Journal: Bioactive Materials

Article Title: An axolotl limb regeneration-inspired strategy to enhance alveolar bone regeneration

doi: 10.1016/j.bioactmat.2025.02.020

Figure Lengend Snippet: Prrx1 + stem cells demonstrated a critical role in BMP-2-induced osteogenesis . ( A ) Schematic illustration of a mouse distraction osteogenesis model. ( B ) Genes of osteogenic stem cells sorted by time of peak expression during jaw regeneration.( C ) Expression patterns of Bmpr1a and Prrx1 . ( D ) Co-localization of Prrx1 and BMPR1A in hPDLSCs. ( E ) Schematic diagram of cell migration experiment using a three-channel microfluidic chip. ( F ) After hPDLSCs adhered in the central channel, culture medium containing 200 ng/mL BMP-2 was perfused in the upper channel while the standard culture medium in lower channel; cell migration is observed after 8 h. ( G ) Schematic of semi-open silk sponges subcutaneously implanted with open sides facing either the epithelial or muscular region in Mice. ( H ) Sp7/ALP co-staining was performed to assess osteogenic effects in the localized region on day 7. ( I ) Schematic of silk sponges for dorsal subcutaneous transplantation in Prrx1-cre; R26R tdTomato mice. ( J ) ALP immunofluorescence staining was utilized to trace the osteogenic differentiation of BMP-2-recruited Prrx1 + stem cells. n = 3 biological replicates. Illustration created with BioRender.

Article Snippet: In immunofluorescence staining procedure, the cells or decalcified sections were incubated overnight at 4 °C with primary antibodies: Prrx1 (ab211292, Abcam, USA), BMPR1A (ab264043, Abcam, USA), α-SMA (ab124964, Abcam, USA),CD31 (AF3628, R&D systems, USA), MyD88 (ab2064, Abcam, USA), ALP (AF2910, R&D systems, USA), Sp7 (ab22552, Abcam, USA) and OCN (23418-1-AP, Proteintech, China).

Techniques: Expressing, Migration, Staining, Transplantation Assay, Immunofluorescence

Journal: Bioactive Materials

Article Title: An axolotl limb regeneration-inspired strategy to enhance alveolar bone regeneration

doi: 10.1016/j.bioactmat.2025.02.020

Figure Lengend Snippet: Channel structure and BMP-2 induced ectopic recruitment of Prrx1 + stem cells for osteogenesis . ( A ) Schematic illustration of the single-channel PDMS film for dorsal subcutaneous transplantation in rats. ( B ) Evaluation of tissue infiltration and collagen formation within the channel structure using HE staining and Masson's trichrome staining on days 4 and 7. ( C ) Prrx1/ALP co-staining detects the osteogenesis of BMP-2 ectopically recruited Prrx1 + stem cells on day 7. ( D and E ) SP7 (D) and OCN (E) immunofluorescence staining for ectopic osteogenesis within channel structure on day 7. n = 4 biological replicates. Illustration created with BioRender.

Article Snippet: In immunofluorescence staining procedure, the cells or decalcified sections were incubated overnight at 4 °C with primary antibodies: Prrx1 (ab211292, Abcam, USA), BMPR1A (ab264043, Abcam, USA), α-SMA (ab124964, Abcam, USA),CD31 (AF3628, R&D systems, USA), MyD88 (ab2064, Abcam, USA), ALP (AF2910, R&D systems, USA), Sp7 (ab22552, Abcam, USA) and OCN (23418-1-AP, Proteintech, China).

Techniques: Transplantation Assay, Staining, Immunofluorescence

Journal: Bioactive Materials

Article Title: An axolotl limb regeneration-inspired strategy to enhance alveolar bone regeneration

doi: 10.1016/j.bioactmat.2025.02.020

Figure Lengend Snippet: Synergistic osteogenic effects of BMP-2 and channel structure in PDMS cube and subgingival tissue . ( A ) Schematic illustration showing the PDMS cube implanted subcutaneously in rats as a BCS model. ( B ) Evaluation of collagen formation within the PDMS cube via Masson's trichrome staining on day 14. ( C ) Assessment of the sequential induction of stem cell infiltration and osteogenesis by BMP-2 and channel structures through Prrx1/ALP co-staining on day 14. ( D to F ) Whole-sample scan of CD31 staining on day 14 showing overall vascularization within the PDMS cubes (D), along with magnified views of the channel region (E) and the central region (F). ( G ) Schematic illustration of the single-channel PDMS film for subgingival transplantation in rats. ( H ) HE staining and Masson's trichrome staining were used to assess tissue infiltration and collagen formation within channel structures on days 4 and 7. ( I ) Prrx1 and OCN immunofluorescence staining for ectopic osteogenesis within channel structure on day 7. n = 4 biological replicates. Illustration created with BioRender.

Article Snippet: In immunofluorescence staining procedure, the cells or decalcified sections were incubated overnight at 4 °C with primary antibodies: Prrx1 (ab211292, Abcam, USA), BMPR1A (ab264043, Abcam, USA), α-SMA (ab124964, Abcam, USA),CD31 (AF3628, R&D systems, USA), MyD88 (ab2064, Abcam, USA), ALP (AF2910, R&D systems, USA), Sp7 (ab22552, Abcam, USA) and OCN (23418-1-AP, Proteintech, China).

Techniques: Staining, Transplantation Assay, Immunofluorescence

Journal: bioRxiv

Article Title: The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

doi: 10.1101/2023.02.12.528214

Figure Lengend Snippet: Representative immunohistochemistry staining of hypertrophic chondrocyte markers ALPL and SOX9 in 12-month-old WT and Col2a1- Bmal1 KO mice. Arrows highlight expression of ALPL and SOX9 in the peripheral edge of the heterotopic ossification region in 12-month-old Col2a1- Bmal1 KO lumbar intervertebral discs. ALPL and SOX9-positive cells are prolific throughout this region, particularly towards the peripheral edges. N=3.

Article Snippet: Primary antibodies for ALPL (R&D Systems, AF2910), SOX9 (Cell Signalling) were diluted at 1:1000 and 1:500, respectively, in blocking buffer.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: bioRxiv

Article Title: The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc

doi: 10.1101/2023.02.12.528214

Figure Lengend Snippet: A) ALPL and SOX9-positive cells were prolific throughout this region, particularly towards the peripheral edges. Cells showed a rounded morphology. B) x40 magnification regions highlighted in A).

Article Snippet: Primary antibodies for ALPL (R&D Systems, AF2910), SOX9 (Cell Signalling) were diluted at 1:1000 and 1:500, respectively, in blocking buffer.

Techniques:

Journal: Journal of Bacteriology

Article Title: TrhA, a bacterial progestin and adiponectin receptor homolog, couples membrane energetics homeostasis and unsaturated fatty acid metabolism

doi: 10.1128/jb.00397-23

Figure Lengend Snippet: TrhA has a periplasmic C-terminus. (A) C-terminal alkaline phosphatase (PhoA) activity of DH5α (pTrc99a) empty vector (ev), DH5α (pTrc99a tolQ-phoA), DH5α (pTrc99a dgkA-phoA), and DH5α (pTrc99a trhA-phoA). Each point represents the average PhoA activity of one biological replicate with technical replicates. Error bars represent the standard deviation. For qualitative analysis of PhoA activity, cells were spotted and dried on Luria Broth (LB) agar containing PhoA substrate X-Pho (5-bromo-4-chloro-3-indolyl-phosphate) (final concentration of 100 µg/mL) (left). Blue colonies have active PhoA reporter fusions, while colorless colonies lack PhoA activity. (B) C-terminal yellow fluorescent protein fluorescence of DH5α (pRH005), DH5α (pRH005 tolQ), DH5α (pRH005 dgkA), and DH5α (pRH005 trhA). Each point represents the YFP fluorescence intensity of an individual cell (N = 25 cells). Error bars represent the standard deviation. Scale bars represent 1 µm.

Article Snippet: PhoA-tagged proteins were detected using anti-alkaline phosphatase antibody (OriGene) at a 1:2,000 dilution.

Techniques: Activity Assay, Plasmid Preparation, Standard Deviation, Concentration Assay, Fluorescence

Journal: Journal of Bacteriology

Article Title: TrhA, a bacterial progestin and adiponectin receptor homolog, couples membrane energetics homeostasis and unsaturated fatty acid metabolism

doi: 10.1128/jb.00397-23

Figure Lengend Snippet: Residues conserved in bacterial and/or Class III PAQRs affect membrane energetics homeostasis in TrhA. (A) C-terminal alkaline phosphatase (PhoA) activity of the following strains was assayed as a proxy for functionality and stability of each TrhAEC SSM: DH5α (pTrc99a) ev control, DH5α (pTrc99a tolQ-phoA), DH5α (pTrc99a dgkA-phoA), DH5α (pTrc99a TrhAWT-phoA), DH5α (pTrc99a S68A-phoA), DH5α (pTrc99a H72F-phoA), DH5α (pTrc99a H76F-phoA), DH5α (pTrc99a H72F/H76F-phoA), DH5α (pTrc99a D87L-phoA), DH5α (pTrc99a G181P-phoA), DH5α (pTrc99a H194F-phoA), and DH5α (pTrc99a H198F-phoA). Data represent the PhoA activities of two biological replicates with three technical replicates. Error bars represent standard deviation. Legend: a, significantly different from negative controls ev and TolQ-PhoA (P < 0.0001); b, significantly different from positive control DgkA-PhoA (P < 0.0001); c, significantly different from TrhAWT-PhoA (P < 0.005). For qualitative analysis of PhoA activity, cells were spotted and dried on LB agar containing PhoA substrate X-Pho (5-bromo-4-chloro-3-indolyl-phosphate) (final concentration of 100 µg/mL) (left). Blue colonies have active PhoA reporter fusions, while colorless colonies lack PhoA activity. (B) ∆trhA strain was transformed with pTrc99a (ev) control, pTrc99a S68A-phoA, pTrc99a H72F-phoA, pTrc99a H76F-phoA, pTrc99a H72F/H76F-phoA, pTrc99a D87L-phoA, and pTrc99a H198F-phoA. Total lysates were then analyzed by Western blotting using anti-PhoA antibody. (C) TrhA-PhoA variant protein stability was determined as the ratio between the TrhA-PhoA and the PhoA bands in at least two Western blot images for each strain and quantitated using ImageJ. * indicates a significant difference compared to TrhAWT at P < 0.05.

Article Snippet: PhoA-tagged proteins were detected using anti-alkaline phosphatase antibody (OriGene) at a 1:2,000 dilution.

Techniques: Membrane, Activity Assay, Control, Standard Deviation, Positive Control, Concentration Assay, Transformation Assay, Western Blot, Variant Assay