Journal: bioRxiv

Article Title: Phosphatase-independent activity of smooth-muscle calcineurin orchestrates a gene expression program leading to hypertension

doi: 10.1101/2023.11.26.568733

Figure Lengend Snippet: ( A ) Experimental design: 10-12-week-old mice were treated with tamoxifen for 5 consecutive days (open arrowheads) during the first week of HFD feeding. After 12 weeks of HFD, Ang-II osmotic minipumps were implanted for 4 weeks; control mice were operated without minipump implantation. Maximum aortic diameter and BP were measured (Eco-BP) at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( B,C ) AbAo diameter in ( B ) Cn-Ctl (n=16), EC-Cn -/- (n=12) Ang-II-treated and Cn-Ctl (n=7), EC-Cn -/- (n=8) control mice, and in ( C ) Cn-Ctl (n=18), SM-Cn -/- (n=16) Ang-II-treated and Cn-Ctl (n=8), SM-Cn -/- (n=6) control mice. Data are presented as mean ± s.e.m. ****p<0.0001, ***p<0.001; repeated-measurements (RM) two-way ANOVA with Tukey’s post hoc test. ( D ) Representative ultrasound images of AbAo from mice before and after 4 weeks of Ang-II treatment. Yellow lines mark the lumen boundary. Scale bar, 1 mm. ( E ) Representative images of AAA. Scale bar, 1 mm. ( F ) Medial area in AbAo sections from Cn-Ctl (n=23), EC-Cn -/- (n=9), and SM-Cn -/- (n=9) Ang-II-treated mice and from Cn-Ctl (n=10), EC-Cn -/- (n=7), and SM-Cn -/- (n=5) control mice. Scale bar, 100µm. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. ****p<0.0001, ***p<0.001, *p<0.05; RM two-way ANOVA with Tukey’s post hoc test. ( G ) Representative images of hematoxylin-eosin (HE), Masson trichrome (Masson) and Van Giemson (VG) staining on AbAo sections from 4 Cn-Ctl, 3 EC-Cn -/- , and 4 SM-Cn -/- mice. Scale bar, 100µm.

Article Snippet: Ang-II (Sigma-Aldrich), CsA (Novartis), NE (Sigma-Aldrich), and TM5441 (MedChemExpress) were administered at 1 μg/kg/min, 5mg/kg/d, 10 mg/kg/d, and 20mg/kg/d, respectively, using subcutaneous osmotic minipumps (Alzet Corp, model 2001 or 2004).

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Phosphatase-independent activity of smooth-muscle calcineurin orchestrates a gene expression program leading to hypertension

doi: 10.1101/2023.11.26.568733

Figure Lengend Snippet: ( A ) Experimental design: 10-12-week-old mice were treated with tamoxifen for 5 consecutive days (open arrow heads) and, after 6 weeks, Ang-II osmotic minipumps were implanted for 4 weeks in one group of mice; control mice were operated without minipump implantation. Maximum aortic diameter and BP were measured (Eco-BP) at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( B ) Systolic BP and ( C ) maximal AbAo diameters at the indicated times in Cn-Ctl (n=22), EC-Cn -/- (n=10), and SM-Cn -/- (n=18) Ang-II-treated mice and in Cn-Ctl (n=5), EC-Cn -/- (n=3), and SM-Cn -/- (n=5) control mice. Data are presented as mean ± s.e.m. ****p<0.0001, ***p<0.001, **p<0.01 vs SM-Cn -/- Ang-II, #### p<0.0001 vs Cn-Ctl control, # p<0.05 vs SM-Cn -/- or Cn-Ctl control; RM two-way ANOVA with Tukey’s post hoc test. ( D ) Experimental design: 10-12-week-old mice were fitted with CsA osmotic minipumps; control mice were operated without minipump implantation. After 48h, Ang-II minipumps were implanted in a group of CsA-treated mice (CsA+Ang-II) and in a group of CsA control mice (Ang-II). The remaining CsA control mice were operated without minipump implantation (Control). Maximal aortic diameter and BP were measured (Eco-BP) at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( E ) Systolic BP and ( F ) maximal AbAo diameters at the indicated times in Ang-II-(n=10), CsA+Ang-II-(n=8), Control-(n=8), and CsA-(n=4) treated mice. Data are presented as mean ± s.e.m. ***p<0.001, **p<0.01, *p<0.05 vs CsA+Ang-II, #### p<0.0001, ### p<0.001 vs control, $$$$ p<0.0001 vs CsA; RM two-way ANOVA with Tukey’s post hoc test.

Article Snippet: Ang-II (Sigma-Aldrich), CsA (Novartis), NE (Sigma-Aldrich), and TM5441 (MedChemExpress) were administered at 1 μg/kg/min, 5mg/kg/d, 10 mg/kg/d, and 20mg/kg/d, respectively, using subcutaneous osmotic minipumps (Alzet Corp, model 2001 or 2004).

Techniques: Control

Journal: bioRxiv

Article Title: Phosphatase-independent activity of smooth-muscle calcineurin orchestrates a gene expression program leading to hypertension

doi: 10.1101/2023.11.26.568733

Figure Lengend Snippet: (A) Experimental design: 10-12-week-old mice were inoculated with LxVPmut-or LxVP-encoding lentivirus (LV) and, after 6 weeks, Ang-II osmotic minipumps were implanted for 4 weeks. BP was measured at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( B ) Systolic and diastolic BP in mice transduced with LV-LxVPmut (n=7) and LV-LxVP (n=7), shown as mean ± s.e.m. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 and ### p<0.001, ## p<0.01 vs beginning of treatment (0 weeks) for LV-LxVPmut-and LV-LxVP-transduced mice, respectively; RM two-way ANOVA with Tukey’s post hoc test. ( C ) Representative images of GFP immunostaining in AbAo and AsAo cross sections from the indicated mice. ( D ) Representative images of southwestern histochemistry with an NFAT probe of AbAo and AsAo cross sections from the indicated mice and ( E ) quantification of the relative staining (NFAT activity) in the AbAo (left panel) and the AsAo (right panel). Dashed lines indicate the medial layer. Only mice with positive aortic GFP staining and consistent NFAT activity were included. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. Mann-Whitney test **p<0.01. Scale bars, 100 μm.

Article Snippet: Ang-II (Sigma-Aldrich), CsA (Novartis), NE (Sigma-Aldrich), and TM5441 (MedChemExpress) were administered at 1 μg/kg/min, 5mg/kg/d, 10 mg/kg/d, and 20mg/kg/d, respectively, using subcutaneous osmotic minipumps (Alzet Corp, model 2001 or 2004).

Techniques: Transduction, Immunostaining, Staining, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Phosphatase-independent activity of smooth-muscle calcineurin orchestrates a gene expression program leading to hypertension

doi: 10.1101/2023.11.26.568733

Figure Lengend Snippet: ( A ) Experimental design: 10-12-week-old mice were treated with tamoxifen for 5 consecutive days (open arrow heads) and, after 6 weeks, Ang-II osmotic minipumps were implanted for 2h and 24h. BP was measured at the indicated time points (purple arrows), and mice were euthanized and analyzed when indicated. ( B ) Systolic BP values in the indicated mice (n=9 mice per group and time point). Each data point denotes an individual mouse, and the horizontal bars denote the mean (long bar) and s.e.m. ****p<0.0001 vs baseline; RM two-way ANOVA with Tukey post hoc test. #### p<0.0001 vs 2h or 24h Ang-II Cn-Ctl; RM two-way ANOVA with the Šídák post hoc test. ( C ) Experimental design: 10-12-week-old mice were fitted with CsA osmotic minipumps (CsA-pretreated); control mice were operated without minipump implantation (Control). After 5 days, Ang-II minipumps were implanted in CsA-pretreated and Control mice for 2h and 24h. BP was measured at the indicated time points (purple arrows), and mice were euthanized and analyzed when indicated. ( D ) Systolic BP in the indicated mice (n=9 mice per group and time point). Each data point denotes an individual mouse, and the horizontal bars denote the mean (long bar) and s.e.m. ****p<0.0001 vs baseline; RM two-way ANOVA with Tukey’s post hoc test. ( E ) Experimental design: 10-12-week-old mice were fitted with Ang-II osmotic minipumps for 4 weeks. After 7 days of Ang-II infusion, tamoxifen was administered for 5 consecutive days (open arrow heads). BP was measured at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( F ) Systolic BP measurements at the indicated times in Cn-Ctl (n=10) and SM-Cn -/- (n=12) mice. Each data point denotes an individual mouse, and the horizontal bars denote the mean (long bar) and s.e.m. ****p<0.0001, ***p<0.001 vs Cn-Ctl Ang-II; RM two-way ANOVA with Šídák post hoc test. Tx indicates tamoxifen administration time points.

Article Snippet: Ang-II (Sigma-Aldrich), CsA (Novartis), NE (Sigma-Aldrich), and TM5441 (MedChemExpress) were administered at 1 μg/kg/min, 5mg/kg/d, 10 mg/kg/d, and 20mg/kg/d, respectively, using subcutaneous osmotic minipumps (Alzet Corp, model 2001 or 2004).

Techniques: Control

Journal: bioRxiv

Article Title: Phosphatase-independent activity of smooth-muscle calcineurin orchestrates a gene expression program leading to hypertension

doi: 10.1101/2023.11.26.568733

Figure Lengend Snippet: ( A ) List of the 30 genes with the highest hypertension score (HS), showing their gene symbol, HS, and expression fold change (FC) after 2h of Ang-II treatment relative to untreated mice. ( B ) RT-qPCR analysis of mRNA expression in aortic extracts from untreated control (3 Cn-Ctl plus 3 WT), SM-Cn -/- (n=5), and CsA-pretreated (n=6) mice and from Ang-II-treated control (3 Cn-Ctl plus 3 WT), SM-Cn -/- (n=5), and CsA-pretreated (n=6) mice. Two-way ANOVA with Šídák post hoc test; p values and comparisons are indicated. ( C ) Representative immunoblot analysis of PAI-1 and tubulin (loading control) and ( D ) quantification of their relative expression in protein extracts from untreated control (n=4) and SM-Cn -/- (n=2) mice and from Ang-II-treated control (n=6), SM-Cn -/- (n=6), and CsA-pretreated (n=6) mice. Molecular weights (kDa) are indicated. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. One-way ANOVA with Tukey post hoc test; p values and comparisons are indicated. ( E ) Representative images (left panels) and normalized surface area of fixed collagen gels (right panel) under the indicated conditions. Each data point denotes the mean of each experiment, and data in histograms are presented as mean ± s.e.m. (n=4 independent experiments with with 3% FBS and 50 µM TM5441). ****p<0,0001, unpaired Student t-test. Scale bars, 5 mm. ( F ) Experimental design: 10-12-week-old mice were fitted with Ang-II osmotic minipumps (Ang-II) 24h before implantating TM5441 minipumps in a group of Ang-II-pretreated mice (Ang-II+TM5441). BP was measured at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( G ) Systolic BP at the indicated times in 10 Ang-II-treated mice (Ang-II) and 8 mice treated with Ang-II+TM5441. Data are presented as mean ± s.e.m. ****p<0.001, vs Control RM two-way ANOVA with Šídák post hoc test. ( H ) Experimental design: 10-12-week-old mice were fitted with Ang-II osmotic minipumps (Ang-II) 7 days before intraperitoneally injecting TM5441 or vehicle to a group of Ang-II-pretreated mice. BP was measured at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. ( I ) Systolic BP at the indicated times in 5 Ang-II + vehicle (Control) and 6 mice treated with Ang-II+TM5441 (Control + TM5441). Data are presented as mean ± s.e.m. ****p<0.001, vs Control RM two-way ANOVA with Šídák post hoc test.

Article Snippet: Ang-II (Sigma-Aldrich), CsA (Novartis), NE (Sigma-Aldrich), and TM5441 (MedChemExpress) were administered at 1 μg/kg/min, 5mg/kg/d, 10 mg/kg/d, and 20mg/kg/d, respectively, using subcutaneous osmotic minipumps (Alzet Corp, model 2001 or 2004).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: NPJ Biofilms and Microbiomes

Article Title: Nonlinear rheological characteristics of single species bacterial biofilms

doi: 10.1038/s41522-020-0126-1

Figure Lengend Snippet: Plots show the variation in the ratio of third ( I 3 ) to first ( I 1 ) order harmonic as a function of strain amplitude for a B. subtilis b C. denitrificans c P. fluorescens and d P. aeruginosa at a frequency of 1 Hz. Lines with slopes of 1.8, 1.5, 1.8 and 1.6 are indicated as reference in the corresponding plots. Blue dotted lines denotes the MAOS region within which the lines of particular slope are observed.

Article Snippet: Single species biofilms of Bacillus subtilis ( B. subtilis 168), Pseudomonas fluorescens (DSMZ-50090) Comamonas denitrificans (DSMZ-17887) and Pseudomonas aeruginosa (DSMZ-22644) were grown overnight in LB Miller (Molecular Dimensions MD12-103, 25g/L), Nutrient (Sigma-Aldrich N7519, 8g/L), Tryptic Soy (Sigma-Aldrich 22092, 30g/L) and Nutrient (Sigma-Aldrich N7519, 8g/L) broth respectively.

Techniques: