EMBO/SourceData huggingface dataset:
Figure 1Blistered samples at P1. (B) Hematoxylin and eosin (H&E, top) and alkaline phosphatase (AP, bottom) staining. Hair follicles (HFs) remaining in the dermis (indicated by arrows). Scale bar: 500 μm. (C) α6 Integrin (ITGA6, indicated by arrowheads) and type IV collagen (COL4, arrows) labeling (left). Laminin 332 (L332, arrows) staining (right). Scale bar: 100 μm. (D) Ultrastructural findings of blistered skin (left image: blister roof, right image: blister bottom) . Hemidesmosomes (white arrowheads) and lamina densa (arrows) are indicated. Scale bar: 1 μm.Keratin 14 (K14) and keratin 10 (K10) staining of the nonlesional (intact) and lesional (blistered) skin at P2 (upper images and inlets: sections, lower images: whole-mount imaging). HFs are indicated by arrows in the whole-mount images. Scale bar: 30 μm.H&E (left), PCK (middle), and LOR staining (right) at P4. Scale bar: 200 μm.
Figure 2Scatter plots of differentially expressed genes in the regenerated epidermis. The red dots represent upregulated genes, and the blue dots represent downregulated genes. The gray dotted lines indicate |logFC|>1.Hair canals in the regenerated (lesional) and nonlesional epidermis at P4 (indicated by asterisks). Scale bar: 300 μm.
Figure 3BrdU labeling of blistered samples at P2. Scale bar: 100 μm. BrdU-positive cells are indicated by arrows. Blisters are indicated by stars. (Bottom) Quantification of BrdU-positive cells in the epidermis (left) and HFs (right) (n=4 biological replicates). The data are shown as the mean ± SE. *0.01 test, followed by Tukey's test. NS, no significance. α5 integrin (ITGA5) labeling at P2 (left image: section, right image: whole-mount). Scale bar: 100 μm. Blister edges (epidermal tongue) and HFs are indicated by arrowheads and arrows, respectively. Blisters are indicated by stars.Sections of K14CreER:H2B-mCherry mice at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3).. The data from individual mice are connected by lines. Student's t-test. NS, no significance. Whole-mount imaging of K14CreER:R26R-confetti samples at P4. Scale bar: 200 μm. Sections of Lrig1CreER:H2B-mCherry mouse skin at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3). The data from individual mice are connected by lines. *0.01 test. Whole-mount imaging of Lrig1CreER:R26R-confetti mouse samples at P4. Scale bar: 200 μm.
Figure 4H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm.
Figure 5K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.
Figure 1C Overabundance of MIAA in LAM plasma. Top panel shows each control and patient group (number (n) of samples are indicated); bottom panel shows aggregation of related pulmonary diseases. The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; top panel, control-LAM P = 0.048, LAM-Langerhans P = 1x10-4, LAM-Sjögren P = 1x10-4, LAM-lupus P = 1x10-3, LAM-emphysema P = 0.041; bottom panel, LAM-Langerhans P = 7x10-6 and control-other P = 0.031). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test P = 6x10-4 (top panel) and P = 3x10-5 (bottom panel).E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin-treated (number (n) of samples are indicated). The asterisk indicates significant difference based on two-sided Mann-Whitney test (P = 0.043). Average values are indicated with lilac-colored lines.G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and/or between LAM and related pulmonary diseases (individual samples indicated in panel C). The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, and ****P < 0.0001; 4-HPA, control-LAM P = 0.024 and LAM-other P = 0.089; HVA, control-LAM P = 0.008 and LAM-other P = 0.013; and VMA, control-LAM P = 1x10-4 and LAM-other P = 0.10). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test; 4-HPA P = 0.095; HVA P = 0.013; and VMA P = 0.005.
Figure 2A Overabundance of MIAA in LAM plasma (UK cohort not treated with rapamycin) relative to healthy women. The number (n) of individuals in each group is indicated; one of 20 healthy controls did not pass the quality controls for LC-MS/MS. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.018). Average values are indicated with lilac-colored lines. B Overabundance of MIAA in plasma of LAM patients with AMLs. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.042). The number (n) of individuals in each group is indicated. Average values are indicated with lilac-colored lines.
Figure 3A 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
Figure 4A 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).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.
Figure 5A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines.D Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal settingE Representative images of immunohistochemical detection of HRH1 in LAM lung lesions.F Evaluation of cell viability inhibition by loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD.H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5% FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10% FBS, P = 0.017; 0.5% FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.
Figure 6B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM.C Further reduction of Tsc2-deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM.E Inhibition of Tsc2-deficient 105K tumor growth with Maoa expression depletion using shRNAs. Asterisks indicate significant reductions relative to control pLKO (two-way ANOVA; P = 1x10-3). Each data point represents the mean and SEM.F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM. G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM.
Figure 7A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1 expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines. B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1, loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines. E Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown.F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.
Figure 1(B) Violin plots showing the expression of prostaglandin (PG) receptors in pre-adipocytes and mature adipocytes from human deep-neck BAT(E-MTAB-8564).
Figure 2(A) Oil Red O staining of mouse BAs differentiated from normal or EP3-deficient brown pre-adipocytes. Scale bar: 50 μm. (B) Mean lipid droplet area of differentiated BAs from normal or EP3-deficient brown pre-adipocytes. *P < 0.05 vs WT (n = 6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests (B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05(D) Representative image of hematoxylin and eosin (H&E) staining for iBAT from EP3F/F and EP3F/FMyf5Cre mice. Scale bar: 50 μm. (E) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates Statistical significance was evaluated by Mann-Whitney U tests E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(K) Representative image of H&E staining of iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD. Scale bar: 50 μm. (L) Mean adipocyte area of BAs in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are pooled from two independent experiments with biological replicates L, Statistical significance was evaluated by Mann-Whitney U tests L, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).
Figure 6(A) Western blotting analysis of ubiquitination of WTAP in the control and EP3-deficient brown pre-adipocytes with or without sulprostone treatment. Data information: Data are representative of two independent experiments with biological replicates(B) Western blotting analysis of ERK1/2 phosphorylation in the control and EP3-deficient brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(C) Effect of ERK1/2 inhibitor, ravoxertinib, on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(D) Effect of wortmannin (Wor), pertussis toxin (PTX), U73122, barbadin, and melittin on ubiquitin-dependent degradation of WTAP in sulprostone-treated brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(E) Analysis of PKA activity in the control and EP3-deficient brown pre-adipocytes (n = 4). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates(F) Effect of PKA inhibitor, H89, on ubiquitin-dependent degradation of WTAP in EP3-/- brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(G) Effect of ERK1/2 phosphorylation site mutation (HA-WTAP-2A) on EP3-mediated ubiquitin-dependent degradation of WTAP in brown pre-adipocytes. Data information: Data are representative of two independent experiments with biological replicates(H) Representative picture of oil red O-stained BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates(I) Western blotting assay of ERK1/2, WTAP, ZFP410, PRDM16, and UCP1 in BAs differentiated from sulprostone-treated brown pre-adipocyte with treatment of PTX, dbcAMP, or ravoxertinib. Data information: Data are representative of two independent experiments with biological replicates
Figure 7(B)Representative pictures of nile red-stained human BAs differentiated from hESC-derived brown progenitors. Scale bar: 50 μm.(C) Time course analysis of the relative mRNA levels of EP3 receptors in hESC-derived BAs during BA differentiation detected by RT-qPCR (n = 7-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates (C, Statistical significance was evaluated by Welch ANOVA test followed by Tamhane's T2 test for post-hoc comparisons (C) *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(D) Representative pictures of hESC-derived BAs treated with L798106 alone or combined with H89. Scale bar: 50 μm. Data information: Data are representative of two independent experiments with biological replicates(E) Effect of H89 on the relative mRNA level of brown marker gene in hESC-derived BAs treated with L798106 detected by RT-qPCR (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for multiple comparisons (E, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(F) Effect of H89 on WTAP mRNA expression in hESC-derived BAs treated with L798106 (n = 6-7). (G) Effect of H89 on ZNF410 mRNA expression in hESC-derived BAs treated with L798106 (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by , two-way ANOVA followed by Tukey's test for multiple comparisons F, G). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(H) Western blotting assay of ERK1/2, WTAP, ZNF410, PRDM16, and UCP1 in hESC-derived BAs treated with L798106 alone or combined with H89.
Figure 1(A) Comparison of migration and invasion abilities between T- and B- lines. PC3 T- and B-line cells were incubated for migration (16h) and invasion (24h) assay. Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).(B) Comparison of cell proliferation rate between T- and B- lines. Cell proliferation assay was performed by MTS reagents. Data are the means ± SD (n=3 biological replicates).(C) Western blot analysis of the endogenous CTH in PC3 orthotopically implanted primary tumor-derived (T2 and T3-lines) and bone-metastases-derived cancer cells (B2 and B3-lines).(D) Top: H2S catalyzing activity of cell lysates from PC3 T2, B2, T3, and B3 lines. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).(E, F) 1x106 PC3-T2, B2, T3, and B3 cells were orthotopically injected into mouse prostate for 15-20 days. Incidences of paraaortic lymph nodes metastasis (E) and bone metastasis (F) are shown (n=6-8 mice per group). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01).(G) Top: expression of CTH in prostate tumors by immunocytochemistry analysis of commercial tissue arrays. Samples were sub-grouped by TNM stages. The representative images from different stages are shown. S: Stroma; Scale bars: 250μm. Bottom: The statistical significance was determined using the χ2 testThe RNA-seq data of CTH was downloaded from the TCGA database (H) The mRNA expression of CTH in PC specimens and normal tissues. Data are presented as means ± SD (n=50 cases for adjancent non-tumor parts and 469 cases for PC tumors). Student's t-test was used for the statistical analysis (**P<0.01).(I) Kaplan-Meier survival analysis of PC patients with high or low CTH expression. The statistical significance was determined using the χ2 test.
Figure 2(A) Western blot analysis of the CTH level in PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1.(B) Top: The H2S production capacity of cell lysates from PC3-T2 cells with CTH overexpression by pCMV-CTH-HA or PC3-B2 cells with CTH knockdown by siCTH-1. Lead acetate-soaked paper strips show a PbS brown stain as a result of reaction with H2S. Bottom: The level of H2S production was quantified by densitometry, and the histograms represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis (*P<0.05).(C) PC3-B2 cells were transfected with control or siCTH-1 and then were subjected to migration (16h) and invasion (24h) assays. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).(D) Cell proliferation rates of PC3-B2 cells with CTH knockdown. Cell proliferation assay was performed by MTS reagent. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).(E) Western blot analysis of the CTH levels in PC3 cells transfected with HA-CTH or pCMV-HA vector control.(F) PC3 cells were transfected with HA-CTH or pCMV-HA vector control and then incubated for migration (16h) and invasion assay (24h). Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).(G) PC3 cells were pre-treated with 10μM PAG for 16 hrs, and incubated for migration (16h) and invasion assay (24h) in the presence of 10μM PAG. Data information: Data shown represent the means ± SD (n=3 biological replicates). Student's t-test was used for the statistical analysis (*P<0.05; **P<0.01; ***P<0.001).