EMBO/soda-nel huggingface dataset:
A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress (2021)
Figure 3

Figure 3: Mec1-S1991 phosphorylation promotes replication on HU.

(A) Flow-cytometry analysis of DNA content. Asynchronous cells from the indicated strains were treated for 2h with 0.2M HU. After HU removal, recovery from replication stress was monitored by FACS (time in min). Red arrows highlight detected delays.

(B-D) Analysis of replication fork progression at the single-molecule level by DNA combing. (B) Scheme for the experimental procedure: exponentially growing cells were synchronized in G1 with α-factor and released in S phase in the presence of 0.2M HU. Newly replicated DNA was labelled with BrdU for 90 and 180 min. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. Box, 25-75 percentile range. Whiskers, 10-90 percentiles range. Median is indicated in kb. *** p value < 10-3, by Mann-Whitney rank sum test. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).

(E) Analysis of DNA polymerase ε progression by ChIP-seq in S phase + HU 0.2M. Median Pol2 (DNA polymerase ε) signal at early origins (n=32) is plotted over a 30kb distance. The rate of progression is inferred from the peak at given times.

(F) Graph depicting the distribution of inter-origin distances determined by DNA combing after 90 min in S phase + 0.2M HU. Box, Whiskers and Median as in D. ns (p value > 0.01), by Mann-Whitney rank sum test. WT (n=370) and mec1-S1991A (n=374).
A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress (2021)
Figure 5

Figure 5: Mec1-S1991 phosphorylation participates to RNAPII degradation on HU.

(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin. The value at the starting point was set to 100% (black dashed line). SEM (at least n=2 biological replicates) is indicated.

(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.
A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress (2021)
Figure 6

Figure 6: Decreased chromatin-bound RNAPII levels triggered by RBP3-TAP tag rescue mec1-S1991A defects on HU.

(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP (Table EV1). Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control. SEM is indicated (n=3 biological replicates).

(B) G1 synchronized cells were released into S phase with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1-S2P, Rpb1-S5P and Tubulin antibodies.

(C) Enrichment of the RNAPII subunit Rpb1 on chromatin in G1 synchronized cells was assessed by ChIP-qPCR at indicated genes. ChIP signal of Rpb3-TAP strain was normalized to the corresponding untagged strain. The dashed line indicates the amount of Rpb1 in the untagged strain. SEM (n=3 biological replicates) is indicated. FIG2 is a mating pheromone-induced gene used as a control to compare inducible gene and constitutively expressed genes (PYK1, YEF3, PMA1, PDC1 and snR13).

(D) mRNA levels measured by RT-qPCR in G1 synchronized cells in the indicated strains. Expression is normalized to either snR13 or FIG2. Data are expressed as a ratio of Rpb3-TAP/WT in percentage. Individual data points are indicated (n=2 biological replicates).

(E) Analysis of replication fork progression (BrdU track lengths) at the single-molecule level by DNA combing in the indicated strains. Box, Whiskers and Median as in Figure 3D. *** p value < 10-3 ; ns= p value > 0.05, by Mann-Whitney rank sum test using RPB3-TAP as a reference. RPB3-TAP 90 min (n=367) and 180 min (n=490), mec1-S1991A 90 min (n=309) and 180 min (n=477), mec1-S1991A RPB3-TAP 90 min (n=490) and 180 min (n=691).

(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.
A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress (2021)
Figure 7

Figure 7: Mec1-S1991 phosphorylation controls Mec1 kinase activity on HU.
(A) Phosphopeptide abundances (log2 ratio [WT/mec1-S1991A]) in S phase cells treated with 0.2M HU for 60 min. Colored dots are factors with significantly different phosphopeptide fulfilling a combined criteria [WT/mec1-S1991A] >4 (blue) or <4 (red) and p value<0.05 (Student's paired t-test, biological replicates n=3) in HU-treated cells (significant phosphopeptides, n= 25 and n=129, respectively).

(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted. Full list (n=25) in Dataset EV4.

(C) Phosphotargets and subunits with decreased abundance on chromatin identified under HU-induced replicative stress from the chromatome (Figure 1A,B) and the phosphoproteome analyses (Figures 1C,D, 7A,B). Common complexes are indicated in blue or red.

(D) GAL1 and GAL10 mRNA levels measured by RT-qPCR in asynchronous culture in 2% galactose and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. Repression is expressed as a ratio of HU treated/asynchronous cells in percentage. Mean and individual data point (n=2 biological replicates) are indicated.

(E) Position relative to the nuclear envelope (Zone 1, see Figure EV4F) of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition. *p-value < 10-3, by two-sided Fischer's exact test. ns, not significant (p>0.01).
A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress (2021)
Figure 8

Figure 8: Mec1-dependent RNAPII degradation occurs during an unchallenged S phase

(A-B) RNAPII occupancy on chromatin in S phase was assessed by ChIP-qPCR. (A) The level of Rpb1 was measured at snR13-TRS31 in both G1 and S phase (70 min at 16°C). Data is expressed as a percentage of input. SEM (n=3 biological replicates) is indicated.

(B) Quantification of the data from (A) depicting the mean retention of Rpb1 in S phase over G1.

(C, D) Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control. (D) Quantitation of blots in C. Rpb1 levels in S phase are expressed as a percentage of the starting level in G1 (100%, black dashed line). SEM for biological replicates (n=4 for WT, n=2 for mec1Δsml1Δ) is indicated.

(E) Quantitation of Rpb1-GFP fluorescent intensity expressed as percentage over the G1 level. SEM is indicated (n=106 for WT and n=102 for mec1Δsml1Δ).
Inhibition of double-strand DNA sensing cGAS ameliorates brain injury after ischemic stroke (2020)
Figure 1

Figure 1. Accumulation of dsDNA and up-regulation of cGAS after brain ischemia. (A) Representative single immunofluorescent staining for dsDNA at 6 h, 24 h, and 3 d of reperfusion after MCAO in the penumbra as well as in the sham brain. Scale bars, 5 μm. (B) Representative images of double-immunofluorescent stained for 53BP-1 and dsDNA (upper panel), Iba-1 and dsDNA (middle panel), and for GFAP and dsDNA (lower panel). Scale bars, 5 μm. (C) Western blot analysis of cGAS and STING using lysates prepared from the indicated tissues of mice brain following MCAO. (D) Quantitative analysis for Western blot analysis. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test. (E) Immunohistochemical staining for cGAS at 3 d of reperfusion after tMCAO in peri-ischemic area as well as in the corresponding regions of sham control brains. Scale bars, 50 μm, 20 μm in insets. (F) Quantitative analysis of cGAS immunohistochemical staining. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test. (G) Representative double immunofluorescent stainings for cGAS (red) and 53BP1 (green). n=6 mice per group. Scale bars, 50 μm. Data information: Data are presented as mean ± SEM. P‐values are reported in Appendix Table S2.
Inhibition of double-strand DNA sensing cGAS ameliorates brain injury after ischemic stroke (2020)
Figure 2

Figure 2. A151 inhibits MCAO-induced expression of cGAS, AIM2 inflammasome- and pyroptosis-associated molecules. Mice received daily intraperitoneal (IP) injections of A151 (300 μg) or an equal volume of vehicle for 3 consecutive days after MCAO induction. (A) The mRNA expression levels of the cGAS and STING after MCAO were determined using qRT-PCR. n = 6 mice per group. **P<0.01, one-way ANOVA followed by Tukey post hoc test. (B) The expression levels of cGAS, STING, and NF-κB proteins in the brains of indicated groups were measured using Western blot analysis. (C) Bar graph shows the relative protein expression levels of cGAS, STING, and NF-κB in the brains of indicated groups. n = 6 mice per group. *P < 0.05, **P<0.01, one-way ANOVA followed by Tukey post hoc test. (D) The mRNA expression levels of the AIM2 inflammasome components (AIM2, ASC, and caspase-1), effector of pyroptosis GSDMD, IL-1β, and IL-18 after MCAO were determined using qRT-PCR. n = 6 mice per group. *P < 0.05, **P<0.01, one-way ANOVA followed by Tukey post hoc test. (E) The expression of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups was measured using Western blot analysis. (F) Bar graph shows the relative protein expression levels of AIM2 inflammasome components, GSDMD, and IL-1β in the brains of indicated groups. n = 6 mice per group. *P < 0.05, **P<0.01, one-way ANOVA followed by Tukey post hoc test. Data information: Data are presented as mean ± SEM. P‐values are reported in Appendix Table S2.
Inhibition of double-strand DNA sensing cGAS ameliorates brain injury after ischemic stroke (2020)
Figure 3

Figure 3. A151 suppresses microglial pyroptosis after focal ischemic stroke. (A) Representative images of immunohistochemical staining of IL-1β, caspase-1, and GSDMD in the brains of indicated group. Scale bars, 50 μm. (B) Quantitative analysis for immunohistochemical staining. n = 6 mice per group. **P<0.01, one-way ANOVA followed by Tukey post hoc test. (C) Representative images of double-immunofluorescent stained for GFAP and GSDMD, for Iba-1 and GSDMD, and for NeuN and GSDMD in post-ischemic brains after tMCAO. Scale bars, 20 μm. (D) Representative images of double-immunofluorescent stained for Iba-1 and GSDMD, for Iba-1 and caspase-1, and for Iba-1 and IL-1β of sham+vehicle, MCAO+vehicle, and MCAO+A151 groups. Scale bars, 50 μm. (E) Bar graph shows the relative immunofluorescence intensity of GSDMD, caspase-1, and IL-1β in the brains of indicated groups. n = 6 mice per group. **P<0.01, one-way ANOVA followed by Tukey post hoc test. DAPI staining: nuclei. Data information: Data are presented as mean ± SEM. P‐values are reported in Appendix Table S2.
Inhibition of double-strand DNA sensing cGAS ameliorates brain injury after ischemic stroke (2020)
Figure 4

Figure 4. A151 alleviates cGAS activity and expression of pyroptosis associated proteins in vitro. (A) Expression of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 (FL), and GSDMD in primary microglia lysates was detected using western blot. (B) Quantitative analysis for Western blot analysis of cGAS, STING, NF-κB, ASC, IL-1β (FL), caspase-1 and GSDMD. n = 3 per group. P‐values are reported in Appendix Table S3, one-way ANOVA followed by Tukey post hoc test. (C) The protein levels of caspase-1 p20 and IL-1β (p17) in the supernatant of cultured primary microglia were determined by western blot. (D) Quantitative analysis for Western blot analysis of caspapse-1 and IL-1β. n = 3 per group. P‐values are reported in Appendix Table S3, one-way ANOVA followed by Tukey post hoc test. *P < 0.05, **P<0.01 compared with the untreated group, #P < 0.05, ##P < 0.01 compared with the LPS+poly(dA:dT) group. (E-F) Immunofluorescence was conducted to detect the expression levels of cGAS, NF-κB, and pyroptosis associated molecules (GSDMD, caspase-1, and IL-1β) in BV2 cells. n = 3 in each group. Scale bars, 50 μm. Data information: Data are presented as mean ± SEM. P‐values are reported in Appendix Table S2.
Inhibition of double-strand DNA sensing cGAS ameliorates brain injury after ischemic stroke (2020)
Figure 5

Figure 5. A151 attenuates neutrophil infiltration and production of proinflammatory factors in microglia after tMCAO. (A) Gating strategy of brain-infiltrating immune cells including CD4+ T cells (CD3+ CD4+), CD8+ T cells (CD3+CD8+), B cells (CD3-CD19+), NK cells (CD3-NK1.1+), monocyte/macrophages (CD11b+CD45highF4/80+), neutrophils (CD11b+CD45highLy6G+), and microglia (CD11b+CD45int) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-4. FMO, fluorescence minus one. (B) Quantitative analysis of the counts of brain-infiltrating leukocytes in the brains of MCAO mice receiving indicated treatment. (C) Bar graph shows counts of leukocytes subtype in the spleens of MCAO mice receiving indicated treatment. n = 6 mice per group. *P < 0.05, **P<0.01, two-tailed unpaired Student's t test. (D) Quantitative analysis of the microglia in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *P < 0.05, **P<0.01, two-tailed unpaired Student's t test. (E) Bar graph shows percentages of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of MCAO mice receiving indicated treatment. n = 6 mice per group. *P<0.05, **P<0.01, two-tailed unpaired Student's t test. (F) Representative images of immunofluorescent staining for ly6G in peri-infarct areas of MCAO mice receiving indicated treatment. (G) Bar graph shows the number of ly6G+ neutrophils. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test. (H) Representative images of immunofluorescent staining for Iba-1 in peri-infarct areas of MCAO mice receiving indicated treatment. (I) Bar graph shows the number of Iba-1+ microglia. n = 6 mice per group. **P<0.01, two-tailed unpaired Student's t test. Scale bar, 50 μm. Data information: Data are presented as mean ± SEM. P‐values are reported in Appendix Table S2.
Inhibition of double-strand DNA sensing cGAS ameliorates brain injury after ischemic stroke (2020)
Figure 7

Figure 7. Microglia-specific cGAS knockout diminishes the protective effects of A151. (A) Representative double immunofluorescent stainings for Iba-1 and cGAS, and for GFAP and cGAS. Scale bars, 10 μm. (B) Gene strategy for preparing cGAS-conditional knockout (cKO) mice. (C) Breeding scheme for CX3CR1CreER mice crossed with cGAS-cKO mice, in which cGAS was deleted in microglia. (D) Representative genotyping analysis for WT, cGAS-KD, and cGAS-KO mice. (E) Flow chart illustrates A151 administration and experimental design. Mice received daily i.p. injections of A151 (300μg) or an equal volume of PBS vehicle for 3 consecutive days. (F) Representative images of TTC staining of brain sections from vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice on day 3 after tMCAO. Three representative rostrocaudal levels of TTC staining are shown. (G) Bar graph shows percentages of infarct volume of indicated group. n = 6 mice per group. **p < 0.01, one-way ANOVA followed by Tukey post hoc test. (H) Bar graph shows modified Neurological Severity Score (mNSS) in vehicle-treated WT MCAO mice, vehicle-treated cGAS-KO mice, and A151-treated cGAS-KO mice n = 10 mice per group. **p < 0.01, one-way ANOVA followed by Tukey post hoc test. Data information: Data indicate mean ± SEM, P‐values are reported in Appendix Table S2.
Modified Atkins diet induces subacute selective raggedred-fiber-lysis in mitochondrial myopathy patients (2016)
Figure 1

Figure 1. Modified Atkins diet induces ragged-red-fiber-lysis in patients with mitochondrial myopathy.

A. Modified Atkins diet (mAD) study protocol. PEO patients followed mAD for 4-11 days; the healthy control subjects for four weeks. Sampling times of controls were matched with their corresponding PEO patients.

B. Beta-hydroxybutyrate (ketone body) concentrations, plasma.

C. Creatine kinase (CK) concentrations, plasma.

D. Myoglobin concentrations, plasma. Dashed line: upper limit of control range. The individual patients are shown separately, and controls as mean with standard deviation (n=10). Arrow: mAD endpoint in PEO patients; lines under graphs: duration of mAD.

E-G. PEO patient (P1) muscle before diet:

E. Ultrathin section of abnormal muscle fiber (arrowhead) with subsarcolemmal accumulations of mitochondria. Scale bar 50 µm

F. Electron micrograph of subsarcolemmal accumulation of abnormal mitochondria (same cell as in E).

G. Enlargement of the area marked in F; mitochondria with paracrystalline “parking lot” inclusions (arrow) and concentric cristae (asterisk). Scale bar 1µm

H-J. PEO patient (P1) muscle after mAD (H-J):

H. Ultrathin section, lytic muscle fiber with centrally located organellar debris (arrow). Scale bar 50 µm

I. Electron micrograph of a degrading muscle fiber with an invaded macrophage (arrow) (same cell as in H).

J. Enlargement of the fiber in I: paracrystalline inclusions within mitochondria (asterisk). Scale bar 1µm

K. Quantification of necrotic or apoptotic cells in PEO muscle from ultrathin sections. n.a., not available.
Modified Atkins diet induces subacute selective raggedred-fiber-lysis in mitochondrial myopathy patients (2016)
Figure 2

Figure 2. Mitochondrial myopathy patients show caspase-3 cleavage, satellite cell activation and stalled autophagy, not affected by modified Atkins diet

A-B. Cleaved caspase-3 positive muscle fibers (*).

A. PEO patient (P3) on normal diet (inset: caspase staining in healthy control individual)

B. PEO patient (P3) after modified Atkins diet. Arrowhead: caspase negative necrotic fiber.

C. Western blot analysis of autophagy receptor p62 and pax7 marking muscle satellite cell activation; left, blot; right, signal quantification in each patient.

D – E. Immunohistochemical analysis of p62 and mitochondrial mass marker, consecutive sections. Asterisks indicate p62-positive fibers and corresponding affected ragged-red fibers.

F – G. p62 and mitochondrial mass after mAD; consecutive sections. Arrowheads indicate fibers that show p62 positivity but have normal mitochondrial mass.

H. p62 and LC3 positive ragged-red fibers (*) in PEO patient (P2), normal diet.

I. Muscle fibers showing granular LC3-positivity, PEO patient (P1), after mAD. Immunohistochemistry on frozen sections; bars: 50 µm, except for H, 100 µm.

Abbreviations: PEO; progressive external ophthalmoplegia, ctr; healthy control individual, ND; normal diet, mAD; modified Atkins diet, Tom20; mitochondrial outer membrane transporter 20, p62; autophagy receptor protein 62, pax7; paired box protein 7.
Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis (2021)
Figure 3

Figure 3. Expression and function of ALDHs and MAOs in LAM tissue and cell model.

A Representative images of immunohistochemical positivity of MAO-A/B in LAM lung lesions (top panels, two patients) and lung tissue from healthy (non-LAM) individuals (bottom panels). In total, seven LAM patients and three healthy controls were analyzed. Brown-stained cells, counter-stained with hematoxylin, are considered positive. Scale bars are shown.

B Representative images of immunofluorescence detection of MAO-A/B and αSMA in LAM lung lesions, nuclei stained with DAPI (merged).

C Graph showing gene expression differences (log10-fold changes) of defined genes (X-axis) in Tsc2-deficient relative to wild-type MEFs grown in DMEM 10% FBS. The asterisks indicate significant differences with two-sided t-test (**P < 0.01, ***P < 0.001, and ****P < 0.0001; Aldh1a3 P = 0.006, Aldh2 P = 2x10-5, Aldh3a1 P = 0.004; Aldh3b1 P = 0.006; Aldh3b2, P = 0.17, Maoa P = 7x10-5, and Maob P = 7x10-4; replicates/condition n = 3, assays n = 4). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).

D Western blot results (independent experiments n = 4) from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% FBS. Loading controls are shown. The inferred protein expression level is indicated by the ratio between the corresponding signal and loading control, standardized to the basal setting (noted as 1 (reference (ref))).

E Flow cytometry results (independent experiments n = 3) showing cell percentages for ALDEFLUOR-positive MEFs grown in DMEM 10% FBS.

F Quantified MAO basal activity (Y-axis) in MEFs as depicted in the inset (grown in DMEM 10% FBS; replicates/condition n = 3 and independent experiments n = 2). The inhibitors (depicted on the X-axis) were added to cell extracts prior to activity assay to assess the contribution of each MAO isoform. The significant difference corresponds to two-way ANOVA test (P = 8x10-4). CPM: counts per minute. The bars indicate mean ± standard error of the mean (SEM).
Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis (2021)
Figure 4

Figure 4. Enhanced mitochondrial activity and H2O2 in the LAM cell model, and consequences for LAM lesions.

A No differences of Vdac1/VDAC1 expression between MEF cell lines (replicates/condition n = 3 and independent experiments n = 2). The graph shows fold change expression (mean ± SEM) in Tsc2-deficient relative to wild-type MEFs.

B Flow cytometry results (independent experiments n = 3) showing a higher percentage of MitoSOX red-positive cells in Tsc2-deficient MEFs.

C Flow cytometry results (independent experiments n = 3) showing higher intensity (X-axis, single channel intensity FL3 670 nm) of MitoTracker red-positive cells in Tsc2-deficient MEFs.

D Higher basal cell respiration (as measured by oxygen consumption, Y-axis) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 5x10-4; replicates/condition n = 5, and independent experiments n = 3).

E Higher hydroxide peroxide levels in Tsc2-deficient MEFs across different numbers of seeded cells (X-axis). Significance corresponds to two-way ANOVA test (P = 1x10-4; replicates/condition n = 2, independent experiments n = 2). The bars indicate mean ± SD.

F Left panel, western blot results showing overexpression of catalase (CAT) in Tsc2-deficient MEFs, and unaffected by exposure to everolimus (independent experiments n = 2). The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting (noted as 1(ref)). Right panel, Cat overexpression (log10-fold change) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 1x10-2; replicates/condition n = 3, and independent experiments n = 2). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).

G Representative images of immunohistochemical detection of acrolein in LAM lung lesions. Seven LAM patients were analyzed and assay controls are shown in Appendix Fig S2.