Journal: Stem Cells International

Article Title: Dot1l Regulates the Spontaneous Bone Regeneration of Periosteum-Derived Stem Cells by Regulating Chac1 Expression

doi: 10.1155/sci/1508850

Figure Lengend Snippet: Mandibular defect repair and periosteal PDSCs isolation: (A) animal experimental design, (B) surgical image of a rat model of jaw defect with periosteum preserved, (C) X-ray images of the defect area in rats at day 7 and 8 weeks later, (D) morphologic photographs of periosteum-derived stem cells in sham and model rats, (E) the proportions of CD44+, CD90+, CD31+, and CD45+ cells in PDSCs isolated from the jaws of sham-operated rats were analyzed using flow cytometry, and (F) the proportion of CD44+, CD90+, CD31+, and CD45+ cells in PDSCs isolated from jaws of model rats were analyzed using flow cytometry.

Article Snippet: For the identification of PDSCs cells, the cells were washed and incubated with CD44 antibody (15675-1-AP, Proteintech), CD90 antibody (66766-1-Ig, Proteintech), CD3 antibody (17617-1-AP, Proteintech), and CD45 antibody (20103-1-AP, Proteintech) to label PDSCs cells (CD44+CD90+CD3-CD45-).

Techniques: Isolation, Derivative Assay, Flow Cytometry

Journal: Stem Cells International

Article Title: Dot1l Regulates the Spontaneous Bone Regeneration of Periosteum-Derived Stem Cells by Regulating Chac1 Expression

doi: 10.1155/sci/1508850

Figure Lengend Snippet: Expression pattern of Dot1l during mandibular defect healing, and Dot1l is important for the osteogenesis of PDSCs. (A) The volcano plot showed the differential genes in PDSCs isolated from the sham operation group and the periosteum-preserved jaw defect model group. (B) GO functional enrichment analysis showing the top 30 significantly enriched biological functions and roles. (C) The mRNA expression level of differential genes was detected using RT-qPCR assay, data were analyzed by using one-way ANOVA, with the Tukey's post hoc test. ⁣ ∗∗∗∗ p < 0.001. (D) The protein expression level of Dot1l was detected using western blotting assay. (E) The proportion of CD44, CD90, CD31, and CD45 positive expression cells in PDSCs from 4-week-old SD rats was analyzed by flow cytometry. (F) ARS staining was used to detect calcium deposition in PDSCs cells, ALP staining was used to detect ALP activity of PDSCs cells, oil red O staining was used to detect the lipogenic ability of PDSCs cells. (G) Detection of the Dot1l mRNA in PDSCs treated with Dot1l ctrl/overexpression via RT-qPCR assay, data were analyzed by t -test analysis, ⁣ ∗∗∗ p < 0.001. (H) Detection of the Dot1l mRNA in PDSCs treated with Dot1l ctrl/overexpression via western blot assay. (I) Detection of the mRNA expressions of osteogenic differentiation related genes in PDSCs treated with Dot1l ctrl/overexpression via RT-qPCR assay, data were analyzed by using one-way ANOVA, with the Tukey's post hoc test. ⁣ ∗∗∗∗ p < 0.001. (J) ALP staining was used to detect ALP activity of PDSCs cells treated with Dot1l ctrl/overexpression. (K) ARS staining was used to detect calcium deposition of PDSCs cells treated with Dot1l ctrl/overexpression.

Article Snippet: For the identification of PDSCs cells, the cells were washed and incubated with CD44 antibody (15675-1-AP, Proteintech), CD90 antibody (66766-1-Ig, Proteintech), CD3 antibody (17617-1-AP, Proteintech), and CD45 antibody (20103-1-AP, Proteintech) to label PDSCs cells (CD44+CD90+CD3-CD45-).

Techniques: Expressing, Isolation, Functional Assay, Quantitative RT-PCR, Western Blot, Flow Cytometry, Staining, Activity Assay, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: Deciphering the CAF-LCN2 axis: Key to overcoming anti-PD-L1 immunotherapy resistance in lung cancer

doi: 10.3892/ijmm.2026.5735

Figure Lengend Snippet: Influence of LCN2 on the growth of lung cancer in mice. (A) Simplified flowchart outlining the establishment of an anti-PD-L1 immunotherapy tolerance model in mice with lung cancer. (B) Western blot analysis of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice. (C) Immunohistochemical detection of LCN2, COL1A1 and THY1 expression in tumor tissues from drug-resistant and drug-sensitive mice (scale bar, 100 µm). (D) Establishment of a model for anti-PD-L1 immunotherapy in LCN2-silenced mice. (E) Evaluation of LCN2 mRNA expression in tumor tissues after LCN2 gene intervention in mice. (F) Assessment of LCN2 protein expression in tumor tissues following LCN2 gene intervention in mice. (G) Tumor size in LCN2 gene intervention mice (n=10 in each group). *P<0.05, **P<0.01 and ***P<0.001. Unpaired two-tailed Student's t-test was used for (B) and (C) and one-way ANOVA with Tukey's multiple comparisons test was used for (E) and (G). LCN2, lipocalin 2; PD-L1, programmed death-ligand 1; sh-, short hairpin; NC, negative control; COL1A1, collagen α-1(I) chain; THY1, Thy-1 membrane glycoprotein; i.p., intraperitoneal.

Article Snippet: The following primary antibodies were used: Rabbit anti-LCN2 (cat. no. ab125075; 1:100; Abcam), rabbit anti-COL1A1 (cat. no ab34710; 1:15; Abcam), rabbit anti-Thy-1 membrane glycoprotein (THY1) (cat. no 13801; 1:1,000; Cell Signaling Technology, Inc.), rabbit anti-E-cadherin (cat. no 3195; 1:400; Cell Signaling Technology, Inc.), rabbit anti-Vimentin (cat. no. 5741; 1:4,000; Cell Signaling Technology, Inc.), rabbit anti-IL-6 (cat. no. ab6672; 1:400; Abcam), rabbit anti-TNF-α (cat. no. ab307164; 1:1,000; Abcam), rabbit anti-Ki-67 (cat. no. ab16667; 1:200; Abcam) and rabbit anti-cleaved caspase-3 A (cat. no. 9661; 1:400; Cell Signaling Technology, Inc.).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Two Tailed Test, Negative Control, Membrane

Journal: International Journal of Oncology

Article Title: The regulatory effect of the YY1/miR-HCC2/BAMBI axis on the stemness of liver cancer cells

doi: 10.3892/ijo.2023.5507

Figure Lengend Snippet: MiR-HCC2 is upregulated in liver cancer tissues and promotes stem cell-like properties in human liver cancer cells. (A) Sequencing reads and statistics of five representative miRNAs identified by deep sequencing. (B) Relative levels of miR-HCC2 in liver cancer tissues and adjacent non-tumor tissues (n=13). U6 RNA was used for normalization. Relative (C) mRNA and (D) protein expression levels of stemness markers in transiently transfected HepG2 and Huh7 cells were assessed by RT-qPCR and western blotting assays, respectively. The arrows indicated nonspecific bands. (E) The mRNA and protein expression levels of miR-HCC2 and stemness markers in stable miR-HCC2-overexpressing Huh7 cells. The samples were derived from the same experiment and the blots were processed in parallel. (F) Tumor sphere-formation assays indicated an enhanced sphere formation ability in stable miR-HCC2-overexpressing Huh7 cells. Scale bar=100 µ m. (G) Evaluation of the proportion of CD90 + cells among transfected Huh7 cells. All experiments were performed at least in triplicate. Data are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 compared with the corresponding control group (miR-HCC2 vs. pcDNA3, ASO-miR-HCC2 vs. ASO-NC and stable miR-HCC2 vs. pcDNA3). miR, microRNA; Nanog, nanog homeobox; Sox2, sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4; CD90, cluster of differentiation 90; EpCAM, epithelial cellular adhesion molecule; ASO, antisense oligonucleotide; NC, negative control.

Article Snippet: Cells were incubated in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min, then incubated with fluorescein isothiocyanate-conjugated CD90 antibody (1 μ g/test; cat. no. FC01818; Boster Biological Technology) at room temperature in the dark for 30 min.

Techniques: Sequencing, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Derivative Assay, Standard Deviation, Control, Binding Assay, Negative Control

Journal: International Journal of Oncology

Article Title: The regulatory effect of the YY1/miR-HCC2/BAMBI axis on the stemness of liver cancer cells

doi: 10.3892/ijo.2023.5507

Figure Lengend Snippet: MiR-HCC2 enhanced the proliferation, metastasis and stemness of liver cancer cells in vivo . A nude mouse tumor xenograft model was used to evaluate the effects of miR-HCC2 on tumor growth and tumor metastasis in vivo . (A) Tumors were isolated from subcutaneously injected mice. The pcDNA3 vector and scrambled miRNA were used as control vector and control miRNA. (B) Tumor weights were quantified after tumors had been isolated from mice. (C) Tumor volumes were assessed after injection. (D) The RNA expression levels of miR-HCC2, BAMBI and stemness markers in mouse tumor nodules. (E) H&E staining and immunohistochemical staining of Sox2 in xenograft tumors from mice that had been injected with Huh7 cells. Scale bar=50 µ m. (F) Gross anatomical assessments of lung and liver tissues isolated from intravenously injected mice. Tumor nodules in histological sections of lung and liver tissues were counted. (G) H&E staining and immunohistochemical staining of Sox2 in lungs and livers. Scale bar=50 µ m. (H) RNA expression levels of miR-HCC2, BAMBI and stemness markers in lungs and livers. Scale bars=50 µ m. All results were compared with corresponding control groups. Data are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001. miR, miRNA; miRNA, micro RNA; Nanog, nanog homeobox; Sox2, sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4; CD90, cluster of differentiation 90; EpCAM, epithelial cellular adhesion molecule; AFP, α-fetoprotein; BAMBI, bone morphogenic protein and activin membrane-bound inhibitor homolog; H&E, hematoxylin and eosin.

Article Snippet: Cells were incubated in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min, then incubated with fluorescein isothiocyanate-conjugated CD90 antibody (1 μ g/test; cat. no. FC01818; Boster Biological Technology) at room temperature in the dark for 30 min.

Techniques: In Vivo, Isolation, Injection, Plasmid Preparation, Control, RNA Expression, Staining, Immunohistochemical staining, Standard Deviation, Binding Assay, Membrane

Journal: International Journal of Oncology

Article Title: The regulatory effect of the YY1/miR-HCC2/BAMBI axis on the stemness of liver cancer cells

doi: 10.3892/ijo.2023.5507

Figure Lengend Snippet: BAMBI promotes stem cell-like properties in human liver cancer cells. The (A and B) mRNA and (C and D) protein expression levels of BAMBI and stemness markers in transfected liver cancer cells were assessed using reverse transcription-quantitative PCR and western blotting assays, respectively. The samples were derived from the same experiment and the blots were processed in parallel. The arrows indicated nonspecific bands. (E) Spheroid formation assays were used to evaluate the impact of BAMBI on tumor sphere-formation ability. Scale bar=100 µ m. (F) The proportion of CD90 + cells in liver cancer cells was assessed using flow cytometry analysis. All experiments were performed at least in triplicate. Data are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 compared with the corresponding control group (BAMBI vs. pcDNA3 and shR-BAMBI vs. pSilencer-NC). Nanog, nanog homeobox; Sox2, sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4; CD90, cluster of differentiation 90; EpCAM, epithelial cellular adhesion molecule; shR, short hairpin RNA; BAMBI, bone morphogenic protein and activin membrane-bound inhibitor homolog; NC, negative control.

Article Snippet: Cells were incubated in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min, then incubated with fluorescein isothiocyanate-conjugated CD90 antibody (1 μ g/test; cat. no. FC01818; Boster Biological Technology) at room temperature in the dark for 30 min.

Techniques: Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay, Flow Cytometry, Standard Deviation, Control, Binding Assay, shRNA, Membrane, Negative Control

Journal: International Journal of Oncology

Article Title: The regulatory effect of the YY1/miR-HCC2/BAMBI axis on the stemness of liver cancer cells

doi: 10.3892/ijo.2023.5507

Figure Lengend Snippet: Suppression of BAMBI expression abrogates the effects of miR-HCC2 on stem cell-like properties in human liver cancer cells. The (A and B) mRNA and (C and D) protein expression levels of stemness markers in HepG2 and Huh7 cells were determined after shR-BAMBI transfection in the presence of miR-HCC2. The samples were derived from the same experiment and the blots were processed in parallel. The arrows indicated nonspecific bands. (E) The miR-HCC2-mediated increase in the tumor sphere-forming ability was abrogated by suppression of BAMBI. Scale bar=100 µ m. (F) The miR-HCC2-mediated increase in the proportion of CD90 + cells was reduced by suppression of BAMBI. All experiments were performed at least in triplicate. Data are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 compared with the corresponding control group (miR-HCC2 + pSilencer-NC vs. pcDNA3 + pSilencer-NC and miR-HCC2 + shR-BAMBI vs. pcDNA3 + pSilencer-NC). Nanog, nanog homeobox; Sox2, sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4; CD90, cluster of differentiation 90; EpCAM, epithelial cellular adhesion molecule; shR short hairpin RNA; BAMBI, bone morphogenic protein and activin membrane-bound inhibitor homolog; NC, negative control; miR, microRNA; ns, not significant.

Article Snippet: Cells were incubated in phosphate-buffered saline (PBS) containing 2% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 30 min, then incubated with fluorescein isothiocyanate-conjugated CD90 antibody (1 μ g/test; cat. no. FC01818; Boster Biological Technology) at room temperature in the dark for 30 min.

Techniques: Expressing, Transfection, Derivative Assay, Standard Deviation, Control, Binding Assay, shRNA, Membrane, Negative Control

Journal: PLoS ONE

Article Title: The effect of S53P4-based borosilicate glasses and glass dissolution products on the osteogenic commitment of human adipose stem cells

doi: 10.1371/journal.pone.0202740

Figure Lengend Snippet: The surface marker expression of undifferentiated hASCs at passage 1, cultured in BM.

Article Snippet: CD90 , Thy-1 (T cell surface glycoprotein) , 98.9 ± 1.2 , APC , BD Biosciences.

Techniques: Marker, Expressing, Cell Culture, Binding Assay, Activation Assay