EMBO/SourceData huggingface dataset:
Figure 4GAG binding capacity of wildtype GCP-2, and mutants (K101E single mutant, K105E single mutant, GCP-2-D = K101E_K105E double mutant and GCP-2-T = triple mutant K100E_ K101E_K105E) was assessed by (B) a heparin microtiter plate binding assay measuring the interaction of biotinylated-heparin (b-heparin) with immobilized proteins(C) heparin affinity chromatography equilibrated and run in PBS, pH 7.4. In (C) the proteins are eluted with a salt gradient from 0-2 M NaCl monitored by the conductivity (Cond), where 20 and 90 mS/cm correspond to 193 and 1360 mM NaCl, respectively; GCP-2-T, GCP-2-D, K101E, K105E and WT proteins elute at 530 mM, 617 mM, 815 mM, 850 mM and 1024 mM NaCl, respectively.D, E Maximum migratory activity of WT GCP-2 (50nM) was compared to GCP-2 mutants (50nM) in (D) chemotaxis and (E) trans-endothelial migration assays in CXCR2-expressing 300-19 pre B cells. WT = wildtype GCP-2; K101E = GCP-2_K101E single mutant; K105E = GCP-2_K105E single mutant; D = GCP-2_K101E_K105E double mutant; T = GCP-2_K100E_K101E_K105E triple mutant; n=4; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed for (F) AKT phosphorylation levels by western blotting; V = vehicle treated samples, GCP-2 = WT GCP-2 treated samples, GCP-2-T = triple mutant GCP-2 treated samples, pAKT = phosphorylated AKT, tAKT = total AKT. On the right, quantification of the density of the bands using ImageJ. Five independent experiments were analyzed. The data were scaled and p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed G spectrophotometric quantification of proteoglycan content by Alcian blue staining. p values were determined by Kruskal-Wallis test with Dunn's post-hoc test; (n=8).C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed H ACAN expression levels by qPCR; ACAN = Aggrecan; p values were determined by ANOVA with Tukey's HSD post-hoc test; n=4.I-J Real time quantitative PCR of COL2A1 (I) and COL10A1 (J) mRNA normalized to β actin. p values were determined by fitting a generalised linear model followed by comparison of the estimated marginal means; n=4 control, 5 GCP-2 and GCP-2-T for COL2A1 and n=6 per group for COL10A1.K Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra-articular injection of adenovirus encoding GFP (n= 3), GCP-2 (n=3) and GCP-2-T (n=3); p values were determined by fitting a generalised linear model (family=poisson) followed by pairwise comparison of the estimated marginal means.
Figure 5B Weekly pain measurement by incapacitance, shown as the percentage of bodyweight loaded on the operated leg in GFP (n= 15) vs GCP-2 (n=14) and GFP vs GCP-2-T (n=15) treated mice. Circles show the mean, and error bars show 95% confidence intervals.C Incapacitance at week 10 (final time-point); red line indicates 50% loading (no pain); p values were determined by unpaired, two-tailed one sample Student's t test testing the hypothesis that mice loaded 50% body weight on the operated limb (n=13 in the GFP group and n=14 in the GCP-2 and GCP-2-T groups). D The area under the curve (AUC) of incapacitance calculated starting from week 6; p values were determined by ANOVA with Tukey's HSD post-hoc test (n=13 in the GFP group and 14 in the GCP-2 and GCP-2-T group).E, F Osteoarthritis severity assessed using (E) OARSI scoring system 10 weeks after MLI; GFP (n= 7), GCP-2 (n=8) and GCP-2-T (n=7); p values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means. F representative image (Safranin O staining) for each treatment; arrows indicate the tidemark;G Quantification of osteophytes area; GFP (n= 7), GCP-2 (n=7) and GCP-2-T (n=8); H Representative images with osteophyte highlighted I Osteophyte maturity score (n=7); p values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means.
Figure 1B. Single-cell lengths were measured from time-lapse microscopy images upon beta-lactam antibiotic treatment. Initial cell length was measured in the first time frame. Elongation ends with cell length shrinkage due to bulging. The longest length of each cell across the time frames was used as the final length. Tracked intermediate cell lengths were shown over time, while initial and final lengths were shown in larger circles. Linear regression of log-scaled lengths over time showed a constant rate of exponential elongation (solid gray line).
Figure 2A. Probability density distributions of final lengths of MG1655 cells exposed to carbenicillin. Individual cell lengths were measured from populations exposed to three doses of carbenicillin (20, 50, and 100 µg/ml) at two temperatures (27°C in blue and 37°C in red). Line plots show the measured initial and final lengths over time in log scale, and the first 15 cells were picked in each dataset for presentation among n ≥ 100 of the tracked cells in each condition Probability density distributions (ρL) of final lengths were collected with the same number of bins within the same final cell length range (bottom panels). The peak of ρL shifted to the left with increasing carbenicillin dose.C. The probability of lysis increased with the filamentation length of bacteria treated with other beta-lactams. MG1655 cells were treated with cefotaxime (20 and 100 µg/ml) and amoxicillin (6.25 and 25 µg/ml, shown in gray text) at two temperatures (27 °C in blue and 37 °C in red).D. The probability of lysis increased with the filamentation length of E. coli pathogens treated with a beta-lactam. E. coli pathogens expressing extended beta-lactam resistance were treated with amoxicillin (6.25 or 25 µg/ml, shown in gray text) and clavulanic acid (50 µg/ml) simultaneously at 37 °C. Cells were sensitized by clavulanate acid, which inhibits beta-lactamases.
Figure 3C. Experimental measurements confirm that τE decreases with the increase of antibiotic dose and growth rates. For growth rate modulation, casamino acid at 0.02% (left panel) and 0.2% (right panel) were added to minimal media. Cells were first cultured without carbenicillin for 3h (dashed line) and then exposed to carbenicillin (antibiotic doses are marked near each plot, µg/ml). The average of four technical repeats (gray lines) was plotted in a colored line. τE points were marked by finding the first time point at which the averaged OD became equal to or less than the OD at the start of carbenicillin exposure.
Figure 5B-C. Stochastic simulations and measurements of survivor length. Simulated survivor lengths were plotted using the predicted LC of lower doses of carbenicillin (left panels) at 2h (B) and 6h (C). The corresponding single-cell length measurements in vitro were plotted (right panels). In the simulation, only non-antibiotic treated cells were set to divide, when the cell length reaches the division length in the linear model (Ld= aLi + b, a = 0.871, b = 2.7. In both simulations and measurements, cells in carbenicillin-treated conditions were further elongated than control cells at all time points. At 2h exposure, antibiotic-exposed cells showed similar lengths across antibiotic concentrations since lysis is not highly probable for short filaments. At 6h exposure, survivor lengths decreased with increasing carbenicillin dose. Independent t-tests between non-treated to treated group showed extreme significance, **** P < 0.0001. The box represents the middle 50%, the central line represents the median, and the whiskers represent the lower and upper quartile of the data points (n>300).
Figure 1(A) Representative images of ILEI immunohistochemistry on healthy (upper panels) and psoriatic (lower panels) skin sections, scale bar 100 µm. Inlets show a magnification of the marked regions, scale bar 50 µm.(B) Distribution of ILEI protein content per keratinocyte (n=5000-15000 cells per skin sample) in healthy and psoriatic skin. Box and whiskers plot: Central band shows median, box extends from the 25th to 75th percentiles and whiskers go from the smallest (min) to the largest (max) value.(C) Median ILEI content of keratinocytes per person shown as mean ± SEM of healthy (n=6) and psoriasis (n=5) conditions. Data information: (C, Statistical significance was determined by Student's t-test and marked with asterisks (**p<0.01).
Figure 2(C) hematoxylin-eosin staining with scale bar of 50 µm of the back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days (n=2-5; 3 independent experiments).(D) mean epidermal thickness ±SEM of the back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days (n=2-5; 3 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; ***p,0.001).(E) Representative images of ILEI immunohistochemistry on sections of back skin of ILEIind and K5-ILEIind mice kept on normal or doxycycline diet upon 5 days of treatment with acetone or TPA. Scale bar 50 µm. Inlets show a magnification of the marked regions, scale bar 20 µm.
Figure 3(A) Representative images of Ki67 immunohistochemistry on back skin sections of K5-ILEIind and ILEIind mice kept on normal or doxycycline diet and treated with acetone or TPA for 5 days. Scale bar: 100 µm. (B) Percentage of Ki67 positive cells in the epidermis shown as mean ±SEM and quantified on samples described in panel A (n=1-5; 3 independent experiments). Data information: Statistical significance was determined by one-way ANOVA (B at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).(C) Immunofluorescence for Keratin 5 (K5; red) and Keratin 10 (K10; green) expression on TPA-treated skin sections of mice described in panel A. Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. Inlets show a magnification of the marked regions. Scale bar: 20 µm.(D) Keratin 16 (K16) western blot analysis (left) and quantification (right) of proteins extracted from back skin of ILEIind and K5-ILEIind mice kept on doxycycline and treated with acetone or TPA for 5 days (n=3). Vinculin was used as loading control. Data information: Statistical significance was determined by one-way ANOVA at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).(G) Mean fold change in mRNA expression ±SEM of K5 and K10 in primary keratinocytes described in panel F (n=5-6; 2 independent experiments). Data information: Statistical significance was determined by two-way ANOVA (G) at ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p,0.001).
Figure 4Mean number ±SEM of (C) neutrophils (MPO+ cells)/mm2 epidermal-dermal area and (D) epidermal CD8+ T-cells/cm skin section of mice described in A (n=3-5 C), n=1-5 (D); 3 independent experiments). N.A., not analyzed. Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.Mean fold change ±SEM in mRNA expression of Tnfa, Il1α and Cxcl1 (E) in freshly sorted keratinocytes enriched for the inter-follicular epithelium (IFE) and hair follicles (HF) from acetone and TPA treated back skin in mice supplemented with doxycycline and treated with acetone or TPA for 96 hours (n=3 (E), independent experiments). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.(G-I) Mean fold change ±SEM in mRNA expression of (G) Tnfa, (H) Il1α and (I) Cxcl1 in primary keratinocytes treated with acetone and TPA and with increasing concentrations (100ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml) of murine recombinant ILEI (mILEI) or dimerization-disabled ILEI (mILEICA) or with empty-vector (empty) for 72 hours (n=3, standing for independent keratinocyte cultures from three mice). Data information: statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.
Figure 5(A-C) Representative images of (A) Phospho-STAT3 (Tyr727), (B) Phospho-Erk1/2 and (C) Phospho-Akt immunofluorescence on thin sections of acetone and TPA treated back skin of ILEIind and K5-ILEIind mice kept on doxycycline diet. Scale bar, 50 µm.(E) Mean STAT3 (Ser727 and Tyr705), Akt and Erk1/2 phosphorylation levels ±SEM over time in primary keratinocytes upon TPA and recombinant ILEI treatment as described in panel A (n=3, standing for independent keratinocyte cultures, each a pool of isolates from two mice).(F) Western blot analysis of STAT3 (Ser727 and Tyr705), Akt and Erk1/2 phosphorylation levels in primary Dox-induced K5-ILEIind keratinocytes treated with acetone or TPA for 4 hours in the presence of the inhibitors STATTIC (10µM), LY92004 (10µM) and UO126 (10µM). DMSO was used as vehicle control. Vinculin was used as loading control. Lanes are from non-continuous parts of the same gel.
Figure 6(B) hematoxylin-eosin staining with a scale bar of 50 µm and (C) mean epidermal thickness ±SEM of the back skin of ILEIfl/fl and ILEI∆Ep mice treated with acetone or TPA for 5 days (n=3-8; 3 independent experiments). Data information: In C statistical significance was determined one-way ANOVA with Tukey multiple comparison test (C marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.(F-H) (F) Representative images of MPO+ microabscesses and neutrophils, (G) mean number ±SEM of MPO-positive microabsesses/cm skin section and (H) mean number of neutrophils (MPO+ cells)/mm2 epidermal-dermal area ±SEM on thin sections of acetone and TPA treated back skin of ILEIfl/fl and ILEI∆Ep mice (n=6-8 (G) and n=2-8 (H); 3 independent experiments). Scale bar, 100 µm. Data information: In G and H, statistical significance was determined one-way ANOVA with Tukey multiple comparison test H) or by Student's t-test (G) and marked with asterisks (*p<0.05; **p<0.01; ****p<0.0001). If significance levels were different for the pairwise comparisons with combined marking, asterisks, valid only for a subset of the pairs were put into brackets.
Figure 7Mean fold change ±SEM in mRNA expression of uPA and uPAR (D) in freshly sorted keratinocytes enriched for the interfollicular epithelium from acetone and TPA treated back skin, (E) in primary keratinocytes isolated from ILEIind and K5-ILEIind mice, supplemented with doxycycline and treated with acetone or TPA for 96 hours (n=3 (D), n=6 (E 1-2 independent experiments). Data information: In D,E Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).Mean fold change ±SEM in mRNA expression of uPA and uPAR (F) in primary wild type keratinocytes treated with acetone and TPA and with murine recombinant wild type ILEI (mILEI) for 8 and 96 hours n=6 F); 1-2 independent experiments). Data information: Statistical significance was determined by one-way ANOVA with Tukey multiple comparison test and marked with asterisks (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).(L) ILEI Western blot analysis of conditioned media harvested from primary keratinocytes of ILEIind and K5-ILEIind mice after 48-hours doxycycline induction and treatment with TPA and DMSO or indicated concentrations of UK371804. Loading was normalized to cell count, numbers indicate relative intensities. Lanes are from non-continuous parts of the same gel.
Figure 1(A and B) One hundred human keratinocytes (YF29, passage V) were cultured in duplicate for twelve days in individual Thermo Scientific Heracell 150 incubators with the temperature set at 35oC, 36oC, 37oC, or 38oC. Temperature of each incubator was monitored with independent thermo probes In parallel, the temperature of the culture medium was recorded every ten seconds using immerged RFID temperature sensors Sharp spikes resulted from wireless communication artifacts. (B) Cells were fixed and stained with 1% Rhodamine B. Temperature did not affect colony-forming efficiency (CFE) but impacted colony size; colonies were smaller when temperatures were below 37oC and larger at 38oC. Asterisks indicate thermo probes for monitoring the temperature.(C and D) Human keratinocytes (YF29) were cultured in mass for seven days at different temperatures before the cultures were dissociated. (C) Colony size was quantified using an image processing software (ImageJ). (D) The number and the size of the cells were determined using a TaliTM cytometer. Temperature impacted both cell number and size. Data information: In C and D, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05, ** p < 0.01 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.
Figure 2(A) The transcripts of eight thermoTRP channels were quantified by qPCR in two different strains of human diploid keratinocytes (YF29 and OR-CA). Data are presented as the mean ± SEM (n = 3 biological replicates).(F and G) YF29 cells were cultured for seven days at 37oC in a Nuaire 8700E culture incubator. Cells were then maintained at 37oC or switched to an adjacent incubator with temperature set at 32oC. Temperature was monitored with independent thermo probes in the presence or absence of rapamycin (100 nM), or a cold agonist (menthol - 500 μM), a warm agonist (2-APB - 200 μM) and the vehicle (control - ethanol 70%) for 30 and 60 minutes. (F) mTORC1 kinase activity decreases in the presence of rapamycin and when the temperature is switched from 37oC to 32oC. (G) mTORC1 kinase activity decreases in the presence of menthol and increases in presence of 2-APB, indicating that ThermoTRP and mTOR signaling are connected. Rapamycin (Rapa), menthol, and 2-APB were added in the keratinocyte cultures 1 h before starting the temperature changes.
Figure 3(A) Colony-forming efficiency (CFE) of keratinocytes was unaltered when keratinocytes were cultured at 35oC or in the presence of 100 nM rapamycin. However, the mean area of colonies was smaller when compared to cells cultured at 37oC. (B) Culture at 35oC and treatment with rapamycin decreased keratinocyte proliferation as shown by the lower total number of cells after 7 days of culture compared with in the control condition. (C) Culture at 35oC and treatment with rapamycin also reduced cell growth as shown by a smaller mean diameter of keratinocytes compared with that in the control condition after 7 days of culture. Data information: In B and C, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.(D) Long-term exposure to temperatures below 37oC decreased mTORC1 kinase activity.
Figure 4(B) Proteins were extracted from different YF29 subcellular components with ProteoExtractTM Subcellular Proteome Extraction kit (Calbiochem). The purity of each subcellular fraction was confirmed using specific markers, GAPDH for the cytosolic fraction, calnexin (CANX) for the membrane and organelles fraction, keratin 5 (KRT5) for the cytoskeletal fraction and HDAC2 for the nuclear fraction. mTOR, p-mTOR, RICTOR and RAPTOR were identified both in the membranes/organelles and nuclear fractions by western blotting. RICTOR and RAPTOR were also identified in the cytosolic fraction.(D) CHO cells engineered to transiently express SNAP-mTOR, SNAP-RAPTOR, and SNAP-RICTOR (Gautier et al., 2008) were incubated with the nuclear export inhibitor leptomycin B (LMB) (6.4 ng/mL) for six hours at 37°C. Cells were labeled with TMR-Star (1 μM) for 30 min, washed and then imaged. This experiment demonstrated that mTOR, RAPTOR, and RICTOR accumulated in the nucleus after LMB treatment. Scale bars, 10μm.
Figure 5A genome-wide expression screen was performed in parallel on normal human epidermal keratinocytes (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin (100 nM). (C) Quantitative PCR analysis of epidermal differentiation-associated genes. Data are presented as the mean ± SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Dunnett's multiple comparisons test vs 37oC).
Figure 6Comprehensive miRNA expression was performed in parallel on normal human epidermal keratinocytes (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin (100 nM). (B) The MA plot showing all miRNAs without low expression or log2FC = 0. miRNAs indicated in pink: upregulated (≥ 1 log2(FC)), blue: downregulated (≤ -1 log2(FC)), grey: others (-1 < log2(FC) < 1) and black: miRNAs related to epidermal differentiation. log2(FC) values are calculated using the edgeR package.
Figure 7(B) Cultured human keratinocytes (YF29 passage III) treated with rapamycin maintained a basal phenotype as shown by the expression of several basal (KRT14, BNC1) and terminal differentiation markers (SPINK5, IVL, KRT1, KRT10) using quantitative PCR (left panel) and Western blotting (right panel). Data are presented as the mean (n = 2 biological replicates).(E) Rapamycin-treated keratinocytes (YF29 passage IV) can fully regenerate an epidermis when transplanted onto SCID mice; grafts were harvested after 71 days and immunostained with an HLA-1 antibody to identify the human cells (white dotted line indicates the boundary between mouse and human epidermis). Scale bar, 100 μm.
Figure 1A Lung function data of COPD (II-IV) and no COPD (ex-smoker controls) donors used in this study. The lung function between the two groups is significantly different. In (A), data points represent each donor values and horizontal bars the group median. An unpaired non-parametric t test (Mann-Whitney test, GraphPad Prism software, version 8.0.1) was employed to compare the lung function (FEV1 and FEV1/FVC values) between control (no COPD, n=3) and COPD II-IV donors (n=5), *p-value<0.05. Exact p-values are: FEV1, p-value= 0.0357; FEV1/FVC, p-value= 0.0357. FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity;T-WGBS data of primary fibroblasts from no COPD and COPD (II-IV) patients was analyzed DMRs E Detailed view of a representative hypo- (top) and hypermethylated (bottom) DMR (grey box). CpG methylation levels of each individual donor (dots) and the group average (lines) methylation profile of three no COPD (blue) and five COPD (II-IV) (dark green) donors are displayed. RefSeq annotated genes and CpG islands are indicated.