EMBO/soda-nel huggingface dataset:
Hyperoxidation of mitochondrial peroxiredoxin limits H2O2-induced cell death in yeast (2019)
Figure 3

Fig 3. Prx1 efficiently transfers oxidative equivalents from H2O2´to glutathione

(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.

(B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91.

(C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura).

(D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase.

(E) Model illustrating the role of Prx1 in coupling H2O2 to oxidation of the mitochondrial glutathione pool.

Data information: In all panels, OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed). Significance was assessed with a Student's 2-tailed, unpaired, t-test. ***p < 0.001; ****p < 0.0001.
Hyperoxidation of mitochondrial peroxiredoxin limits H2O2-induced cell death in yeast (2019)
Figure 5

Fig 5. Hyperoxidation of Prx1 promotes cell survival upon acute H2O2 stress

(A) Structure of ScPrx1 (PDB ID: 5YKJ). The positions of relevant amino acids are indicated and the applied modifications in Prx1-P233stop and Prx1-C91A are shown. The C-terminal region absent in the Prx1-P233stop mutant is highlighted in green.

(B) The response of a mitochondrial matrix-localized roGFP2-Prx1-P233stop probe expressed in wild-type cells to the indicated concentrations of exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).

(C) Maleimide-based gel-shift assay to assess Prx1 cysteine redox state. Wild-type cells transformed with an empty plasmid or ∆prx1 cells with either an empty plasmid or a plasmid encoding wild-type Prx1, the hyperoxidation-resistant Prx1-P233stop or the inactive Prx1-C91A variants, were grown to exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24.

(D) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe, expressed in Δprx1 cells containing a plasmid encoding either wild-type Prx1, Prx1-P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2. Cells had been pre-treated with either 10 mM H2O2 or with water as a control. For these experiments, cells were grown in SGal (-Leu, -Ura) medium and harvested at early exponential phase. OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).

(E) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with empty vector or with a vector encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SD (-Ura) and treated with the indicated concentrations of H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 5-8 biological replicates, with cells taken from independent cultures for each individual biological replicate).

Data information: In all relevant panels significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.
Hyperoxidation of mitochondrial peroxiredoxin limits H2O2-induced cell death in yeast (2019)
Figure 6

Fig 6. Modulating the coupling between H2O2 and matrix glutathione links matrix glutathione oxidation to cell death

(A) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with an empty plasmid or a plasmid encoding Prx1, Tsa1, PRDX3, PRDX5, PRDX6, PfAOP or PfAOP-L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre-treated with 0, 1, 5, 10, 25 mM H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume was plated on YPD plates. The number of viable colonies was counted after 2 days of growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3-8 biological replicates, with cells taken from independent cultures for each individual biological replicate). Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

(B) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress. Data from viability and matrix Grx1-roGFP2 responses during acute H2O2 stress were correlated.
Hyperoxidation of mitochondrial peroxiredoxin limits H2O2-induced cell death in yeast (2019)
Figure 7

Fig 7. Mitochondrial matrix GSSG accumulation drives cell death during acute H2O2 stress.

(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to 1 mM H2O2. in BY4742 wild-type cells with an empty vector, in Δglr1 cells transformed either with an empty vector or a vector encoding wild-type Glr1 or the cytosolic form of Glr1, where the MTS-encoding region was removed, or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1. Cells were grown to exponential phase in SGal (-Leu, -Ura) medium. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).

(B) Growth curve of wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids (n = 3 biological replicates). Significance for the difference in the time the cultures reach 50% of their maximal OD600 was assessed with the t-test. Error bars (as ribbon) represent the standard deviation (n = 4 biological replicates, with cells taken from independent cultures for each individual biological replicate).

(C) H2O2 'acute stress' assay. Wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells pre-grown in SGal medium complemented with all amino acids to early exponential phase. Cells were treated with H2O2 at the indicated concentrations for 30 mins. Subsequently cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate). An inset with a different scale on the y-axis is presented for the 1 and 5 mM concentration to allow better interpretation.

(D) H2O2 'acute stress' assay. Δprx1Δglr1 cells co-transformed with an empty plasmid or with a plasmid encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SGal (-Ura) medium to early exponential phase. Cells were treated with the indicated amounts of H2O2 for 30 mins. Subsequently, the cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate).

(E) Model. Prx1 drives oxidation of glutathione upon exposure to hydrogen peroxide. During acute H2O2 stress, Prx1 becomes hyperoxidized, which effectively uncouples the glutathione pool from H2O2. Prx1 hyperoxidation thus helps to limit GSSG accumulation and contributes to cell survival.

Data information: Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.
Infection and transmission of SARS-CoV-2 depends on heparan sulfate proteoglycans (2021)
Figure 1

Fig 1. SARS-CoV-2 pseudovirus binds to heparan sulfates

(A) Huh7.5 cells were pre-incubated with neutralizing antibody to ACE2 and
SARS-CoV-2 pseudovirus was pre-incubated with patient isolated mAb COVA1-18, COVA1-21 and COVA2-15 (10 µg/mL) or UF heparin (250 IU/mL) for 30 min at 37°C.
SARS-CoV-2 pseudovirus alone or with blocks was added to the cells for 4h at 4°C and binding was determined by ELISA. (B) Heparan sulfates were removed from Huh7.5 cells by enzymatic treatment with heparinase III for 1 hour at 37°C, then washed, and exposed to SARS-CoV-2 pseudovirus for 4h at 4°C. Treated and untreated cells were subsequently lysed and binding was determined by ELISA. (C) Flow cytometry analysis of cell surface expression of heparan sulfates (HS) in control transduced cells or upon CRISPR/Cas9-mediated EXT1 KO (EXT1-/-). (D) Control and EXT1-/- XG1 cells were exposed to SARS-CoV-2 pseudovirus or SARS-CoV-2 pseudovirus pre-treated with 250 IU/mL UF heparin for 30 min at 37°C. After incubation for 4h at 4°C, cells were lysed and binding was measured by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) ordinary one-way ANOVA with Tukey multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001(n=3), (B) unpaired Student's t-test with Welch's correction. *P≤0.05, **P≤0.01(n=3), (D) two-way ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n=3).
Infection and transmission of SARS-CoV-2 depends on heparan sulfate proteoglycans (2021)
Figure 3

Fig 3. SARS-CoV-2 infection of polarized epithelial cells is blocked by UF heparin and LMWH

(A) Barrier integrity was analysed by TEER measurements of Caco-2 and Calu-3 over a period of 20 days (n=3 donors measured in quadruplicates). (B) ACE2 cell surface expression on Caco-2 and Calu-3 was determined by quantitative real-time PCR. (C, D) SARS-CoV-2 binding was measured in polarized Caco-2 (C) and Calu-3 (D) cells in presence or absence of antibodies against ACE2 (cell incubation), and UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) (virus incubation). (E) Polarized Calu-3 were inoculated with 0.5 TCID/mL of a SARS-CoV-2 isolate (hCoV-19/Italy) either directly or in presence of antibodies against ACE2 or upon pre-treatment with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Virus was detected by lysing after 48 hours through quantitative real-time PCR of viral RNA (E) and virus production was determined by detection of viral RNA in supernatant using quantitative real-time PCR (F). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (B) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01 (n=5 Caco-2 donors measured in monoplo), *p=0.0460 (n=5 Calu-3 donors measured in monoplo), (C, D) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3 Caco-2 donors measured in triplicate), (D)(n=3 Calu-3 donors measured in triplicate), (E, F) ordinary one-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n=3 donors measured in monoplo). TEER: Transepithelial electrical resistance.
Infection and transmission of SARS-CoV-2 depends on heparan sulfate proteoglycans (2021)
Figure 4

Fig 4. Heparan sulfate proteoglycans Syndecan 1 and 4 are important for SARS-CoV-2 binding

(A) Namalwa cells ectopically expressing either Syndecan 1 or 4 were exposed to either SARS-CoV-2 pseudovirus alone or SARS-Cov-2 pseudovirus pre-treated with UF heparin (250 IU/mL) or LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Binding was measured after 4 hours at 4°C by ELISA. (B) SARS-CoV-2 isolate (hCoV-19/Italy) was pre-incubated with LMWH enoxaparin (250 IU/mL) for 30 min at 37°C. Namalwa cells expressing Syndecan 1 and 4 were exposed to either SARS-CoV-2 isolate (100 TCID/mL) or SARS-CoV-2 isolate (100 TCID/mL) pre-treated with LMWH LMWH enoxaparin (250 IU/mL) for 4 hours at 4°C and binding was determined by quantitative real-time PCR. Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) 2way-ANOVA with Dunnett's multiple-comparison test. *P≤0.05, **P≤0.01 (n = 7), (B) 2way-ANOVA with Sidak's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (n = 3 measured in triplicate).
Infection and transmission of SARS-CoV-2 depends on heparan sulfate proteoglycans (2021)
Figure 7

Fig 7. SARS-CoV-2 attaches to and infects primary nasal cells via heparan sulfate proteoglycans

(A) Flow cytometry analysis of single cell suspensions from the nasal epithelium. Cells were labelled with EpCAM and CD45 antibodies and gated accordingly. (B) ACE2, Syndecan 1 and Syndecan 4 cell surface expression on nasal epithelial cells, compared to polarized epithelial Calu-3 was confirmed by quantitative real-time PCR. (C, D) Nasal epithelial cells were exposed to SARS-CoV-2 isolate (hCoV-19/Italy, 100 TCID/mL) either directly or after pre-treatment with antibodies against ACE2 cell surface receptors (1 hour at 37°C )or after pre-treatment with LMWH enoxaparin
(250 IU/mL) for 30 min at 37°C. Detection of viral binding after 4 hours at 4°C (C) and persistently-infected cells lysed after 24 hours at 37°C (D) was determined by quantitative real-time PCR. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (C, D) ordinary on-way ANOVA with Tukey's multiple-comparison test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001 (C) (n=3), (D) (n=3 in duplicates).
Kaposiform lymphangiomatosis effectively treated with MEK inhibition (2020)
Figure 2

Figure 2. Mutation analysis for the KLA patient uncovered a novel variant in the CBL gene.

A. Deep coverage exome sequencing Integrative Genomics Viewer (IGV) view of CBL c.2322T>G in DNA sample that was derived from CD31 selected cells from pleural effusion fluid. The alternate allele fraction was 4% (20/493).

B. Droplet digital polymerase chain reaction (ddPCR) result with 5 mutant positive droplets (circle) from a low concentration left over sample which resulted in ~0.005% fractional abundance.
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 1

Figure 1. MSCs accelerate healing of CD18-/- wounds.

A. Four full-thickness excisional wounds were produced on each of CD18-/- or WT mice by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or PBS control. Each wound region was digitally photographed at the indicated time points, and wound areas were analyzed using Adobe Photoshop. Depicted are representative macroscopic pictures of PBS injected or AT-MSCs injected WT or CD18-/- wounds at days 0, 3, 5, 7 and 9 post-wounding.

B. Quantitative analysis of 20 wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value of 20 wounds from 5 mice. *p<0.05, **p<0.01, ***p<0.001 by Student's t-test with Welch's correction.
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 2

Figure 2. Injection of MSCs restores TGF-β1 concentration in CD18-/- wounds.

A. AT-MSCs or PBS were intradermally injected to WT or CD18-/- wounds and the wound tissues were harvested at days 2, 5, 7 post-wounding. Depicted are representative photographs of wound cryosections at days 2 and 5 post-wounding stained for human-specific β2M (green). Nuclei were stained with DAPI (blue). Scale bars: 100 µm.

B. Genomic DNA was isolated from the wound tissues, and the amount of human-specific Alu DNA was quantified by real time PCR, and then converted to the number of AT-MSCs based on a standard curve. Data are expressed as mean ± SEM, n = 3 at each time point, *p<0.05 by two-tailed unpaired t-test.

C. Total TGF-β1 concentrations in wound lysates were measured by TGF-β1 specific ELISA. Results are expressed as mean ± SEM, n = 3 at each time point, *p<0.05, **p<0.01 by one-way ANOVA with Tukey's test.

D. The expression of human TGF-β1 in cultured AT-MSCs and wound tissues of WT or CD18-/- wounds received AT-MSCs injection was quantified by qPCR with primers specifically amplify human TGF-β1, and normalized on the numbers of MSCs in each condition shown in (B). Data are expressed as mean ± SEM, n = 8 wounds (2 wounds x 4 mice) per time point, *p<0.05 by one-way ANOVA with Tukey's test. AU, arbitrary unit.
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 3

Figure 3. Injection of MSCs induces granulation tissue formation in CD18-/- wounds.

A. Cryosections of PBS or AT-MSCs injected CD18-/- wounds at days 5 and 7 post-wounding were immunostained for α-SMA (red). Nuclei were stained with DAPI (blue). The dashed lines separate epidermis and wound bed. e, epidermis; wb, wound bed. Scale bars: 100 µm.

B. Quantification of α-SMA immunofluorescence shown in (A), mean ± SEM, n = 3 wounds per group, *p<0.05 by two-tailed unpaired t-test.

C. Cryosections of AT-MSCs injected CD18-/- wounds at day 5 post-wounding were co-immunostained for human β2M (green) and TGF-β1 (red, upper panel) or α-SMA (red, lower panel). The depicted results are representative photographs of three independent experiments. Scale bars: 50 µm.
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 4

Figure 4. TGF-β1 released by MSCs is responsible for accelerated healing in CD18-/- wounds.

A-F. Representative photographs of immunofluorescent staining for α-SMA (red) and human β2M (green) on cultured human AT-MSCs (A), murine primary dermal fibroblasts (mFB) (B), mFB treated with 2 ng/ml recombinant human TGF-β1 (C), and co-cultures of mFB with AT-MSCs (D) or TGF-β1 siRNA transfected AT-MSCs (E) or control siRNA transfected AT-MSCs (F) after 48 h culture. Nuclei were counterstained with DAPI (blue). Scale bars: 100 µm.

G. Quantification of α-SMA immunofluorescence shown in (A-F), mean ± SEM, n ≥ 3 wells per group, **p<0.01; ***p<0.001; n.s., not significant, by one-way ANOVA with Tukey's test.

H. Four full-thickness excisional wounds were produced on each of CD18-/- mouse by 6 mm biopsy punches. Each wound received intradermal injection of 2.5x105 AT-MSCs or human dermal fibroblasts (HDF) or TGF-β1 siRNA transfected AT-MSCs or control siRNA transfected AT-MSCs or PBS. Each wound region was digitally photographed at days 0, 3, 5, 7 and 10 post-wounding, and wound areas were analyzed using Adobe Photoshop. The depicted result is the quantitative analysis of all wound areas per group, expressed as percentage of the initial wound size at day 0. The line in each group represents the mean value. *p<0.05, **p<0.01, ***p<0.001 by two-tailed unpaired t-test with Welch's correction.
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 5

Figure 5. TGF-β1 released by MSCs contributes to induced myofibroblast differentiation and granulation tissue formation

A-C. 2.5x105 of TGF-β1 siRNA or control siRNA transfected AT-MSCs were intradermally injected around each of CD18-/- murine wound. PBS mock injection served as negative control. Wound tissue were harvested at day 2, 5, and 7 post-wounding for quantification of human TGF-β1 mRNA (A) at day 2 by qPCR, total TGF-β1 (B) and active TGF-β1 (C) protein at day 5 by ELISA. Data are expressed as mean ± SEM, n = 3 wounds per group, **p<0.01, by one-way ANOVA with Tukey's test.

D-G. Expression of α-SMA (D, E) and CD31 (F, G) at day 5 and 7 by immunostaining on tissue sections. The dashed lines indicate the border of the wound bed and epidermis or eschar. e, epidermis; es, eschar; wb, wound bed. Scale bars: 100 µm. Quantification data are expressed as mean ± SEM, n = 3 wounds per group, *p<0.05, ***p<0.001, by one-way ANOVA with Tukey's test.
MSCs Rescue Impaired Wound Healing in a Murine LAD1 Model by Adaptive Responses to Low TGF-β1 Levels (2020)
Figure 6

Figure 6. TGF-β1 signaling in MSCs is regulated by environmental TGF-β1 concentration.

A. AT-MSCs were cultured in the labelling media containing [35S]-Met/Cys and increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for overnight. Supernatants were harvested and incubated with TGF-β1 capture antibody. After washing the captured TGF-β1 was transferred to glass filter membrane, and the radioactivity was counted with a scintillation counter (black bars). The supernatants were incubated with new TGF-β1 capture antibody, and the radioactivity of captured TGF-β1 was counted again (white bars). The data is given as mean ± SD of 5 repeated measurements. This experiment was performed four times with comparable results. CPM, count per minute.

B-C. AT-MSCs (B) or HDFs (C) were cultured in media containing increasing concentrations of r.h. TGF-β1 at 0, 0.1, 0.3, 1, 3 and 10 ng/ml for 24 h, before protein lysates or total RNAs were harvested. Western blot analysis of Smad7 was performed with protein lysates and semi-quantitatively analyzed with densitometry. β-actin served as loading control. Data is given as mean ± SEM of three independent blots. ** p<0.01, by by one-way ANOVA with Tukey's test. AU, arbitrary unit.

D. From total RNA, the mature miR-21 and endogenous control RNU6B were converted to cDNA, and quantified by qPCR based TaqMan microRNA assays. The expression of miR-21 was normalized on RNU6B. Data is given as mean ± SEM of three independent experiments.