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: Microbial Cell Factories

Article Title: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

doi: 10.1186/s12934-019-1078-0

Figure Lengend Snippet: Identification of signal peptide sequences for enhanced secretory expression of SUMO-fused heterologous proteins in Bacillus subtilis. a Visualisation of alkaline phosphatase activity of representative B. subtilis KO7A colonies on solid medium containing the chromogenic substrate BCIP and IPTG as an inductor of PhoA reporter-gene expression. The colonies derived from a transformation of B. subtilis KO7A with plasmid pJHS03 with the SP amyQ encoding sequence replaced by a DNA-library of 173 different B. subtilis signal peptide encoding sequences. b Extracellular PhoA activities of three B. subtilis KO7A clones isolated from the signal peptide screen. KO7A/pJHS02 served as a negative control (−), KO7A/pJHS03 (SP amyQ ) and KO7A/pJHS04 (SP aprE ) served as positive controls. The signal peptides were identified as SP yopL (pJHS05), SP yncM (pJHS06) and SP yoaW (pJHS07) by DNA sequencing of re-isolated plasmids. IPTG was added to each 50 ml bacterial culture shaking at 37 °C at a final concentration of 1 mM at an OD 600 of 0.8. Samples were withdrawn after 4 h and PhoA activities were measured as described in “ ” section. c In parallel to PhoA activity estimation (see above), the cell free culture supernatants were subjected to protein precipitation with trichloroacetic acid. Precipitated proteins were dissolved in one tenth of the volume of Laemmli-buffer and 10 µl were analysed via SDS-PAGE. The StrepII-SUMO-PhoA protein is labelled with a star (*). d Western blot analysis of culture supernatants (CS) precipitated with TCA and whole-cell lysate samples (CE). Blots were developed with anti-PhoA antibodies. CS: 10 µl of a 10-fold dilution of the samples used for Coomassie staining (see above) were loaded; CE: 10 µg whole-cell protein per lane

Article Snippet: Blots were developed with polyclonal antibodies against E. coli alkaline phosphatase PhoA (OriGene Technologies, Inc.) at a dilution of 1:5000 and polyclonal secondary anti Rabbit-HRP antibodies (Carl Roth, #4750.1) according to the manufacturer’s specifications.

Techniques: Expressing, Activity Assay, Gene Expression, Derivative Assay, Transformation Assay, Plasmid Preparation, Sequencing, Clone Assay, Isolation, Negative Control, DNA Sequencing, Concentration Assay, SDS Page, Western Blot, Staining

Journal: Microbial Cell Factories

Article Title: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

doi: 10.1186/s12934-019-1078-0

Figure Lengend Snippet: GFP-specific scFv purification and antibody-antigen binding assay. a SDS-Page representing purification steps for the GFP-specific scFv antibody fragment from culture supernatants of B. subtilis strain KO7A/pJHS11, including immobilized metal ion affinity (IMAC) purification via the polyhistidine-tag and NiNTA, affinity purification via the StrepII-tag and StrepTactin agarose, and removal of the StrepII-SUMO part via SenP protease treatment. Cells were induced with IPTG at an OD 600 of 0.8 and incubated for further 14 h at 30 °C. StrepII-SUMO-scFv fusion protein is marked with one star (*), GFP-specific scFv is marked with two stars (**), and StrepII-SUMO is marked with three stars (***). Lane 1 and 2: culture supernatant concentrated by TCA precipitation before (B) and after (A) NiNTA-agarose treatment. Lane 3: sample of wash fraction from first IMAC (W); lane 4: sample of elution fraction from first IMAC (E); lane 5: sample of elution fraction from StrepII-tag affinity purification; lane 6: sample of elution fraction from StrepII-tag affinity purification incubated with SenP-protease for 1 h at 37 °C; lane 7: sample of flow-through fraction of second IMAC (F); lane 8: sample of elution fraction from second IMAC (E). For details, see “ ” section. b SDS-Page analysis of pull down assay using NiNTA magnetic agarose beads. GFP-specific scFv antibody fragments were bound to magnetic beads via the C-terminal polyhistidine tag (lanes are marked with a plus: +). Unloaded magnetic beads served as a control (marked with a minus: −). GFP was then added to the magnetic beads in excess as a purified native protein (lanes 3–6) or as a cleared lysate from E. coli cells overproducing GFP (lanes 7–10). As a negative control, no GFP was added (lanes 1 and 2). After 1 h incubation at 4 °C allowing interaction complex formation, the magnetic beads were removed, and a sample of each remaining supernatant (S) submitted to SDS-Page (lanes 1, 3, 5, 7 and 9). The beads were then washed and protein was eluted using 250 mM imidazole. The elution fractions (E) were submitted to SDS-Page (lanes 2, 4, 6, 8 and 10). GFP-specific scFv is marked with two stars (**)

Article Snippet: Blots were developed with polyclonal antibodies against E. coli alkaline phosphatase PhoA (OriGene Technologies, Inc.) at a dilution of 1:5000 and polyclonal secondary anti Rabbit-HRP antibodies (Carl Roth, #4750.1) according to the manufacturer’s specifications.

Techniques: Purification, Binding Assay, SDS Page, Affinity Purification, Incubation, TCA Precipitation, Pull Down Assay, Magnetic Beads, Control, Negative Control

Journal: Microbial Cell Factories

Article Title: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

doi: 10.1186/s12934-019-1078-0

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Blots were developed with polyclonal antibodies against E. coli alkaline phosphatase PhoA (OriGene Technologies, Inc.) at a dilution of 1:5000 and polyclonal secondary anti Rabbit-HRP antibodies (Carl Roth, #4750.1) according to the manufacturer’s specifications.

Techniques: Plasmid Preparation, Expressing, Purification, Clone Assay

Journal: Stem Cell Discovery

Article Title: Alkaline phosphatase-positive cells isolated from human hearts have mesenchymal stem cell characteristics

doi: 10.4236/scd.2011.13008

Figure Lengend Snippet: Figure 3. Purification of the ALPL+ population from auri- cle-derived cell cultures. Isotype control, TAD cells (and the ALPL+-enriched population after selection on micromagnetic beads and evalution by flow cytometry, a representative ex- periment (a) Markers for pericytes (b) Markers for endothe- lium (c) MSC-positive Markers (d) and MSC-negative mark- ers (e) Imunnofenotyping was evaluated in TAD (total au- ricle-derived cell population), white bars and in ALPL+ cells (tissue nonspecific alkaline phosphatase positive cell popula- tion), black bars. Data are expressed as medians and standard error (n = 3). Von Willebrand factor (vWF).

Article Snippet: For the selection of tissue nonspecific alkaline phosphatase-positive cells (ALPL+), the total auricle-derived cell population (TAD) was incubated with a biotinylated antibody directed against the human tissue nonspecific alkaline phosphatase (R&D Systems, USA).

Techniques: Purification, Derivative Assay, Control, Selection, Flow Cytometry

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of AS-IV on proliferation and osteogenic differentiation of PDLSCs. ( A ) CCK-8 analysis of AS-IV effects on PDLSCs on day 1, 3, and 5. ( B and C ) ALP activity ( B ) and extracellular matrix mineralization ( C ) quantification of PDLSCs affected by AS-IV. ( D ) ALP staining. Scale bar: 500 μm. ( E ) Alizarin Red staining. Scale bar: 500 μm. ( F – H ) qRT-PCR analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV. ( I – L ) Western blot and semi-quantitative analysis of COL-1 (J), ALP(K), and RUNX-2 ( L ) in PDLSCs cultured with AS-IV. 20 μM AS-IV group had a significant promoting effect. Data are presented as mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: CCK-8 Assay, Activity Assay, Staining, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of LY294002 on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and D ) ALP staining( A ) and ALP activity quantification( D ) of PDLSCs affected by LY294002. Scale bar: 500 μm. ( B and E ) Immunofluorescence staining( B ) of COL-1 (green) and DAPI staining (blue) and quantification(E). Scale bar: 50 μm. ( C and F ) Immunofluorescence staining( C ) of eNOS (green) and DAPI staining (blue) and quantification(F). Scale bar: 50 μm. ( G ) NO2- concentration represented the level of NO in cell supernatants. ( H–K ) Western blot and semi-quantitative analysis of COL-1(I), ALP(J), and RUNX-2 ( K ) in PDLSCs cultured with AS-IV and LY294002. ( L–O ) Western blot and semi-quantitative analysis of eNOS(M), iNOS( N ) and p-AKT/AKT ( O ) after addition of AS-IV at different time points. ( P–S ) Western blot and semi-quantitative analysis of eNOS(Q), iNOS( R ) and p-AKT/AKT ( S ) in PDLSCs cultured with AS-IV and LY294002. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Concentration Assay, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of L-NAME on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and B ) ALP staining( A ) and ALP activity quantification( B ) of PDLSCs affected by L-NAME. Scale bar: 500 μm. ( C and D ) Immunofluorescence staining( C ) of COL-1 (green) and DAPI staining (blue) and quantification(D). Scale bar: 50 μm. ( E–H ) Western blot and semi-quantitative analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV and L-NAME. ( I–K ) Western blot and semi-quantitative analysis of eNOS( J ) and p-AKT/AKT ( K ) in PDLSCs cultured with AS-IV and L-NAME. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ns: P> 0.05. Each experiment was repeated five times.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Hematoxylin and eosin staining and immunohistochemical staining. ( A ) HE staining of the tension-side periodontal tissues of the control group (A-a) and AS-IV group (A-b) after OTM for 14 days. Scale bar: 50 μm. ( B ) Expression of ALP of the control group (B-a) and AS-IV group (B-b) after OTM for 14 days and quantitative analysis (B-c). Scale bar: 20 μm. ( C ) Expression of COL-1 of the control group (C-a) and AS-IV group (C-b) after OTM for 14 days and quantitative analysis (C-c). Scale bar: 20 μm. ( D ) Expression of eNOS of the control group (D-a) and AS-IV group (D-b) after OTM for 14 days and quantitative analysis (D-c). Scale bar: 20 μm. Data are presented as mean ± SD (n=8 rat/group). * P <0.05, ** P <0.01.

Article Snippet: Incubate the membrane with primary antibodies toward target proteins including ALP (Proteintech, 11187-1-AP, 1:1000), RUNX-2 (ImmunoWay, YM8347, 1:2000), COL-1 (Proteintech, 67288-1-Ig, 1:5000), eNOS (Abways, AB3537, 1:500), iNOS (Abways, CY3425, 1:500), p-AKT (Abways, CY6569, 1:1000), AKT (Abways, CY5561, 1:1000) and GAPDH (Proteintech, 10494-1-AP, 1:5000) overnight at 4°C.

Techniques: Staining, Immunohistochemical staining, Control, Expressing