Journal: Cells

Article Title: A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

doi: 10.3390/cells11142168

Figure Lengend Snippet: The domain structure of the human TFAM and a general outline of the GeneSwap approach. ( A ), Upward vertical arrow, a crossover point in chimeras. Here, we refer to the TFAM portion preceding the crossover point as N-terminal domain (NTD), and the portion following HMG2 as C-terminal domain (CTD). Domain boundaries are given in aa coordinates. MTS, matrix targeting sequence, Link, linker region. ( B ), Engineering of a GeneSwap cell line using TFAM GeneSwap 143B#6 as an example. The three main steps include: (1) A gene of interest ( TFAM ) is inactivated by CRISPR-Cas9, (2) the resulting ρ0 cells are transduced with a retrovirus encoding a wt hTFAM gene “floxed” with loxP sites for Cre recombinase, (3) mtDNA is reintroduced into these transduced ρ 0 cells by fusing them with enucleated cells. ( C ), To implement the GeneSwap approach, 143B#6 cells (the top construct) are co-transduced with retroviruses encoding Cre recombinase and altered TFAM (TFAMvar, central panel.). This results in the excision of the wtTFAM and simultaneous re-expression of the TFAMvar . In the resulting co-transductants, mtDNA is retained only if TFAMvar is functional in hmtDNA replication. In that case the functionality is further validated by transducing cells with PhiC31 recombinase, which effects the loss of TFAMvar and hmtDNA (the bottom construct). ( D ), 143B cells KO for hTFAM , hPolRmt , hTFB2M, hPolG1, hPolG2 , and hSSBP1 , are viable, but lose hmtDNA. ( E ), Validation of the hTFAM, hPolRmt, hTFB2M, hPolG1 , and hSSBP1 KO by Western blotting. ( F ), 143B cells KO for for hTFAM, hPolRmt, hTFB2M, hPolG1, hPolG2 , and hSSBP1 survive in +UP media, but die in −UP media. A representative of two independent experiments, each with three technical replicates. ( G ), A diagram for PCR-genotyping of ΔhTFAM (panel ( I )). ( H ) A diagram for PCR genotyping of hTFAM, Cre , and PhiC31 (panels ( I , J) ). Primer sequences and fragment sizes as in . ( I ) A PCR genotyping and Western blotting verification of the main steps in the engineering and validation of the 143B#6 TFAM GeneSwap cell line. KO, CRISPR-Cas9 hTFAM KO; KO/WTlox, TFAM KO cells transduced with rv.4000 encoding wt hTFAM ; KO/WTlox = cybrid, reintroduction of hmtDNA in KO cells complemented with rv.4000; Cybrid/Cre, Cre/lox deletion of the wt hTFAM introduced with rv.4000; GeneSwap, GeneSwap of the wt hTFAM encoded by rv.4000 for wt hTFAM encoded by rv.5460. HSP60, loading control for Western blotting. PCR subpanels (top to bottom): Top, duplex PCR for nDNA and mtDNA. ΔhTFAM, diagnostics for excision of hTFAM encoded by rv.4000, see diagram in ( G ); hTFAM, detection of the cDNA for wt hTFAM encoded by rv.4000 and rv.5460, see diagram in ( H ); Cre, detection of Cre recombinase gene encoded by rv.3442, see diagram in ( H ). ( J ) GeneSwap/PhiC31, PhiC31-mediated excision of the wt hTFAM encoded by rv.5460 is accompanied by the loss of mtDNA. nDNA, mtDNA, duplex PCR for nDNA, and mtDNA. Note that due to high copy number, mtDNA suppresses amplification of the nDNA. NTC, no template control. Subpanels as in ( I ) plus: Excision/PhiC31-PCR diagnostic of hTFAM excision from rv.5460, see the middle and the bottom of panel C; PhiC31, PCR detection of PhiC31 gene, see diagram in ( H ). See for primer sequences and predicted fragment sizes.

Article Snippet: Antibodies used were: anti-MT-CO1 and anti-MT-CO2 (Abcam, Cambridge, MA, USA, Cat# ab14705 and ab91317, RRID:AB_2084810 and AB_10712683, respectively); anti-hTFAM, N-terminal (Cell Signaling Technology Cat# 7495, RRID:AB_10841294); anti-hTFAM, C-terminal (Santa Cruz Biotechnology Cat# sc-376672, RRID:AB_11150497); anti m + h TFAM (PhosphoSolutions Cat# 2001-TFAM, RRID:AB_2492259); anti-TFB2M (Proteintech Cat# 24411-1-AP, RRID:AB_2879530), anti-PolRmt (Abcam Cat# ab32988, RRID:AB_873619); anti-SSBP1 (Proteintech Cat#12212-1-AP, RRID:AB_2195320), anti-PolG1 (Cell Signaling Technology Cat# 13609, RRID:AB_2750886); anti-β-actin (Sigma-Aldrich Cat# A5441, RRID:AB_476744), anti-α-actinin (Santa Cruz Biotechnology Cat# sc-17829, RRID:AB_626633); Goat Anti-Mouse IgG (H + L), HRP Conjugate (Boster Biological Technology, Cat# BA1050, RRID:AB_2904507); Goat-anti-rabbit IgG (H + L), HRP conjugate (Advansta, Cat# R-05072-500, RRID:AB_10719218).

Techniques: Sequencing, CRISPR, Transduction, Construct, Expressing, Functional Assay, Biomarker Discovery, Western Blot, Control, Amplification, Diagnostic Assay

Journal: Cells

Article Title: A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

doi: 10.3390/cells11142168

Figure Lengend Snippet: Genotyping cells transduced with TFAM orthologs. ( A – C ) The three alternative strategies for geno-typing excision of oTFAM (PCR subpanel ΔPhiC31 in ( D )): The Main strategy with forward internal primer ( A ), the Main with reverse internal primer ( B ), and Alternative strategy ( C ). ( D ), PCR-genotyping of the clones in which hmtDNA replication is supported by oTFAM s. Superscripts a, b, and c correspond to genotyping strategies in ( A – C ). ( E ) PCR genotyping of cells expressing oTFAM s that are unable to support hmtDNA replication. Geno-typing for mtDNA, nDNA, ΔhTFAM , and PhiC31 are essentially as depicted in I,J. oTFAM s were geno-typed as diagramed in H. * These orthologs were excised by transiently transfecting cells with pMA4854 . See for primer sequences and predicted fragment sizes.

Article Snippet: Antibodies used were: anti-MT-CO1 and anti-MT-CO2 (Abcam, Cambridge, MA, USA, Cat# ab14705 and ab91317, RRID:AB_2084810 and AB_10712683, respectively); anti-hTFAM, N-terminal (Cell Signaling Technology Cat# 7495, RRID:AB_10841294); anti-hTFAM, C-terminal (Santa Cruz Biotechnology Cat# sc-376672, RRID:AB_11150497); anti m + h TFAM (PhosphoSolutions Cat# 2001-TFAM, RRID:AB_2492259); anti-TFB2M (Proteintech Cat# 24411-1-AP, RRID:AB_2879530), anti-PolRmt (Abcam Cat# ab32988, RRID:AB_873619); anti-SSBP1 (Proteintech Cat#12212-1-AP, RRID:AB_2195320), anti-PolG1 (Cell Signaling Technology Cat# 13609, RRID:AB_2750886); anti-β-actin (Sigma-Aldrich Cat# A5441, RRID:AB_476744), anti-α-actinin (Santa Cruz Biotechnology Cat# sc-17829, RRID:AB_626633); Goat Anti-Mouse IgG (H + L), HRP Conjugate (Boster Biological Technology, Cat# BA1050, RRID:AB_2904507); Goat-anti-rabbit IgG (H + L), HRP conjugate (Advansta, Cat# R-05072-500, RRID:AB_10719218).

Techniques: Transduction, Clone Assay, Expressing

Journal: Cells

Article Title: A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

doi: 10.3390/cells11142168

Figure Lengend Snippet: Coelacanth TFAM promotes spontaneous loss of hmtDNA. ( A ), Upon cultivation in +UP media, mtCN in cells expressing coelTFAM is reduced. Cells expressing coelTFAM were incubated in +UP media for up to 35 days, cell aliquots were collected at indicated intervals, and mtCN was determined by qPCR. ( B ), coelTFAM promotes spontaneous loss of mtDNA. Cells were grown in +UP media for 5 weeks, plated by limiting dilution, the resulting colonies were picked and tested for the presence of mtDNA. Red vertical arrows indicate clones that lost mtDNA. ( C , D ), in the experiment described in ( B ), clones representative of various mtCNs were analyzed by conventional ( C ) and dddPCR. ( D ). ( E ), Transduction with hTFAM augments mtCN in cells expressing coelTFAM. Cells expressing coelTFAM were selected in –UP media and transduced with a retrovirus rv.5132 , which expresses hTFAM . ****, p < 0.0001. ( E ), One-way ANOVA with post hoc Tukey test. A representative of two independent experiments. ( F – H ), Changes in OXPHOS and mtDNA-encoded OXPHOS subunits expression correlate with expression of the MT-RNR2. ( F ), OXPHOS deficiency in cells expressing coelTFAM can be partially rescued with hTFAM. ****, p < 0.0001. One-way ANOVA with post hoc Tukey test. A representative of two independent experiments. ( G ), coelTFAM does not significantly affect expression of the MT-ND1, MT-CO1 , or MT-ND6 , but affects expression of the MT-RNR2 . Two-way ANOVA with Dunnet correction. ****, p < 0.0001; ns, not significant. ( H ), OXPHOS correlates with expression of mtDNA-encoded subunits MT-CO1 and MT-CO2. A representative of two independent experiments.

Article Snippet: Antibodies used were: anti-MT-CO1 and anti-MT-CO2 (Abcam, Cambridge, MA, USA, Cat# ab14705 and ab91317, RRID:AB_2084810 and AB_10712683, respectively); anti-hTFAM, N-terminal (Cell Signaling Technology Cat# 7495, RRID:AB_10841294); anti-hTFAM, C-terminal (Santa Cruz Biotechnology Cat# sc-376672, RRID:AB_11150497); anti m + h TFAM (PhosphoSolutions Cat# 2001-TFAM, RRID:AB_2492259); anti-TFB2M (Proteintech Cat# 24411-1-AP, RRID:AB_2879530), anti-PolRmt (Abcam Cat# ab32988, RRID:AB_873619); anti-SSBP1 (Proteintech Cat#12212-1-AP, RRID:AB_2195320), anti-PolG1 (Cell Signaling Technology Cat# 13609, RRID:AB_2750886); anti-β-actin (Sigma-Aldrich Cat# A5441, RRID:AB_476744), anti-α-actinin (Santa Cruz Biotechnology Cat# sc-17829, RRID:AB_626633); Goat Anti-Mouse IgG (H + L), HRP Conjugate (Boster Biological Technology, Cat# BA1050, RRID:AB_2904507); Goat-anti-rabbit IgG (H + L), HRP conjugate (Advansta, Cat# R-05072-500, RRID:AB_10719218).

Techniques: Expressing, Incubation, Clone Assay, Transduction

Journal: Cells

Article Title: A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication

doi: 10.3390/cells11142168

Figure Lengend Snippet: Screening oTFAM chimeras for their ability to support replication of hmtDNA. ( A , B ), PCR genotyping strategies for cells expressing oTFAM chimeras. ( C , D ), PCR genotyping strategies for cells expressing dmTFAM-hTFAM and hTFAM-dmTFAM chimeras, respectively. ( E , F ), PCR genotyping of oTFAM chimeras in which both NTD and CTD support hmtDNA replication ( G , H ), PCR genotyping of oTFAM chimeras in which only NTD supports hmtDNA replication ( I ), PCR genotyping of oTFAM s chimeras in which neither NTD, nor CTD supports hmtDNA replication. MTS, matrix targeting sequence of the human ornithine transcarbamylase was appended in front of oTFAM. *, excised by transiently transfecting cells with pMA4854 . **, a spurious PCR product in hTFAM-oTFAM PCR due to homology between hTFAM and tdTFAM . mtDNA, nDNA, ΔTFAM , and PhiC31 genotyping as in . Subpanel oTFAM-hTFAM, detection of the corresponding chimeras (see diagrams ( A , C )). Subpanel hTFAM-oTFAM, detection of the corresponding chimeras (see diagrams ( B , D )). Subpanels F + R1 and F + R3, detection of chimera excision (see diagrams ( A – D )). See A for chimera design and for primer sequences and predicted amplicon sizes.

Article Snippet: Antibodies used were: anti-MT-CO1 and anti-MT-CO2 (Abcam, Cambridge, MA, USA, Cat# ab14705 and ab91317, RRID:AB_2084810 and AB_10712683, respectively); anti-hTFAM, N-terminal (Cell Signaling Technology Cat# 7495, RRID:AB_10841294); anti-hTFAM, C-terminal (Santa Cruz Biotechnology Cat# sc-376672, RRID:AB_11150497); anti m + h TFAM (PhosphoSolutions Cat# 2001-TFAM, RRID:AB_2492259); anti-TFB2M (Proteintech Cat# 24411-1-AP, RRID:AB_2879530), anti-PolRmt (Abcam Cat# ab32988, RRID:AB_873619); anti-SSBP1 (Proteintech Cat#12212-1-AP, RRID:AB_2195320), anti-PolG1 (Cell Signaling Technology Cat# 13609, RRID:AB_2750886); anti-β-actin (Sigma-Aldrich Cat# A5441, RRID:AB_476744), anti-α-actinin (Santa Cruz Biotechnology Cat# sc-17829, RRID:AB_626633); Goat Anti-Mouse IgG (H + L), HRP Conjugate (Boster Biological Technology, Cat# BA1050, RRID:AB_2904507); Goat-anti-rabbit IgG (H + L), HRP conjugate (Advansta, Cat# R-05072-500, RRID:AB_10719218).

Techniques: Expressing, Sequencing, Amplification

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: