EMBO/soda-nel huggingface dataset:
A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress (2020)
Figure 8

Figure 8. In vivo role of C. elegans HLH-30 Cys284 residue.

A, Lifespan assay of hlh-30 mutants at 25°C. Graph represents Kaplan-Meier survival plots of two independent experiments initiated with 100 animals per strain. (**)p<0.01 and (****)p<0.0001 by Log-rank (Mantel-Cox) test compared to wild type control.

B, Survival of hlh-30 mutants on 7.5 mM NaAsO2. Data are the mean ± SD of three independent experiments initiated with 120 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control.

C, Survival of hlh-30 mutants to infection by Staphylococcus aureus. Data are the mean ± SD of three independent experiments initiated with 100 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control.

D, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP in an eat-2(ad1116) mutant background. Data are from three independent experiments with at least 50 animals per experiment.

E, Synthetic developmental delay phenotype of eat-2(ad1116) worms carrying mutations in the hlh-30 gene. Data are the mean ± SD of three independent experiments with at least 20 animals per assay. (****)p<0.0001 by One-way ANOVA with multiple comparison test.

F, Representative images of worms quantified in (E). Scale bars 200 µm.

G, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP in a daf-2(e1370) mutant background. Data are from three independent experiments with at least 50 animals per experiment.

H, Effect of hlh-30 mutations on the dauer formation phenotype of daf-2(e1370) worms. Data are the mean ± SD of four independent experiments. (***)p<0.001; (****)p<0.0001 by one-way ANOVA with multiple comparison test.

I, Effect of hlh-30 mutations on the dauer recovery phenotype of daf-2(e1370) worms. Data are the mean ± SD of three independent experiments. (****)p<0.0001 by two-way ANOVA compared to wild type control.
Two-step nuclear centring by competing microtubule- and actin-based mechanisms in 2-cell embryos (2022)
Figure 1

Figure 1. Nuclear movement during and after mouse embryonic first mitotic division.

A, B Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex.

C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres).

D A histogram showing the frequencies of maximal d values for individual blastomeres (n = 50 blastomeres).

E Shown are 3 representative examples of nuclear tracking from metaphase (dashed white lines) to late 2-cell interphase. The look-up table shows the colour scale corresponding to nuclear velocities ranging from 0% to 100% of maximum.

F Shown are representative time-lapse images of a zona-free embryo undergoing the first mitotic division depicting a distinct chromosome-induced cortical protrusion (see 1 h post-anaphase-onset and corresponding enlarged region).

Data information: In (A, F), time is shown as h post-metaphase and scale bar = 20 µm. In (C, D), data are shown as mean ± SEM.
Two-step nuclear centring by competing microtubule- and actin-based mechanisms in 2-cell embryos (2022)
Figure 7

Figure 7. Microtubule-based forces antagonise nuclear centring.

A MSD analysis during centre-to-cortex nuclear displacement (n = 18 blastomeres). Power-law slopes of 1 and 2 are drawn for comparison.

B Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos).

C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos).

D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos).

E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group).

F The proportions of normal bipolar and abnormal spindles in DMSO- and dynarrestin-treated blastomeres undergoing the second mitosis.

G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement.

H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM).

I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm.

Data information: In (D, F), time is shown as h post-drug addition. In (B, D, G, I), scale bar = 20 µm. In (C, E, H), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).
In vitro and in vivo identification of clinically-approved drugs that modify ACE2 expression (2020)
Figure 1

Figure 1: The landscape of ACE2 expression levels alterations in response to drug treatments across cell lines in CMAP (Subramanian et al, 2017).

(A-F) Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are given for (A) 48 antihypertensive drugs and (C) 672 clinically approved drugs that are each tested on four carcinoma cell lines from CMAP (Subramanian et al, 2017). The ACE2 expression levels in control and drug-treated groups for the top significant drugs (vorinostat, panobinostat, and isotretinoin) among the 672 clinically approved drugs are shown with boxplots in (D), the ACE2 expression log fold-change values and adjusted P values from limma, as well as the number of samples per group are labeled for each drug. In the boxplot of panel (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots. The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (B) and (E), for the analysis on the 48 antihypertensive drugs and the 672 clinically approved drugs, respectively. We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines (16 cancer and 12 normal cells; this list is provided in Table EV2A with details including primary site, subtype and donor demographics) where each drug may have been tested on a different subset of cells (Methods). and performed enrichment analysis using the WHO ATC drug classification data (World Health Organization, 2006), and top enriched drug classes are shown in (F). The horizontal and the vertical dashed lines denote a P value of 0.05. Drug indication and ATC classification annotations are obtained from the DrugBank database (Wishart et al, 2018). All P values are computed from differential expression analysis using limma (Ritchie et al, 2015) (Methods). Abbreviations: ERAs, endothelin receptor antagonists; CCBs, calcium channel blockers; ARBs, angiotensin II type-I receptor blockers; ACEIs, angiotensin-converting enzyme inhibitors; TKR, tyrosine kinase receptor.
In vitro and in vivo identification of clinically-approved drugs that modify ACE2 expression (2020)
Figure 2

Figure 2: Drug-induced ACE2 differential expression in lung and kidney-derived cells or tissue types from the GEO database.

(A-D) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database, spanning both cancer and non-cancer datasets (Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are displayed in (A) for 74 datasets of cells or tissue samples from lung, involving 42 clinically approved drugs, and in (C) for 35 kidney datasets involving 29 clinically approved drugs. Among these, we focused on the top significant drugs that modulate ACE2 expression in non-cancerous cells or tissue samples from lung and kidney, which are shown in (B) and (D), respectively, where the ACE2 expression (Y-axis) difference between control and treated groups (X-axis) are shown with boxplots. In the title of each boxplot, the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided. Different datasets involving experiments using the same drug were analyzed and presented separately, since the sample types can be different across datasets. All the P values are computed from differential expression analysis using limma (Ritchie et al, 2015) (Methods). In the boxplots of panels (B) and (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots. Abbreviations: HBEC, human primary bronchial epithelial cells.

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Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 2

Figure 2. AG induces expression of MMPs.

(A) Heat map showing RPKM (Reads Per Kilobase per million of mapped reads) mean values of Mmps from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for 24 h or left untreated (NT).

(B) Quantitative polymerase chain reaction (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times.

(C) Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times; GADPH of cell lysates served as a loading control.

(D) qPCR analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of mice at indicated days post intraperitoneal administration of AG (100 μg).

(E) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for 24 h in absence or presence of AG aptamers (0.5 μg/ml).

(F) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times in absence or presence of AG aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control.

(G) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of mice at 3 days post intraperitoneal administration of AG (100 μg) in absence or presence of AG aptamers. AG aptamers (1 μg/mouse) were intranasally administrated per day.

(H) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h (MOI=5) in absence or presence of AG aptamers (1 μg/ml).

(I) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in the absence or presence of AG aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control.

(J) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from lungs of mice intranasally infected with H37Rv (2×106 cfu/mouse) for 4 weeks in absence or presence of AG aptamers. AG aptamers (1 μg/mouse) were intranasally administrated once at a 1 week-interval.

Data information: Data in (B, E, H) are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Data in (D, G, J) are means + SD of indicated numbers of mice from 1 of at least n=2 independent experiments and each symbol represents data from 1 mouse. Data (D, G, J) shown are representative of n=2 (D) or n=3 (G, J) independent experiments. Two-way ANOVA followed by Tukey's post hoc test (B, D, E, G, H, J) was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.
Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 3

Figure 3. AG activates ERK to induce MMPs.

(A) Immunoblots of cell lysates were performed to analyze p-ERK1/2, p-JNK, p-P38 and p-P65 by THP-1 cells stimulated with AG (1 μg/ml) for indicated times, and GADPH as a loading control.

(B) Immunoblots of cell lysates were performed to analyze p-ERK1/2, p-JNK, p-P38 and p-P65 by mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times left untreated or pretreated with AG aptamer (1 μg/ml), and GADPH as a loading control.

(C-E) qPCR analysis of Mmps including Mmp9 (C), Mmp10 (D), and Mmp12 (E) from THP-1 cells stimulated with AG (1 μg/ml) for 24h in absence or presence of different inhibitors targeting NF-κB (PDTC), ERK (PD98059), JNK (SP600125) and p38 (SB203580) at the concentration of 10 μM.

(F) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.

(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.

Data information: Data in (A and B) are representative of n=3 independent experiments. Data in (C-E) are means + SD averaged from n=3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test (C-E) were used for statistical analysis. ns, not significant; *, p<0.05; ****, p<0.0001.
Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 4

Figure 4. Lung injury induced by AG depends on MMPs.

(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (A) and quantification of lung lesion burden from H&E stained sections (B).

Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm.
Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 5

Figure 5. AG interacts with galectin-9.

(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.

(B) Coomassie blue stained SDS-PAGE of carbohydrate recognition domain CRD1 and CRD2 of galectin-9. Data are representative of n=3 independent experiments.

(C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.

(D) FACS assay showing interactions of Mtb H37Rv with different galectins including galectin-3, galectin-8, and galectin-9. The blank control was H37Rv staining with APC-anti-rabbit antibody alone.

(E) FACS assay of interactions of galectin-9 with mycobacteria including M. bovis BCG and M. smegmatis mc2155 as well as E. coli. The blank control was BCG staining with APC-anti-rabbit antibody alone. Data shown are representative of n=3 independent experiments.

(F) FACS assay of interactions of galectin-9 with wild -type M. marinum (Mm_WT) and MMAR-5356 and MMAR-5357 mutants of M.marinum treated with Tet. The blank control was Mm_WT staining with APC-anti-rabbit antibody alone. Data shown are representative of n=3 independent experiments.
Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 6

Figure 6. Galectin-9 is essential for AG-induced production of MMPs.

(A) Immunoblots of cell lysates were performed to analyze galectin-9 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice, and GADPH as a loading control.

(B-E) qPCR analysis of Mmps including Mmp9 (B), Mmp10 (C), Mmp12 (D), and Mmp13 (E) from mouse peritoneal macrophages isolated from either wild-type or Galectin-9 KO mice stimulated with AG (1 μg/ml) for 24h.

(F) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times; GADPH of cell lysates served as the loading control.

(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice infected with H37Rv for indicated times (MOI=5); GADPH of cell lysates served as the loading control.

(H) Immunoblot of lysates of peritoneal macrophages isolated from wild-type and Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments.

(I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments.

(J) qRT-PCR detection of Mmp transcripts including Mmp9, Mmp10, and Mmp12 in wild-type and Galectin-9 KO peritoneal macrophages stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM).

(K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12 in shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM).

Data information: Data in (B to E, J, and K) are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.
Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 7

Figure 7. Galectin-9 mediates TAK1 recruitment to induce production of MMPs.

(A, B) Immunoblots and immunoprecipitation of cell lysates to analyze endogenous interaction of galectin-9 with TAK1 by human THP-1 cells (A) or mouse peritoneal macrophages (B) left unstimulated or stimulated with AG (1 μg/ml) for 1 h.

(C) In vitro glutathione S-transferase (GST) precipitation assay purified histidine (His)-tagged Galectin-9 (+) with GST alone or GST-tagged TAK1.

(D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.

(E) Immunoblots of cell lysates to analyze phosphorylated TAK1 by mouse peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) for indicated times; GADPH of cell lysates served as the loading control. Data are representative of at least n=3 independent experiments.

(F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated times. Data are representative of n=3 independent experiments.

(G-J) qPCR analysis of Mmps including Mmp9 (G), Mmp10 (H), Mmp12 (I), and Mmp13 (J) from WT or Galectin-9 KO mouse peritoneal macrophages stimulated with AG (1 μg/ml) for 24h in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM).

Data information: Data in (G to J) are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001 ****, p<0.0001.
Sensing of arabinogalactan by galectin-9 exacerbates mycobacterial infection (2021)
Figure 8

Figure 8. Galectin-9 is essential for the in vivo effects of AG.

(A) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from the lungs of WT or Galectin-9 KO mice at 3 days post intraperitoneal administration of AG (100 μg).

(B, C) WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (B) and quantification of lung lesion burden from H&E-stained sections (C).

(D, E) WT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E (D) and quantification of lung lesion burden from H&E-stained sections (E).

(F) CFU quantification of the bacterial titers of lung tissue homogenates from WT or Galectin-9 KO mice intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval.

Data information: Data in (B and D) are representative of n=3 independent experiments. Data in (A, C, E, F) are means + SD of the indicated number of mice from 1 of n=3 independent experiments and each symbol represents data from 1 mouse. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001. Scale bar, 200 μm.

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