Journal: The Journal of Neuroscience

Article Title: Somatotopy of Mouse Spinothalamic Innervation and the Localization of a Noxious Stimulus Requires Deleted in Colorectal Carcinoma Expression by Phox2a Neurons

doi: 10.1523/JNEUROSCI.1164-22.2022

Figure Lengend Snippet: Resources and reagents used

Article Snippet: Reagent or resource Source/reference Identifier Mice (MGI notation) Dcc flox Krimpenfort et al. 2012 MGI: 3665466 Phox2a Cre Roome et al. 2022 RRID: NA Ai14 B6;129S6-Gt (ROSA)26Sortm14(CAG- tdTomato ) Hze/J The Jackson Laboratory Catalog #JAX:007908 RRID: IMSR_JAX:007908 Cdx2 FlpO Tg(CDX2-flpo)#Gld Dr. Martyn Goulding, Salk Institute, San Diego MGI: 5911680 RRID: NA Ai65 B6;129S-Gt ROSA)26Sortm65.1(CAG- dTomato) Hze/J The Jackson Laboratory Catalog #JAX:021875 RRID: IMSR_JAX:021875 Ntn1 Bgeo Skarnes et al., 1995 RRID: NA 129S1/SvImJ The Jackson Laboratory Catalog #JAX:002448 RRID: IMSR_JAX:002448 B6C3F1/J The Jackson Laboratory Catalog #JAX: 100010 RRID: IMSR_JAX:100010 Primary antibodies Goat anti-mouse DCC (1:500) R&D Systems Catalog #AF844 RRID: AB_2089765 Rabbit anti-Phox2a (1:10,000 from frozen stock) Jean-François Brunet (École Normale Supérieure, Paris) ( Tiveron et al., 1996 ) RRID: AB_2315159 Rabbit anti-RFP (red fluorescent protein; 1:1000) Rockland Catalog #600-401-379 RRID: AB_2209751 Goat anti-tdT (red fluorescent protein; 1:300) SICGEN Catalog #AB8181-200 RRID: AB_2722750 Rabbit anti-GFP (1:1000) Life Technologies Catalog #A-11122 RRID: AB_221569 Goat anti-netrin-1 (1:200) R&D Systems Catalog #AF1109, RRID: AB_2298775 Rabbit anti-c- fos (1:500) Cell Signaling Technology Catalog #2250S RRID: AB_2247211 Rabbit anti-Pax6 (1:500) Millipore Catalog #AB2237, RRID: AB_1587367 Rabbit anti-Sox2 (1:200) Abcam Catalog #97959 RRID: AB_2341193 Secondary antibodies Alexa 488 Donkey anti-Rabbit (1:500) Jackson ImmunoResearch Laboratories Catalog #711–545-152 Lot #141848 RRID: AB_2313584 Alexa 488 Donkey anti-Goat (1:500) Jackson ImmunoResearch Laboratories Catalog #705–545-147 Lot #136089 RRID: AB_2336933 Cy3 Donkey anti-Rabbit (1:500) Jackson ImmunoResearch Laboratories Catalog #711–165-152 Lot #138270 RRID: AB_2307443 Cy3 Donkey anti-Goat (1:500) Jackson ImmunoResearch Laboratories Catalog #705–165-147 Lot #134527 RRID: AB_2307351 Cy5 Donkey anti-Rabbit (1:500) Jackson ImmunoResearch Laboratories Catalog #711–175-152 Lot #138336 RRID: AB_2340607 Cy5 Donkey anti-Goat (1:500) Jackson ImmunoResearch Laboratories Catalog #: 705–175-147 Lot#: 134531 RRID: AB_2340415 Chemicals ( E )-Capsaicin Tocris Bioscience Catalog #0462 Lot #7A/218361 RRID: NA 4′,6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific Catalog #D1306 RRID: NA Alexa 488-conjugated Choleratoxin B Thermo Fisher Scientific Catalog # C22841 Lot #2038245 RRID: NA X-gal Millipore Sigma Catalog #7240-90-6 RRID: NA MOWIOL Millipore Sigma Catalog #81381 RRID: NA Open in a separate window MGI, Mouse Genome Informatics; NA, Not applicable.

Techniques:

Journal: Nature Communications

Article Title: The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

doi: 10.1038/ncomms11074

Figure Lengend Snippet: ( a ) Immunofluorescence analysis of ACFs isolated from WT and PMCA4 −/− mice. Cells were stained with anti-DDR2 (fibroblasts marker) and anti-PMCA4 antibodies (scale bars, 25 μm). ( b ) qRT–PCR analysis showing a significant reduction in Pmca4 level in ACFs of PMCA4 −/− mice ( n =4; * P <0.05). ( c ) Western blotting to detect PMCA4 protein level in ACFs and quantification of band density showing significant reduction of PMCA4 expression in PMCA4 −/− cardiac fibroblasts ( n =4; * P <0.05). ( d ) Analysis of basal intracellular calcium showed significantly elevated intracellular calcium level in PMCA4 −/− cardiac fibroblasts ( n =4; * P <0.05). ( e ) Real-time RT–PCR and ( f ) western blot analysis, to examine sFRP2 expression in cardiac fibroblasts. ( g ) Quantification of band density supported the qRT–PCR analysis, showing significantly increased sFRP2 expression in PMCA4 −/− fibroblasts ( n =5; * P <0.05. ( h ) Expression of Pax2 was also elevated in PMCA4 −/− fibroblasts ( n =4; * P <0.05). ( i ) Using adenovirus-mediated NF-κB luciferase reporter construct, we detected a higher level of NF-κB activity in fibroblasts lacking PMCA4 ( n =4; * P <0.05). ( j ) Treatment with NF-κB inhibitor (BAY11–7082) at 10 μM for 24 h remarkably reduced sFRP2 mRNA level in PMCA4 −/− fibroblasts as detected by qRT–PCR ( n =4; * P <0.05). Student's t -test was used in all statistical tests above. All error bars represent the s.e.m.

Article Snippet: For TAC experiments involving anti-sFRP2 antibody administration, 8-week-old PMCA4 −/− mice were injected i.p. with either 200 μg per kg control goat IgG or goat anti-mouse sFRP2 antibody (R&D Systems) 1 day before TAC.

Techniques: Immunofluorescence, Isolation, Staining, Marker, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Construct, Activity Assay

Journal: Nature Communications

Article Title: The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

doi: 10.1038/ncomms11074

Figure Lengend Snippet: ( a ) Representative images of NRCMs cultured in conditioned medium of either WT or PMCA4 −/− cardiac fibroblasts. NRCMs were treated with phenylephrine (PE), 30 μM or PE with anti-sFRP2 antibody (0.2 μg ml −1 ) for 72 h. Cells were then stained with α-actinin antibody, to specifically visualize cardiomyocytes. Scale bar, 25 μm. ( b ) Measurement of cell surface area indicated that treatment with PMCA4 −/− fibroblasts conditioned medium (KO) significantly reduced PE-induced hypertrophy (results were from three independent experiments conducted in triplicate; a minimum of 100 cells were measured per replicate, * P <0.05, one-way analysis of variance (ANOVA) followed by post-hoc multiple comparison). Addition of anti-sFRP2 antibody abolished the anti-hypertrophic effect of PMCA4 −/− conditioned medium. ( c ) ELISA analysis showed a significantly higher sFRP2 level in PMCA4 −/− conditioned medium ( n =4 in each group; * P <0.05, Student's t -test). ( d ) Using an adenovirus expressing the TCF/LEF-luciferase construct, we detected activation of the Wnt/β-catenin pathway. Consistent with this observation, conditioned medium from PMCA4 −/− fibroblasts inhibited PE-induced Wnt/β-catenin activation, which was reversed by treatment with anti-sFRP2 antibody ( n =3 independent experiments; * P <0.05, one-way ANOVA followed by post-hoc multiple comparison). All error bars represent the s.e.m.

Article Snippet: For TAC experiments involving anti-sFRP2 antibody administration, 8-week-old PMCA4 −/− mice were injected i.p. with either 200 μg per kg control goat IgG or goat anti-mouse sFRP2 antibody (R&D Systems) 1 day before TAC.

Techniques: Cell Culture, Staining, Comparison, Enzyme-linked Immunosorbent Assay, Expressing, Luciferase, Construct, Activation Assay

Journal: Nature Communications

Article Title: The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

doi: 10.1038/ncomms11074

Figure Lengend Snippet: ( a ) sFRP2 level was detected in heart tissues using qRT–PCR (WT, n =4; PMCA4 −/− , n =3; * P <0.05, Student's t -test). ( b ) Representative image of hearts from WT and PMCA4 −/− mice following TAC (5 weeks) or sham operation (scale bar, 5 mm). ( c ) Measurement of HW/TL ratio showed significant reduction of the hypertrophic response in PMCA4 −/− mice ( n =10 in each group; * P <0.05, one-way analysis of variance (ANOVA) followed by post-hoc multiple comparison). ( d ) Representative heart tissue sections stained with haematoxylin and eosin, and ( e ) measurement of cardiomyocyte cross-sectional area showed smaller cardiomyocyte size in PMCA4 −/− mice after TAC (scale bars, 50 μm,* P <0.05, one-way ANOVA followed by post-hoc multiple comparison). ( f ) Western blot analysis of active β-catenin and ( g ) quantification of band density level indicated a higher level of β-catenin activation in PMCA4 −/− mice after TAC ( n =4 in each group; * P <0.05, one-way ANOVA followed by post-hoc multiple comparison). ( h ) When stimulated with long-term (12 weeks) TAC, more PMCA4 −/− mice survived than WT. All of the sham-treated animals survived at 12 weeks following procedure (WT, n =7; PMCA4 −/− n =5 at the beginning of the experiments). All error bars represent the s.e.m.

Article Snippet: For TAC experiments involving anti-sFRP2 antibody administration, 8-week-old PMCA4 −/− mice were injected i.p. with either 200 μg per kg control goat IgG or goat anti-mouse sFRP2 antibody (R&D Systems) 1 day before TAC.

Techniques: Quantitative RT-PCR, Comparison, Staining, Western Blot, Activation Assay

Journal: Nature Communications

Article Title: The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

doi: 10.1038/ncomms11074

Figure Lengend Snippet: ( a ) ACFs isolated from WT mice were subjected to ATA treatment (1 μM for 48 h). Measurement of basal intracellular calcium using fluo-3 dye indicated a significantly higher calcium level in ATA-treated cells ( n =4; * P <0.05, Student's t -test). ( b ) Analysis of NF-κB activity using the NF-κB-luciferase construct showed a significant increase in NF-κB activity in ATA-treated cells ( n =4; * P <0.05, Student's t -test). ( c ) ACFs were treated with ATA (1 μM) for 3 or 10 days. qRT–PCR analysis showed that sFRP2 level was significantly enhanced in ATA-treated cells ( n =4; * P <0.05, Student's t -test). ( d ) WT mice were injected with ATA (5 mg kg −1 per day for 2 weeks). sFRP2 level was detected in total heart's mRNA. Results showed a significant increase in sFRP2 level ( n =6–8 in each group; * P <0.05, Student's t -test). All error bars represent the s.e.m.

Article Snippet: For TAC experiments involving anti-sFRP2 antibody administration, 8-week-old PMCA4 −/− mice were injected i.p. with either 200 μg per kg control goat IgG or goat anti-mouse sFRP2 antibody (R&D Systems) 1 day before TAC.

Techniques: Isolation, Activity Assay, Luciferase, Construct, Quantitative RT-PCR, Injection