Journal: Stem cells (Dayton, Ohio)

Article Title: CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

doi: 10.1002/stem.2063

Figure Lengend Snippet: Figure 1. CD49f is a stemness marker for BMSCs and sensitive to environmental changes. (A): Phase-contrast images of BMSCs from fetal and adult bone marrow and fluorescence-activated cell sorting (FACS) analysis of the surface markers profiles including CD44, CD73, CD90, CD105, cocktails of the hematopoietic lineage markers (CD45, CD34, CD11b, CD19, and HLA-DR), and CD49f. Scale bar 5 200 mm. (B): FACS analysis of CD49f-positive cells (left) and qRT-PCR of CD49f expression in different cell types (right). The data are presented as the mean- 6 SEM from no less than three donors (except for hESCs from the H1 and H9 cell lines) with independent experiments. **, p < .01; ***, p < .001 (two-tailed t test). (C): Serial passages of fetal BMSCs resulted in the loss of CD49f during long-term culture. (D): Changes in CD49f expression in cultured, FACS sorted CD49f1, and CD49f2 BMSCs. (E): CFU-F assay of sorted CD49f1 and CD49f2 BMSCs. (F): Oil red O staining and alizarin red S staining after adipogenic and osteogenic differentiation of CD49f1 and CD49f2 BMSCs. The positive stain- ing areas were quantified and statistically analyzed. Scale bar 5 400 mm. Data are presented as the mean 6 SEM from independent experi- ments. *, p < .05; **, p < .01. Abbreviations: AEC, amnion epithelial cells; F-BMSC/A-BMSC, mesenchymal stem cells from fetal/adult bone marrow; F-Fibro/A-Fibro, fibroblasts from fetal/adult skin; hESC, human embryonic stem cells.

Article Snippet: For coimmunoprecipitation, Nonidet P40 lysis buffer (Beyotime) was used and cell lysates were incubated overnight with an anti-CD49f antibody (GoH3, Novus Biologicals) or nonspecific rat IgG control.

Techniques: Marker, FACS, Quantitative RT-PCR, Expressing, Two Tailed Test, Cell Culture, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

doi: 10.1002/stem.2063

Figure Lengend Snippet: Figure 2. CD49f is required for the differentiation of bone marrow mesenchymal stem cells (BMSCs). (A): Knockdown of CD49f reduced the differentiation potential of BMSCs. qRT-PCR and Western blot showed that CD49f expression was reduced after CD49f shRNA infec- tion. A scrambled sequence was used as a control (i). Oil red O and alizarin red S staining for adipo- and osteo-differentiation of CD49f knockdown BMSCs (ii). (B): qRT-PCR, fluorescence-activated cell sorting, and Western blot confirmed that CD49f isoforms A and B were specifically overexpressed in BMSCs; LV-GFP was used as a control. (C): CD49f isoforms or a LV-GFP control was overexpressed in BMSCs and the differentiation ability of the specific isoforms was compared and quantified. Scale bar 5 500 mm. All data were presented as the mean 6 SEM. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: LV-GFP, green fluorescent protein control lentivirus; LV-ISO A, len- tivirus containing CD49f isoform A; LV-ISO B, lentivirus containing CD49f isoform B; shCD49f, CD49f shRNA; shScram, scramble shRNA.

Article Snippet: For coimmunoprecipitation, Nonidet P40 lysis buffer (Beyotime) was used and cell lysates were incubated overnight with an anti-CD49f antibody (GoH3, Novus Biologicals) or nonspecific rat IgG control.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Expressing, shRNA, Sequencing, Control, Staining, FACS

Journal: Stem cells (Dayton, Ohio)

Article Title: CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

doi: 10.1002/stem.2063

Figure Lengend Snippet: Figure 3. CD49f expression is downregulated by TNF-a with impaired differentiation of bone marrow mesenchymal stem cells (BMSCs). (A): Fluorescence-activated cell sorting analysis of CD49f and CD106 on BMSC treated with 40 ng/ml TNF-a or 10 ng/ml TGF-b1 for 48 hours. The effects of TNF-a on CD49f and CD106 were verified with two different concentrations of TNF-a. The percentage of CD49f or CD106-positive cells after TNF-a treatment was normalized to the control group. The data are presented as the mean 6 SEM, *, p < .05. (B): qRT-PCR (left) and conventional RT-PCR (right) for CD49f isoform expression in BMSCs treated with different concentrations of TNF- a for a series of time points. The data are presented as the mean 6 SEM, *, p < .05. (C): BMSCs were pretreated with TNF-a at 10 and 40 ng/ml for 48 hours to downregulate CD49f expression and then subjected to adipo- and osteo-induction. Oil red O staining and aliza- rin red S staining are shown and the positive staining areas have been quantified. Scale bar 5 400 mm. Data are presented as the mean- 6 SEM, *, p < .05; **, p < .01. (D): Expression of key genes during adipogenesis and osteogenesis of TNF-a-treated BMSCs was detected by qRT-PCR. The data are presented as the mean 6 SEM, *, p < .05; **, p < .01. Abbreviations: CTL, control, means normal cultured cells; T10, 10 ng/ml TNF-a; T40, 40 ng/ml TNF-a.

Article Snippet: For coimmunoprecipitation, Nonidet P40 lysis buffer (Beyotime) was used and cell lysates were incubated overnight with an anti-CD49f antibody (GoH3, Novus Biologicals) or nonspecific rat IgG control.

Techniques: Expressing, Fluorescence, FACS, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Staining, Cell Culture

Journal: Stem cells (Dayton, Ohio)

Article Title: CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

doi: 10.1002/stem.2063

Figure Lengend Snippet: Figure 4. CD49f mediates the decreased adhesion of bone mar- row mesenchymal stem cells (BMSCs) to laminins after TNF-a treatment. (A): The adhesion of BMSCs to laminin 511 or 521 in a precoated 96-well plate. BSA was used as a control. 4 3 104

Article Snippet: For coimmunoprecipitation, Nonidet P40 lysis buffer (Beyotime) was used and cell lysates were incubated overnight with an anti-CD49f antibody (GoH3, Novus Biologicals) or nonspecific rat IgG control.

Techniques: Control

Journal: Stem cells (Dayton, Ohio)

Article Title: CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

doi: 10.1002/stem.2063

Figure Lengend Snippet: Figure 5. CD49f works together with TG2 to increase the migration of bone marrow mesenchymal stem cells (BMSCs) after TNF-a stim- ulation. (A): Transwell migrated BMSCs pretreated with 10 and 40 ng/ml TNF-a were stained with crystal violet and then photographed and counted. The bars represent the mean 6 SEM from at least five fields under the microscope. Scale bar 5 200 mm. *, p < .05; ***, p < .001. (B): Coimmunoprecipitation of CD49f and TG2 within BMSCs overexpressing specific CD49f isoforms (i) and normal cultured hESC and AEC cells (ii). Confocal microscopy images showed the colocalization of CD49f and TG2 on the membrane of AEC, scale bar 5 50 mm (iii). (C): qRT-PCR and Western blot results of TG2 expression in BMSCs treated with TNF-a at 10 and 40 ng/ml for 48 hours. (D): Fluorescence-activated cell sorting analysis of integrin b1 and b4 on TNF-a-treated BMSCs. Abbreviations: AEC, amnion epi- thelial cells; CD49f iso A-MSC, CD49f isoform A overexpressed fetal BMSCs; CD49f iso B-MSC, CD49f isoform B overexpressed fetal BMSCs; CTL, control, means normal cultured cells; hESC, human embryonic stem cells; T10, 10 ng/ml TNF-a; T40, 40 ng/ml TNF-a.

Article Snippet: For coimmunoprecipitation, Nonidet P40 lysis buffer (Beyotime) was used and cell lysates were incubated overnight with an anti-CD49f antibody (GoH3, Novus Biologicals) or nonspecific rat IgG control.

Techniques: Migration, Staining, Microscopy, Cell Culture, Confocal Microscopy, Membrane, Quantitative RT-PCR, Western Blot, Expressing, Fluorescence, FACS, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

doi: 10.1002/stem.2063

Figure Lengend Snippet: Figure 6. TNF-a activates mTOR rather than the NF-jB pathway to mediate CD49f downregulation in bone marrow mesenchymal stem cells (BMSCs). (A): TNF-a induced IKK phosphorylation and IjBa degradation in BMSCs. Cells were serum starved for 12 hours before stimu- lation with 10 ng/ml TNF-a for a series of time points. The data are expressed as the integral optical density (IOD) of phospho-IKKa/b and normalized against total IKKb and the IOD of IjBa against b-actin as the mean 6 SEM of three independent experiments, *, p < .05; **, p < .01; ***, p < .001 versus control group (i). The NF-jB inhibitory peptide SN50 suppressed p65 nuclear translocation induced by TNF-a. SM50 was used as a control. BMSCs were pretreated with 50 mg/ml SN50 or SM50 for 30 minutes and then stimulated with 10 ng/ml TNF- a for 30 minutes (ii). (B): mTOR and its target proteins pS6K1 and AKT were significantly phosphorylated after TNF-a stimulation. Cells were serum starved for 12 hours before stimulation with 10 ng/ml TNF-a in a series of time points. The data are expressed as the IOD of the tar- get phosphorylated proteins normalized against the total target proteins as the mean 6 SEM of three independent experiments, *, p < .05; **, p < .01; ***, p < .001 versus control group (i). Immunocytochemistry stainings for p-mTOR(S2448) and mTOR after TNF-a treatment and further inhibition by rapamycin and PP242, scale bar 5 100 mm (ii). Rapamycin and PP242 were also shown to inhibit the TNF-a- induced activation of pS6K1 and AKT (iii). The cells were serum starved for 12 hours before treatment with 100 nM rapamycin or 0.5 mM PP242 for another 24 hours (DMSO as a control) and then induced with 10 ng/ml TNF-a for 1 hour. (C): Fluorescence-activated cell sorting analysis of CD49f on BMSCs treated with 10 ng/ml TNF-a with or without pretreatment of rapamycin, PP242, SN50, and SM50. The times for pretreatment are consistent with those shown in panel (B). (D): The effects of rapamycin and PP242 on CD49f expression on BMSCs during passaging. Abbreviations: CTL, control, normal cultured cells; PP, PP242; Rapa, rapamycin; TNF, TNF-a.

Article Snippet: For coimmunoprecipitation, Nonidet P40 lysis buffer (Beyotime) was used and cell lysates were incubated overnight with an anti-CD49f antibody (GoH3, Novus Biologicals) or nonspecific rat IgG control.

Techniques: Phospho-proteomics, Control, Translocation Assay, Immunocytochemistry, Inhibition, Activation Assay, Fluorescence, FACS, Expressing, Passaging, Cell Culture

Journal: Cell reports

Article Title: 2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity

doi: 10.1016/j.celrep.2021.110220

Figure Lengend Snippet:

Article Snippet: Rat monoclonal anti-CD49F (Clone GoH3) , Fluidigm , Cat# 3164006B.

Techniques: Recombinant, Control, Blocking Assay, Suspension, Electron Microscopy, Purification, Whole Genome Amplification, SYBR Green Assay, Labeling, Staining, Methylation Sequencing, Methylated DNA Immunoprecipitation, Biomarker Discovery, Software, Pyromark Assay

Journal: Scientific Reports

Article Title: The secretome from bovine mammosphere-derived cells (MDC) promotes angiogenesis, epithelial cell migration, and contains factors associated with defense and immunity

doi: 10.1038/s41598-018-23770-z

Figure Lengend Snippet: AFDC and MDC are distinct populations. ( a ) Immunofluorescence staining of AFDC and MDC, demonstrating expression of the mammary cell markers vimentin (expressed by both populations), αSMA, CK14 (expressed only in AFDC), ERα and CK18 (expressed only in MDC). Bars = 50 µm. ( b ) Flow cytometry was performed with three mammary cell surface markers: CD29, CD44 and CD49f. The percentage of positive cells was quantified compared to a relevant isotype control. ( c ) Immunofluorescence of acinar structures expressing β-lactoglobulin (green). Inset: enlargement of representative acinar structure. Nuclei were stained with DAPI (blue). Bar = 100 µm; * p < 0.05, *** p < 0.001.

Article Snippet: Cells were collected using accutase (Innovative Cell Technologies, San Diego, CA) and stained with mouse monoclonal TDM29 anti-CD29 (diluted 1:50; Sigma-Aldrich), rat monoclonal IM7 anti-CD44 (diluted 1:20; ThermoFisher Scientific), or rat monoclonal GoH3 anti-CD49f (diluted 1:10; BD Biosciences, San Jose, CA) antibodies diluted in PBS with 1% BSA, for 1 h. Staining with an isotype control antibody (Abcam), diluted 1:10 in PBS with 1% BSA, or no antibody stain were included as controls.

Techniques: Immunofluorescence, Staining, Expressing, Flow Cytometry

Journal: Carcinogenesis

Article Title: Synthetic D-amino acid peptide inhibits tumor cell motility on laminin-5

doi: 10.1093/carcin/bgl005

Figure Lengend Snippet: HYD1 inhibits laminin-5-dependent cellular invasion. (A) Concentration-dependent inhibition of PC3N cell invasion toward laminin-5. Cells were added into the upper chamber of laminin-5-coated transwells for 1 h prior to treatment with 75, 100, 250 or 500 (μg/ml) HYD1 or HYDS in serum-free media as described in Materials and methods. (B) Comparison of HYD1 activity to other known inhibitors of invasion. Cells were added as in (A) and followed by treatment with 250 μg/ml HYD1 or HYDS in serum-free media. The Rho-kinase inhibitor, Y-27632, was used at a concentration of 25 μM in serum-free media. Integrin function-blocking antibodies P1B5 (α3), GoH3 (α6) and AIIB2 (β1) were used at a concentration of 10 μg/ml and were incubated with cells for 15 min at room temperature prior to adding the cells into transwells. Error bars are standard deviation from the mean. (C) Laminin subchain composition of laminin-5 matrix material. The concentrated conditioned media from HaCaT cells were analyzed by 6% PAGE, transferred to PVDF membranes and probed for the α3 chain (BM165, mouse monoclonal antibody), the β3 chain (BM140, mouse monoclonal antibody) or the γ2 chain (GB3, goat monoclonal antibody).

Article Snippet: Antibodies and reagents GoH3, a rat anti-α6 functional-blocking antibody was purchased from Serotech (Raleigh, NC).

Techniques: Concentration Assay, Inhibition, Comparison, Activity Assay, Blocking Assay, Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

doi: 10.1371/journal.pone.0030113

Figure Lengend Snippet: List of antibodies used in the study.

Article Snippet: CD49f , FITC-conjugated, rat monoclonal, clone GoH3 , StemCell Technologies , 1∶20 , Not applied , Not applied , Not applied.

Techniques: Transduction, Labeling, Polymer