Journal: iScience

Article Title: The hypoxia-inducible factor EPAS1 is required for spermatogonial stem cell function in regenerative conditions

doi: 10.1016/j.isci.2023.108424

Figure Lengend Snippet:

Article Snippet: Rat mAB to CD49f, Biotin-conjugated , Miltenyi , 130-097-243; RRID: AB_2658557.

Techniques: Control, Positive Control, Software

Journal: European Journal of Medical Research

Article Title: RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells

doi: 10.1186/2047-783X-18-52

Figure Lengend Snippet: HepG2 and Bel-7402 cells were infected with integrin α6 short hairpin RNA (shRNA) plasmids and control shRNA plasmids respectively. Stable transfectant clones were picked up by puromycin. Integrin α6 expression levels remarkably reduced. (A) Western blot assays yielded the expression level of integrin α6 on protein level in HepG2 and Bel-7402 cell lines. (B) The qRT-PCR assays showed the expression level of integrin α6 on mRNA level in HepG2 and Bel-7402 cell lines. * P < 0.05; *** P < 0.001 compared with control group.

Article Snippet: Integrin α6 shRNA plasmids (sc-43129-sh) were constructed and synthesized by Santa Cruz Biotechnology, Inc., CA, USA.

Techniques: Infection, shRNA, Control, Transfection, Clone Assay, Expressing, Western Blot, Quantitative RT-PCR

Journal: European Journal of Medical Research

Article Title: RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells

doi: 10.1186/2047-783X-18-52

Figure Lengend Snippet: Cells were incubated in 96-well plates coated with laminin (LN) or fibronectin (FN) respectively for 48 hours. The MTT assays showed that the lower-level expression of integrin α6 caused decreased proliferation of HCC cells with significant statistical difference ( P < 0.05) in the cells incubated on LN, while the cells incubated on FN did not show any statistically significant effect ( P > 0.05). * P < 0.05, **** P < 0.0001 compared with control group.

Article Snippet: Integrin α6 shRNA plasmids (sc-43129-sh) were constructed and synthesized by Santa Cruz Biotechnology, Inc., CA, USA.

Techniques: Incubation, Expressing, Control

Journal: European Journal of Medical Research

Article Title: RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells

doi: 10.1186/2047-783X-18-52

Figure Lengend Snippet: Cells were cultured in Transwell chambers with the upper part coated with laminin (LN) or fibronectin (FN) respectively for 48 hours. The invaded cells were numerated under a light microscope (×400). (A) HepG2 infected by control shRNA plasmids invaded LN. (B) HepG2 cells infected by control shRNA plasmids invaded FN. (C) HepG2 cells infected by integrin α6 shRNA plasmids invaded LN. HepG2 had a decreased invasion potential after the knockdown of integrin α6 with significant statistical difference ( P < 0.0001). It also proved that LN can increase invasion potential of HepG2 ( P < 0.05).

Article Snippet: Integrin α6 shRNA plasmids (sc-43129-sh) were constructed and synthesized by Santa Cruz Biotechnology, Inc., CA, USA.

Techniques: Cell Culture, Light Microscopy, Infection, Control, shRNA, Knockdown

Journal: European Journal of Medical Research

Article Title: RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells

doi: 10.1186/2047-783X-18-52

Figure Lengend Snippet: Cells were cultured in 96-well plates coated with laminin (LN) or fibronectin (FN) respectively for 30 minutes and then cultured with acid phosphatase substrate at 37°C. The reaction was stopped two hours later. (A) The adhesion assays showed that lower-level integrin α6 reduced the adhesion of HCC cells with significantly statistical difference ( P < 0.0001) in the cells incubated with LN, while the cells incubated with FN did not show any significant effect ( P > 0.05). (B) Cells cultured in 96-well plates were coated with LN for 150 minutes. The spreading cells were numerated under a light microscope (×200). HepG2 cells with lower level of integrin α6 had a lower spreading rate. (C) The qRT-PCR assay showed that MMP-2 expression reduced significantly at mRNA level ( P < 0.01). (D) The qRT-PCR assay showed that MMP-9 expression reduced significantly at RNA level ( P < 0.01). * P < 0.05, ** P < 0.01, **** P < 0.0001 compared with control.

Article Snippet: Integrin α6 shRNA plasmids (sc-43129-sh) were constructed and synthesized by Santa Cruz Biotechnology, Inc., CA, USA.

Techniques: Cell Culture, Incubation, Light Microscopy, Quantitative RT-PCR, Expressing, Control

Journal: European Journal of Medical Research

Article Title: RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells

doi: 10.1186/2047-783X-18-52

Figure Lengend Snippet: Expression levels determination of PI3K/AKT and MAPK/ERK. (A) Western blot assay revealed the expression level of p-ERK was lower in HepG2 and Bel-7402 cells transfected with integrin α6 short hairpin RNA (shRNA) plasmids, while the expression of ERK remained unchanged. Cells transfected with control shRNA plasmids show no difference on p-ERK and ERK expression. (B) Western blot assay revealed the expression of p-AKT was lower in HepG2 and Bel-7402 cells transfected with integrin α6 shRNA plasmids, while the expression level of AKT remained unchanged. Cells transfected with control shRNA plasmids show no difference in p-AKT and AKT expression. (C) Western blot assay showed that PI3K inhibitor LY294002 reduced the expression of both p-AKT and p-ERK. (D) MTT assay showed that the proliferation of HepG2 cells cultured with LY294002 was dramatically reduced ( P < 0.05). (E) Transwell assay showed that the invasion potential of HepG2 cells cultured with LY294002 was dramatically reduced ( P < 0.05). * P < 0.05 compared with control.

Article Snippet: Integrin α6 shRNA plasmids (sc-43129-sh) were constructed and synthesized by Santa Cruz Biotechnology, Inc., CA, USA.

Techniques: Expressing, Western Blot, Transfection, shRNA, Control, MTT Assay, Cell Culture, Transwell Assay

Journal: Virology Journal

Article Title: HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

doi: 10.1186/s12985-017-0736-2

Figure Lengend Snippet: HaCaT cell line possesses a “progenitor” sub-population. a Flow cytometric analyses for α6-integrin and CD71 expression in HaCaT cell line exhibited three phenotypes: α6-integrin dim (R8), α6-integrin bri /CD71 bri (R7) and α6-integrin bri /CD71 dim (R6). Upper panels correspond to enrichment assays sorting and reseeding only cells from the α6-integrin bri /CD71 dim subpopulation. Lower panels correspond to enrichment assays sorting and reseeding cells from a mix of α6-integrin dim (R8) and α6-integrin bri /CD71 bri (R7) subpopulations. b Bars graph of two rounds enrichment of the α6-integrin bri /CD71 dim subpopulation in self-renewal assays. Bars represent the mean ± SD of three independent assays ( p < 0.05). c Bars graph of the clonogenic assays from the α6-integrin bri /CD71 dim subpopulation seeded after each round of enrichment. Bars represent the mean ± SD of three independent assays ( p < 0.05). d RT-PCR analyses for the expression of stem cell markers ( SOX2 , OCT4 and NANOG ) in α6-integrin bri /CD71 dim subpopulation, Non-α6-integrin bri /CD71 dim subpopulation and total population in HaCaT cell line. e RT-qPCR analyses for the expression of SOX2 , OCT4 and NANOG in total HaCaTwt cells and after sorting in α6-integrin bri /CD71 dim and Non-α6-integrin bri /CD71 dim subpopulations. Values are expressed as the difference in ΔΔ Ct and expression of a housekeeping gene (β-actin). Relative expression of three separated assays expressed as the mean ± SD, p < 0.05, is presented. All images shown are representative of at least three independent experiments

Article Snippet: Cells were pelleted by centrifugation and suspended at 1 × 10 6 /100 μl in ice-cold PBS containing 1% BSA and then processed for single or double staining with PE-Cy5 mouse anti-human CD71 and PE rat anti-human α6 integrin (BD Biosciences, NJ, USA) during 45 min at 4 °C, using the appropriate negative controls to establish the compensation settings on the FACS.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Virology Journal

Article Title: HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

doi: 10.1186/s12985-017-0736-2

Figure Lengend Snippet: HPV16-E2 expression modifies the α6-integrin-CD71 subpopulations profile in HaCaT cell line. a RT-PCR analysis of lentivirus transduced HaCaT cells 5 days post-infection. HPV16-E2 is expressed only in transduced cells HaCaT-HPV16-E2. b Western blot analysis showing the expression of HPV16-E2 protein only in the transduced HaCaT-HPV16-E2 cells. c Representative flow cytometry analysis for the α6-integrin-CD71 subpopulations profile in HaCaT-HPV16-E2 cells. R6 decreases almost 50% while R8 increases at least 5 times in these cells. All images shown are representative of at least three independent experiments

Article Snippet: Cells were pelleted by centrifugation and suspended at 1 × 10 6 /100 μl in ice-cold PBS containing 1% BSA and then processed for single or double staining with PE-Cy5 mouse anti-human CD71 and PE rat anti-human α6 integrin (BD Biosciences, NJ, USA) during 45 min at 4 °C, using the appropriate negative controls to establish the compensation settings on the FACS.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Western Blot, Flow Cytometry

Journal: Virology Journal

Article Title: HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes

doi: 10.1186/s12985-017-0736-2

Figure Lengend Snippet: HPV16-E2 expression alters the level of stem cell markers in HaCaT cells. RT-qPCR analyses for expression of SOX2 , NANOG and OCT4 in HaCaTwt, HaCaT-Vac and HaCaT-HPV16-E2 total cell population and after sorting in α6-integrin bri /CD71 dim and Non-α6-integrin bri /CD71 dim subpopulations. Values are expressed as the difference in ΔΔ Ct compared to non-infected cells and expression of a housekeeping gene (β-actin). Relative expression of three separated assays expressed as the mean ± SD, p < 0.05, is presented

Article Snippet: Cells were pelleted by centrifugation and suspended at 1 × 10 6 /100 μl in ice-cold PBS containing 1% BSA and then processed for single or double staining with PE-Cy5 mouse anti-human CD71 and PE rat anti-human α6 integrin (BD Biosciences, NJ, USA) during 45 min at 4 °C, using the appropriate negative controls to establish the compensation settings on the FACS.

Techniques: Expressing, Quantitative RT-PCR, Infection

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Morroniside promotes skin wound re-epithelialization by facilitating epidermal stem cell proliferation through GLP-1R-mediated upregulation of β-catenin expression

doi: 10.3724/abbs.2024070

Figure Lengend Snippet: Characterization of mouse and human EpSCs (A,D) Morphology of mouse EpSCs (A) and human EpSCs (D) under a light microscope. (B,E) Flow cytometry analysis of α6 integrin and CD71 expression in mouse EpSCs (B) and human EpSCs (E). (C,F) Immunofluorescence staining of cytokeratin 19 (CK19, green) and β1 integrin (red) in mouse EpSCs (C) and human EpSCs (F). Cell nuclei were stained with DAPI. Scale bar: 100 μm. Photographs of mouse and human EpSCs, immunofluorescence staining images, and flow cytometry results are representative of three independent experiments.

Article Snippet: The cultured mouse or human EpSCs were harvested and suspended in FACS buffer (1% goat serum and 5% FBS in PBS) at 25°C for 1 h. The cells were then incubated with FITC-conjugated α6 integrin antibody and PE-labeled CD71 antibody (BD Biosciences, San Jose, USA) at room temperature for 0.5 h. IgG2a labeled with either PE or FITC was used as an isotype control.

Techniques: Light Microscopy, Flow Cytometry, Expressing, Immunofluorescence, Staining

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Morroniside promotes skin wound re-epithelialization by facilitating epidermal stem cell proliferation through GLP-1R-mediated upregulation of β-catenin expression

doi: 10.3724/abbs.2024070

Figure Lengend Snippet: Morroniside promotes EpSC proliferation in mouse skin wound through GLP-1R-mediated activation of downstream molecules Mouse skin wounds were treated with PBS, 50 μg/mL morroniside (Mor), 415 μg/mL Ex (9-39), or 50 μg/mL Mor combined with 415 μg/mL Ex (9-39) for 4 days. Skin around the wound edge was collected and examined for EpSC proliferation by immunohistochemical staining for β1 integrin- and PCNA-expressing cells in serial sections (A,B) and for phosphorylation of AKT and ERK (C,D) and expression of β-catenin, c-Myc, cyclin D1, and cyclin E1 (E,F) by western blot analysis. Scale bars are 40 μm and 20 μm in the left and right panels of the β1 integrin and PCNA immunohistochemistry images, respectively. Arrows indicate β1 integrin and PCNA double-positive cells. n=3 mice/group. The data are presented as the mean±SEM. * P<0.05, ** P<0.01, *** P<0.001, compared with PBS-treated mice. # P<0.05, ## P<0.01, ### P<0.001, compared with Mor-treated mice.

Article Snippet: The cultured mouse or human EpSCs were harvested and suspended in FACS buffer (1% goat serum and 5% FBS in PBS) at 25°C for 1 h. The cells were then incubated with FITC-conjugated α6 integrin antibody and PE-labeled CD71 antibody (BD Biosciences, San Jose, USA) at room temperature for 0.5 h. IgG2a labeled with either PE or FITC was used as an isotype control.

Techniques: Activation Assay, Immunohistochemical staining, Staining, Expressing, Western Blot, Immunohistochemistry