Journal: Bone & Joint Research

Article Title: Down-expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenesis and accelerates age-related bone loss via PTEN/PI3K/AKT pathway

doi: 10.1302/2046-3758.132.BJR-2023-0146.R2

Figure Lengend Snippet: Effects of senescent MLO-Y4 cell-derived exosomes (abbreviated tert-Butyl hydroperoxide (TBHP)-exos in the figure) on osteogenic differentiation of MC3T3-E1 cells. a) Cell Counting Kit 8 (CCK-8) assay of MC3T3-E1 cells treated with different concentrations (20, 100, or 300 μg/ml) of senescent MLO-Y4 cell-derived exosomes for different lengths of time (0, 24, 48, or 72 hours). b) Alkaline phosphatase (ALP) and Alizarin Red S staining of MC3T3-E1 cells on days 7, 14, and 21 after treatment with the indicated concentrations of exosomes obtained from senescent MLO-Y4 cells. c) and d) Western blotting and grayscale analysis of ALP, Runt-related transcription factor 2 (Runx2), and collagen type I α1 (Col-I) expression in MC3T3-E1 cells in different treatment groups. Data are shown as column charts and the p-values were calculated by one-way analysis of variance with Sidak’s multiple comparison tests. The p-values were specified only when p < 0.05 (statistically significant). GD, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The primary antibodies used were P16 (1:1000, ab51243; Abcam, USA), Runt-related transcription factor 2 (Runx2, 1:1,000, 12,556 S; Cell Signaling Technology), collagen type I α1 (Col-I, 1:1,000, 72,026 T; Cell Signaling Technology), ALP antibody (1:1,000, AF2910; R&D Systems, USA), AKT (1:1,000, 60203-2-Ig; Proteintech, USA), phosphorylated (p)-AKT (1:1,000, 66444-1-Ig; Proteintech), PI3K (1:1,000, 20584-1-AP; Proteintech), PTEN (1:1,000, 22034-1-AP; Proteintech), CD9 (1:1,000, 60232-1-Ig; Proteintech), and CD63 (1:1,000, 67605-1-Ig; Proteintech), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000, 60004-1-Ig; Proteintech) was used as a loading control.

Techniques: Derivative Assay, Cell Counting, CCK-8 Assay, Staining, Western Blot, Expressing, Comparison

Journal: Bone & Joint Research

Article Title: Down-expression of miR-494-3p in senescent osteocyte-derived exosomes inhibits osteogenesis and accelerates age-related bone loss via PTEN/PI3K/AKT pathway

doi: 10.1302/2046-3758.132.BJR-2023-0146.R2

Figure Lengend Snippet: miR-494-3p in MLO-Y4 cell-derived exosomes regulated osteogenic differentiation of MC3T3-E1 cells via the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/AKT pathway. a) Alkaline phosphatase (ALP) and Alizarin Red S staining of MC3T3-E1 cells on day 21 in different treatment groups. The magnification is 10× (lower part). b) and c) Western blotting and grayscale analysis of ALP, Runt-related transcription factor 2 (Runx2), collagen type I α1 (Col-I), p-AKT, AKT, PI3K, and PTEN expression in MC3T3-E1 cells in different treatment groups. Data are shown as column charts and the p-values were calculated by one-way analysis of variance with Sidak’s multiple comparison tests. The p-values were specified only when p < 0.05 (statistically significant). GD, glyceraldehyde 3-phosphate dehydrogenase; miR, microRNA; TBHP, tert-Butyl hydroperoxide.

Article Snippet: The primary antibodies used were P16 (1:1000, ab51243; Abcam, USA), Runt-related transcription factor 2 (Runx2, 1:1,000, 12,556 S; Cell Signaling Technology), collagen type I α1 (Col-I, 1:1,000, 72,026 T; Cell Signaling Technology), ALP antibody (1:1,000, AF2910; R&D Systems, USA), AKT (1:1,000, 60203-2-Ig; Proteintech, USA), phosphorylated (p)-AKT (1:1,000, 66444-1-Ig; Proteintech), PI3K (1:1,000, 20584-1-AP; Proteintech), PTEN (1:1,000, 22034-1-AP; Proteintech), CD9 (1:1,000, 60232-1-Ig; Proteintech), and CD63 (1:1,000, 67605-1-Ig; Proteintech), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000, 60004-1-Ig; Proteintech) was used as a loading control.

Techniques: Derivative Assay, Staining, Western Blot, Expressing, Comparison

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: 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: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 2. The C-terminal region 99– 192 of HCV E1 protein determines lo- calization in the ER of the reporter CD8. FRT cells stably expressing CD8 (a–b) or CD8-E199–192 (c–f) were analyzed by double indirect immunofluorescence microscopy as detailed under “Experi- mental Procedures” and “Results.” a, c, e, and f, permeabilized cells; b and d, not permeabilized cells. a, c, and e, anti-CD8 mAb OKT8; b and d, polyclonal antibody anti-CD8; f, polyclonal antibody anti- SSRa; a, c, and e, Texas Red-conjugated anti-mouse secondary antibody; b, d, and f, fluorescein-conjugated anti-rabbit sec- ondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 6. The juxtamembrane region 99–142 of the ectodomain of HCV E1 protein contains another determi- nant for ER localization. FRT cells sta- bly expressing CD8-E199–142 (a–d) and CD8-SV (e–f) were analyzed by double in- direct immunofluorescence microscopy as detailed under “Experimental Proce- dures” and “Results.” a, c–e, permeabi- lized cells; b and f, non-permeabilized cells. a, c, and e, anti-CD8 OKT8 mAb; b and f, polyclonal antibody anti-CD8; d, polyclonal antibody anti-calnexin; a, c, and e, Texas Red-conjugated anti-mouse secondary antibody; b, d, and f, fluores- cein-conjugated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 5. The TMD region of HCV E1 protein determines localization in the ER of the reporter CD8. FRT cells stably expressing CD8-E1155–192 were analyzed by double indirect immunofluorescence microscopy as detailed under “Experimental Procedures” and in the Results. a, c, and d, permeabilized cells; b, non-permeabilized cells. a and c, anti-CD8 mAb OKT8; b, polyclonal antibody anti-CD8; d, polyclonal antibody anti-calnexin; a and c, Texas red-conjugated anti-mouse secondary antibody; b and d, fluorescein-conjugated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 9. CD8-E199–142M protein is ex- pressed on the plasma membrane. HuH-7 cells were transfected with plas- mids expressing CD8-SV (a and b), CD8- E199–142 (c and d), and CD8-E199–142M (e and f) proteins. 36 h post-transfection, the cells were analyzed by single (a and b) and double (c and f) indirect immunoflu- orescence microscopy as detailed under “Experimental Procedures.” a, c, and e, permeabilized cells; b, d, and f, not per- meabilized cells; a–c and e, anti-CD8 mAb OKT8, Texas Red-conjugated anti-mouse secondary antibody; d and f, anti-CD8 polyclonal antibody, fluorescein-conju- gated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 12. The juxtamembrane region 99–142 of the ectodomain of HCV E1 determines intracellular localization of the AP protein. HuH-7 cells were transfected with plasmids expressing AP8 (a and b), AP8-SV (c and d), AP8- E199–142 (e and f), and AP8-E199–142M (g and h) proteins. 36 h post-transfection, the cells were analyzed by double indirect immunofluorescence microscopy as de- tailed under “Experimental Procedures.” a, c, e, and g, permeabilized cells, anti-AP mAb, Texas red-conjugated anti-mouse secondary antibody; b, d, f, and h, not permeabilized cells, anti-AP polyclonal antibody, fluorescein-conjugated anti- rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy