Journal: Bone Reports

Article Title: Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

doi: 10.1016/j.bonr.2024.101763

Figure Lengend Snippet: Effect of ClC3 and ClC5 knockout on bone structure. A-C shows vertical sections of wild type, ClC3 knockout, and ClC5 knockout mice. D-I show micro-computed tomography: In each case, the left bar is wild type, ClC3 −/− is the middle bar, in red, and ClC5 − (males) or −/− (females) are shown in the right bar (green). In each bar, for individual data points males are blue dots and females are red dots. A. Vertical section through L4 of the wild type mouse. B. Vertical section through L4 of the ClC3 −/− animal. The density of bone is clearly less than wild type of ClC5 knockout C. Vertical section through L4 of the ClC5 −/− (male) animal. The difference in bone density with wild type (A) is not clear by visual examination. D. Bone volume/Total volume, %. This is reduced in both knockouts about 30 % on average relative to the wild type; difference in this parameter between ClC3 and ClC5 is not significant. E. Intersection Surface, μ m 2 . This is reduced on average relative to the wild type about 40 % in the ClC3 only. This is marginally increased relative to the wild type in ClC5 knockouts. F. Total Porosity. This is increased by a small amount in both knockouts. G. Trabecular Thickness. This is decreased in both knockouts, more in the ClC3 knockout. H. Trabecular number per cm. This is unaffected by the knockouts, in keeping with trabecular formation involving different mechanisms than growth. I. Bone Surface/Bone Volume. This is increased in the ClC3 knockout and to a minor extent in the ClC5 knockout, on average.

Article Snippet: Cell culture on Transwells were fixed on day 14 with 3.7 % formaldehyde for 10 min followed by permeabilization with 0.1 % Triton X-100 for 20 min. After blocking with 2 % BSA, the cells were incubated with primary antibody against C-terminal regions of ClC3 or ClC5 (Alomone Labs, Israel) at 1:100 at 4 °C overnight and then with Alexa Fluor 488- or 555- conjugated secondary antibodies (Life Technologies) (green or red, respectively) at room temperature for 1 h, except for fluorescent labeling of ClC3 in mouse bone of wild type or ClC3 knockout animals which used rabbit polyclonal anti-ClC3 raised to amino acids 80–125 of human ClC3 (Bioss, Woburn, MA), 1:400 followed by fluorescent secondary antibodies as above.

Techniques: Knock-Out, Micro-CT

Journal: Bone Reports

Article Title: Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

doi: 10.1016/j.bonr.2024.101763

Figure Lengend Snippet: Bone formation in ClC3 and ClC5 knockout mice. A-C. Effect on calcein and xylenol orange labeling in representative bone sections. A minimum of four animals of each genotype were labeled using calcein IP on day 1, xylenol orange IP on day 4, and sacrifice on day 6, sections were cut and analyzed. The sections shown are 450 μm wide. In each case, the top frame shows the calcein label in green and xylenol orange in red; the bottom frame shows the same labels superimposed on a phase image of the trabecular bone at the same site. D-E. Effect on quantitative calcein and xylenol orange labeling. In each case, the left graph shows males (blue) and females (red) superimposed. The middle graph is for males only, the right graph females only. D. Labeled area as percent of bone surface. There are interesting differences but all are quantitatively small, and the only ones significant are slight but significant increase in labeled area in ClC3 −/− males relative to wild type (middle graph) and decreased labeled area in ClC3 −/− females relative either wild type or ClC5 knockouts (right graph). E. Interlabel distance in microns. Detailed μCT showed effects of the ClC5 and ClC3 knockout on interlabel distance were both significantly reduced ( p = 0.01 to 0.02), and by about 30 % in either males or females. These findings were remarkably consistent between the ClC3 and ClC5 knockouts.

Article Snippet: Cell culture on Transwells were fixed on day 14 with 3.7 % formaldehyde for 10 min followed by permeabilization with 0.1 % Triton X-100 for 20 min. After blocking with 2 % BSA, the cells were incubated with primary antibody against C-terminal regions of ClC3 or ClC5 (Alomone Labs, Israel) at 1:100 at 4 °C overnight and then with Alexa Fluor 488- or 555- conjugated secondary antibodies (Life Technologies) (green or red, respectively) at room temperature for 1 h, except for fluorescent labeling of ClC3 in mouse bone of wild type or ClC3 knockout animals which used rabbit polyclonal anti-ClC3 raised to amino acids 80–125 of human ClC3 (Bioss, Woburn, MA), 1:400 followed by fluorescent secondary antibodies as above.

Techniques: Knock-Out, Labeling

Journal: Bone Reports

Article Title: Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

doi: 10.1016/j.bonr.2024.101763

Figure Lengend Snippet: The ClC3 functional knockout and the ClC5 knockout. A. Sequence of ClC3 showing deleted amino acids in the functional knockout. Amino acids deleted in ClC3 are in green (end of exon 6) and yellow (exon 7) by total exon sequencing. The ClCN3 functional knockout had been characterized by PCR and Southern analysis; amino acid sequencing had not previously been performed. The deletion is as expected from the original mouse despite many generations of breeding and changing the host to C57black. B. The ClC5 knockout produces no ClC5 protein. Amount of ClC 5 produced in wild type and knockout animals. This was also done using stromal stem cells, with identical results (not shown). C. Expression of ClC3 protein is reduced in the ClC3 functional knockout. We compared abundance of a unique peptide in the wild type and ClC3 knockout in osteoblasts from stromal stem cells, by mass spectrometry for a peptide unique to ClC3. This confirmed the PCR result that the protein is exported in the knockout, albeit with ~40 % reduction of the wild type quantity.

Article Snippet: Cell culture on Transwells were fixed on day 14 with 3.7 % formaldehyde for 10 min followed by permeabilization with 0.1 % Triton X-100 for 20 min. After blocking with 2 % BSA, the cells were incubated with primary antibody against C-terminal regions of ClC3 or ClC5 (Alomone Labs, Israel) at 1:100 at 4 °C overnight and then with Alexa Fluor 488- or 555- conjugated secondary antibodies (Life Technologies) (green or red, respectively) at room temperature for 1 h, except for fluorescent labeling of ClC3 in mouse bone of wild type or ClC3 knockout animals which used rabbit polyclonal anti-ClC3 raised to amino acids 80–125 of human ClC3 (Bioss, Woburn, MA), 1:400 followed by fluorescent secondary antibodies as above.

Techniques: Functional Assay, Knock-Out, Sequencing, Produced, Expressing, Mass Spectrometry

Journal: Bone Reports

Article Title: Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

doi: 10.1016/j.bonr.2024.101763

Figure Lengend Snippet: Localization of ClC5 and of ClC3 to the matrix surface of osteoblasts. A-D. Antibody fluorescence labeling of bone trabeculae for ClC3 and ClC5. Each frame is 750 μm across. (A) Trabecular structure at this power is shown at the top left in phase contrast. (B) Strong labeling of ClC3 and (C) slightly less labeling of ClC5 at borders of trabeculae, in young wild type mice (12 weeks). (D) Knockout bone using antibody to the internal epitope missing in the ClC3 knockout (Methods and ) does not label ClC3. Both the wild type and ClC3 knockout proteins are exported, although somewhat less in the ClC3 knockout, (see C). E-F. Antibody‑gold labeling of ClC3 (left) and ClC5 (right) at the cytoplasm-matrix interface. Some of the gold indicated by green lines. In the ClC3, gold is 6 nm seen at high power (bar is 100 nm). For clarity, an insert in E shows part of the surface with gold at twice the magnification. The ClC5 is labeled with 20 nm gold and is seen at lower magnification (bar 400 nm).

Article Snippet: Cell culture on Transwells were fixed on day 14 with 3.7 % formaldehyde for 10 min followed by permeabilization with 0.1 % Triton X-100 for 20 min. After blocking with 2 % BSA, the cells were incubated with primary antibody against C-terminal regions of ClC3 or ClC5 (Alomone Labs, Israel) at 1:100 at 4 °C overnight and then with Alexa Fluor 488- or 555- conjugated secondary antibodies (Life Technologies) (green or red, respectively) at room temperature for 1 h, except for fluorescent labeling of ClC3 in mouse bone of wild type or ClC3 knockout animals which used rabbit polyclonal anti-ClC3 raised to amino acids 80–125 of human ClC3 (Bioss, Woburn, MA), 1:400 followed by fluorescent secondary antibodies as above.

Techniques: Fluorescence, Labeling, Knock-Out

Journal: Bone Reports

Article Title: Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

doi: 10.1016/j.bonr.2024.101763

Figure Lengend Snippet: Weights and serum chemistry knockout mice. Recorded in 5 month animals, males and females of wild type and ClC3 or ClC5 knockouts. A. Weights of wild type versus ClC3 and ClC5 knockout animals, Males. Knockout ClC3 animals, male or female, were 15–20 % reduced on average, with p < 0.001 in both cases. B. Weights of wild type versus ClC3 and ClC5 knockout animals, Females. Females were ~ 15 % lighter than males but the pattern was the same as in males. C. Serum Alkaline Phosphatase in Wild Type and ClC3 or ClC5 knockout Mice. Alkaline phosphatase in serum was reduced in the ClC5 knockout, although variability was high with significant overlap. D. Serum TRAP in Wild Type and ClC3 or ClC5 knockout Mice. TRAP did not change significantly relative to wild type in either knockout. E. Sections, 420 μ m square, adjacent to the growth plate antibody labeled for TRAP . On the top is direct labeling of tartrate resistant acid phosphatase near growth plates of wild type. On the bottom, an adjacent section from the same animal in hematoxylin and eosin (H&E) showing bone and marrow. This method is qualitative. However, relative to wild type (left frame) labeled cells in the ClC3 knockout (middle frame) were consistent with greater TRAP in serum (Panel D). Bone mass in the ClC5 knockout (right frame) was consistent with greater average animal weights (Panels A and B).

Article Snippet: Cell culture on Transwells were fixed on day 14 with 3.7 % formaldehyde for 10 min followed by permeabilization with 0.1 % Triton X-100 for 20 min. After blocking with 2 % BSA, the cells were incubated with primary antibody against C-terminal regions of ClC3 or ClC5 (Alomone Labs, Israel) at 1:100 at 4 °C overnight and then with Alexa Fluor 488- or 555- conjugated secondary antibodies (Life Technologies) (green or red, respectively) at room temperature for 1 h, except for fluorescent labeling of ClC3 in mouse bone of wild type or ClC3 knockout animals which used rabbit polyclonal anti-ClC3 raised to amino acids 80–125 of human ClC3 (Bioss, Woburn, MA), 1:400 followed by fluorescent secondary antibodies as above.

Techniques: Knock-Out, Labeling

Journal: Bone Reports

Article Title: Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice

doi: 10.1016/j.bonr.2024.101763

Figure Lengend Snippet: M ineral formation in osteoblasts is hypothesized to depend on H + /Cl − exchange. A model of the hypothesis tested here, that when phosphate is converted to hydroxyapatite with evolution of acid, the acid is removed from the bone by an ion transport mechanism that depends on ClC proton-chloride exchangers, at least in part. The work here shows functional importance of both ClC3 and ClC5. Beyond this, the phosphate is transported by active transport (neutral phosphate transporter 2) and the calcium by passive transport (intercellular). Once acid is taken up by osteoblasts, its removal is by basolateral sodium hydrogen exchangers (NHEs).

Article Snippet: Cell culture on Transwells were fixed on day 14 with 3.7 % formaldehyde for 10 min followed by permeabilization with 0.1 % Triton X-100 for 20 min. After blocking with 2 % BSA, the cells were incubated with primary antibody against C-terminal regions of ClC3 or ClC5 (Alomone Labs, Israel) at 1:100 at 4 °C overnight and then with Alexa Fluor 488- or 555- conjugated secondary antibodies (Life Technologies) (green or red, respectively) at room temperature for 1 h, except for fluorescent labeling of ClC3 in mouse bone of wild type or ClC3 knockout animals which used rabbit polyclonal anti-ClC3 raised to amino acids 80–125 of human ClC3 (Bioss, Woburn, MA), 1:400 followed by fluorescent secondary antibodies as above.

Techniques: Functional Assay

Journal: Cell reports

Article Title: Cell-Type-Specific Gene Regulatory Networks Underlying Murine Neonatal Heart Regeneration at Single-Cell Resolution

doi: 10.1016/j.celrep.2020.108472

Figure Lengend Snippet: (A) UMAP representation of different myeloid cell populations analyzed. (B) Heatmap showing expression of top enriched genes for each myeloid cell sub-population. (C–G) UMAP plots showing expression of Cd86 , an M1-macrophage marker gene (C); Arg1 , an M2-macrophage marker gene (D); Plac8 , a monocyte-enriched gene (E); Ly6c2 , an M1 monocyte-enriched gene (F); and Naaa , a DC-like cell enriched gene (G). (H) Percentage of each immune cell population over total nonmyocytes within each sample. (I) UMAP plot showing expression of Clcf1 among different cell types present in the P1+1 dpi and dps scRNA-seq data. (J) Representative images showing EdU incorporation (magenta) and cardiac troponin T (cTnT) immunofluorescent staining (green) of NRVM cells treated with 200 ng/mL BSA (negative control), 20 ng/mL recombinant insulin-like growth factor 2 (IGF2) (positive control), or different concentrations of recombinant CLCF1 (5, 10, 25, and 50 ng/mL). Scale bar, 100 μm. (K) Quantification showing the proportion of EdU-positive cells among cTnT-positive cells (CM) after treatment of BSA, CLCF1 recombinant protein, or recombinant IGF2 (n = 4 per each group; **p < 0.01, ****p < 0.0001). See also .

Article Snippet: Recombinant human CLCF1 protein (R&D Systems, 962-CL-050), recombinant human RSPO1 protein (Abcam, ab134626) and recombinant mouse IGF-2 protein (R&D Systems, 792-MG-050) were reconstituted in 0.1% BSA/PBS.

Techniques: Expressing, Marker, Staining, Negative Control, Recombinant, Positive Control

Journal: Cell reports

Article Title: Cell-Type-Specific Gene Regulatory Networks Underlying Murine Neonatal Heart Regeneration at Single-Cell Resolution

doi: 10.1016/j.celrep.2020.108472

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Recombinant human CLCF1 protein (R&D Systems, 962-CL-050), recombinant human RSPO1 protein (Abcam, ab134626) and recombinant mouse IGF-2 protein (R&D Systems, 792-MG-050) were reconstituted in 0.1% BSA/PBS.

Techniques: Virus, Plasmid Preparation, Recombinant, Modification, Red Blood Cell Lysis, Protease Inhibitor, Multiplex Assay, Software