduced with 2 different ETV4; ETV5 gRNA combinations, making 6 different organoid lines altogether. Organoid cells were given 8 days to recover after plating and grown into small colonies before treating with Dox and TMP. Organoids were then maintained in Dox and TMP and serially passaged by breaking into pieces every 3-4 days.

(E) Representative images of organoids grown in self-renewing medium (left) and self-renewing medium without RTK ligands (EGF, FGF7, FGF10) (right).

(F) qRT-PCR of ETV4, ETV5 and SOX9 expression level in organoids in self-renewing medium and self-renewing medium without RTK ligands. N = 3 different organoid lines used. Error bars: mean ± SEM. Two-tailed paired T-test: **p < 0.01; n.s. non-significant.

(G) Venn diagrams showing overlap of SOX9-ETV5 TaDa peak regions (upper) and overlapping genes from TaDa peak annotations (lower).

(H) Examples of SOX9 and ETV5 genomic occupancies showing co-regulation. At the CD44 locus (upper), SOX9 binds to an intron, whereas ETV5 primarily occupied the promoter. By contrast, for the LGR5 gene (lower), SOX9 and ETV5 both showed occupancy at the promoter.

(I) Schematic model of SOX9 regulation of tip progenitor cell self-renewal.

Data information: Scale bars denote 50 μm (A, B) and 100 μm (E).

Expanded view figure legends
IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect (2022)
Figure 2

Figure 2. Affected individuals with variants in IQCN exhibited TFF and defects in the acrosomal ultrastructure.

A. The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15-18 h post-injection. The white arrowheads represent the polar body (PB). PN represents pronucleus.

B. The expression and localization of PLCζ in sperm from the affected individuals. Scale bars, 3 μm.

C. H&E staining of spermatozoa from affected individuals. The black arrow represents normal spermatozoa. The black arrowheads represent tapered-head spermatozoa with increased major axis length and normal minor axis length. The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length. Scale bars, 20 μm.

D. The ultrastructure of spermatozoa from normal and affected individual. The white squares represent the perinuclear ring. Ac represents acrosome. N represents nucleus. Scale bars, 500 nm.

E. The range of acrosomes in sperm heads from normal and affected individual (n=7).

Data information: Data represent mean ± SEM. ****P < 0.0001 by unpaired Student's t-test (E).
IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect (2022)
Figure 3

Figure 3. Knockout of Iqcn leads to male infertility and acrosomal defects in a mouse model.

A. Fertility assessment experiments in WT and Iqcn-/- male mice after mating with WT female mice (n = 18).

B. Size of the testes and epididymides in WT and Iqcn-/- male mice.

C. Quantification ratio of the testis weight/body weight in WT and Iqcn-/- male mice (n = 3).

D. Sperm counts in the caudal epididymis from WT and Iqcn-/- male mice (n = 3).

E. H&E staining of sperm from WT and Iqcn-/- male mice. Red arrowheads represent sperm with no sickle shape heads. Scale bars, 20 μm.

F. Percentage of sperm without sickle shape heads from WT and Iqcn-/- male mice (n = 3).

G. Ultrastructure of spermatozoa from WT and Iqcn-/- male mice. Ac represents the acrosome. Scale bars, 500 nm.

H. Immunostaining of lectin (green) in spermatozoa from WT and Iqcn-/- male mice. Scale bars, 3 μm.

I. Percentage of abnormal acrosomes in spermatozoa from WT and Iqcn-/- male mice (n = 3).

J. Percentage of spontaneous and induced acrosome reactions in spermatozoa from WT and Iqcn-/- male mice (n = 3).

Data information: Data represent mean ± SEM. ***P < 0.001, ****P < 0.0001, ns represents no significance by unpaired Student's t-test (A, C, D, F and I), or by one-way ANOVA (J).
IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect (2022)
Figure 4

Figure 4. Iqcn-/- male mice induce TFF with oocyte activation failure.

A. Representative two pronucleus (2PN) zygotes, two-cell embryos, and blastocysts after ICSI in WT and Iqcn-/- male mice. Scale bars, 40 μm.

B. Percentage of 2PN zygotes, two-cell embryos and blastocysts after ICSI in WT and Iqcn-/- male mice (n = 3).

C. Profile of Ca2+ responses induced by oocyte injection with spermatozoa from WT and Iqcn-/- male mice (n = 5). Spermatozoa from WT male mice induced 8-9 Ca2+ spikes over a 3-h period (blue line), while spermatozoa from Iqcn-/- male mice failed to induce spikes (red line).

D. Immunostaining of oocytes injected with spermatozoa from WT and Iqcn-/- male mice. The expression of PLCζ (red) and α-tubulin (green) was detected in oocytes. M represents male nucleus and F represents the female nucleus. Scale bars, 10 μm.

E. Expression and localization of PLCζ in sperm from WT and Iqcn-/- male mice. Scale bars, 3 μm.

F. Expression and localization of PLCζ during acrosomal biogenesis from WT and Iqcn-/- male mice. PLCζ was localized in the acroplaxome in WT spermatids. PLCζ was only localized in the tip of the head in the cap and acrosome phases (white arrowheads) and was lost during the maturation phase in Iqcn-/- mice. Scale bars, 3 μm.

Data information: Data represent mean ± SEM. ****P < 0.0001 by one-way ANOVA (B).
IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect (2022)
Figure 5

Figure 5. Iqcn-/- male mice cause defects in manchette assembly during spermiogenesis.

A. Expression and localization of IQCN during spermiogenesis in the testes of WT male mice. IQCN (red) was co-localized with α-tubulin (green) from steps 8-14. Sb represents late spermatids. Scale bars, 20 μm.

B. Ultrastructure of the testes from WT and Iqcn-/- male mice from steps 8-16. MA represents manchette. PR represents perinuclear ring. CD represents cytoplasmic droplets. Scale bars, 2 μm (steps 8-12; 15-16) and 5 μm (steps 13-14).

C. Immunostaining of α-tubulin (green) in spermatids at steps 13-14. MA represents manchette. PR represents perinuclear ring. Scale bars, 5 μm.

D. Length of the manchette structure in spermatids at steps 13-14 from WT and Iqcn-/- male mice (n = 10).

Data information: Data represent mean ± SEM. ****P < 0.0001 by unpaired Student's t-test (D).
IQCN disruption causes fertilization failure and male infertility due to manchette assembly defect (2022)
Figure 6

Figure 6. IQCN interacts with calmodulin to participate in manchette assembly.

A. Expression and localization of calmodulin during spermiogenesis in the testes of WT and Iqcn-/- male mice. Calmodulin (red) was co-localized with α-tubulin (green) from steps 8-14 in WT mice, but failed to co-localize in steps 13-14 in Iqcn-/- male mice. Scale bars, 3 μm.

B. Interaction between IQCN and calmodulin (CaM) following co-IP by IQCN antibody in testes from WT and Iqcn-/- male mice (n = 3). IQCN interacts with calmodulin in WT mice, the interaction of which is completely lost in Iqcn-/- male mice. GAPDH was used as the internal control.

C. Immunostaining of calmodulin (red) and α-tubulin (green) in HeLa cells. The normal (WT) and mutant (c.910C>T and c.2453_2454del) plasmids of IQCN were transfected into HeLa cells. HeLa cells were treated with an antagonist of calmodulin W-7. Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W-7 treatment. Scale bars, 2 μm.

D. Percentage of multipolar spindles in WT and mutant IQCN transfected HeLa cells. (n = 3 biological replicates)

Data information: Data represent mean ± SEM. ****P < 0.0001 by one-way ANOVA (D).
Proteomic patterns associated with response to breast cancer neoadjuvant treatment (2020)
Figure 4

Figure 4: PYCR1 abundance level is associated with breast cancer relapse.

A) Abundance level of the Eigengene vector of yellowgreen module. Each bar represents a patient and is separated and colored according to relapse status

B) Scatter plot of module membership vs gene significance score for the yellowgreen module that shows highest correlation to relapse. Proline biosynthesis proteins PYCR1 and ALDH18A1 are highlighted. Significance of Pearson correlation was determined using t-test.

C) Diagram of the proline metabolism pathways. Proline biosynthesis genes are PYCRL, Pyrroline-5-Carboxylate Reductase Like; PYCR1, Pyrroline-5-Carboxylate Reductase 1; PYCR2, Pyrroline-5-Carboxylate Reductase 2; ALDH18A1, Aldehyde Dehydrogenase 18 Family Member A1 (Pyrroline-5-Carboxylate Synthetase).

D) Proline biosynthesis protein levels in breast cancer subtypes (Log2 ratios of light vs. SILAC standard). The upper and lower limits of the box show values from the first to the third quartile, with the horizontal line indicating the median. The whiskers extend 1.5 times the interquartile range from the edges of the box. Outlier values were plotted outside the whiskers. Significance was determined using paired Student's t-test and p values were corrected using permutation based FDR . *p < 0.05 and *** p < 0.001.

E) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study (n=3); Scale bar: 200 μm.

F) Quantification of IHC shows percentage of cells with 2+ and 3+ PYCR1 staining intensity. Data is expressed as mean ± SD of three patient samples. Paired samples were compared by a Student's t-test. *p < 0.05 and *** p < 0.001.

G) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data. Cox univariate p value, risk table and hazard ratio with 95% CI indicated.
Proteomic patterns associated with response to breast cancer neoadjuvant treatment (2020)
Figure 5

Figure 5: CRISPR/Cas9 based PYCR1 knockout increases glutamine flux to the TCA cycle.

A) Seahorse experiment measuring oxygen consumption rate (OCR) in MCF7 cells upon addition of oligomycin, FCCP and a mixture of antimycin and rotenone. Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values were indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.

B) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.

C) 13C5 -Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate, malate and citrate in MCF7 cells. Data are presented as mean ± SD of triplicate samples. Isotopologues are represented as M+n where M indicates the mass and n equals number of 13C incorporated

D) Seahorse experiment measuring extracellular acidification rate (ECAR) in MCF7 cells upon addition of glucose, oligomycin and 2-deoxyglucose (2DG). Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.

E) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments.Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.

F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.
Proteomic patterns associated with response to breast cancer neoadjuvant treatment (2020)
Figure 6

Figure 6: PYCR1 knockout results in reduced invasion of MCF7 cells.

A) Representative pictures of transwell migration and invasion after PYCR1 knockout. Scale bar :100 μm.

B) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p-values are reported as follows *** p < 0.001.

C) Representative images of colony formation in soft agar upon PYCR1 KO. Scale bar: 100 μm.

D) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours. Data represents mean ± SE of three biological experiments. Samples were compared using paired Student's t-test.

E-H) Bar plots show percentage of viable cells after treatment with 0.75 mM H2O2 (E), 0.06 µM paclitaxel (F), 0.15 µM doxorubicin (G), 2.5 mM cyclophosphamide (H). Data represent mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05.

I) Tumor volume measurements for 26 days in MCF7 injected NSG mice. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) are shown. Data represents mean ± SE. Pax: Paclitaxel, Dox: Doxorubicin. Groups were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *p < 0.05, ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.

J) Bar plot indicates mean ± SE of tumor volume measurements for day 26. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.

EXPANDED VIEW FIGURE LEGENDS
Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor (2019)
Figure 4

Figure 4. CggR intracellular levels and expression profile with different promoter, strains and conditions. (A) CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments. (B) The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.
Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor (2019)
Figure 5

Figure 5. Biochemical characterization of CggR and respective mutants. (A) Thermal shift assays were used to determine the melting curves of wild-type CggR and mutants. Error bars correspond to the standard deviation of at least five replicates. (B) CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals. (C) Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype. (D) Summary of the biochemical characterization of the wildtype and mutant CggR variants. KD ratio change indicates the ratio between the KD of the CggR mutant variant and the one of the wildtype. A plus sign indicates desired characteristics achieved in the mutants; a minus sign indicates undesired effects of the mutations. The mutant highlighted with grey background is the one we selected for further analyses, as this mutant had the desired characteristics with regards to all three criteria. (E) Ray-traced picture of the wildtype CggR and R250A mutant structure. Carbon atoms of the FBP ligand are in turquoise while the part of the side chain that is eliminated by the R250A mutation is in green. Hydrogen bonds are indicated with dashed lines.