Journal: Science (New York, N.Y.)

Article Title: Tumor-initiating cells establish an IL-33-TGF-β niche signaling loop to promote cancer progression

doi: 10.1126/science.aay1813

Figure Lengend Snippet: (A) Immunolabeling of tumor sections showing efficient IL-33 protein depletion in Il33 shRNA-transduced (YFP+) cells. Note that some YFPneg stromal cells maintain nuclear IL-33 (arrows). (B) HRASG12V-driven tumors in control and Il33 KD mice were sized at the time of euthanasia. Scramble control, n = 46; Il33 KD, n = 31. Approximation curves were drawn by applying the Michaelis-Menten kinetics curve. (C) Hematoxylin and eosin (H&E) staining of tumor sections showing smoother edges in Il33 KD tumors compared with control tumors, suggestive of reduced invasive capacity. Scale bars, 100 μm. (D) Immunolabeling of K10 showing a loss of differentiation property in control tumors, but not in Il33 KD tumors. (E) Detection of EdU incorporated 4 hours before euthanasia. (Graph) Quantification of EdU+ cells in integrin α6+ tumor basal cells of control and Il33 KD tumors (n = 3) (n = 806 to 1747 cells). Data are shown as mean with SEM and were analyzed with unpaired t test, **P = 0.004. (F) Flow cytometry analysis of TGF-β reporter (mScarlet) expression in YFP+ tumor epithelial cells. YFP+ cells in Il33 KD tumors show fewer and reduced TGF-β reporter activity compared with those in control tumors. (Graph) Proportion of TGF-β reporter+ tumor cells. Each dot indicates values from an individual tumor. Scramble control, n = 30; Il33 KD, n = 19. Data are shown as mean with individual values and were analyzed with unpaired t test, **P = 0.007. (G) Tumor-bearing mice were treated with cisplatin (10 mg/kg), and cells undergoing apoptosis (cleaved caspase-3+) in K5+ tumor basal cells were quantified 2 days after treatment (n = 3). Data are shown in box-and-whisker plots (midline, median; box, 25th and 75th percentiles; whiskers, min and max) and were analyzed with unpaired t test, **P = 0.0061, *P = 0.0334. (H) Volume of tumors after cisplatin administration. (Left) Spider plots showing the changes for each tumor. (Right) Normalized tumor volume of each condition. Scramble control, n = 3 (15 tumors), Il33 KD, n = 5 (24 tumors). Tu, tumor. St, stroma. Scale bars, 50 μm [except for (C)].

Article Snippet: The following primary antibodies were used: chicken anti-GFP (to detect YFP) (Abcam, ab13970), chicken anti-keratin 5 (Covance, SIG-3475), rabbit anti–TGF-β (R&D Systems, AB-100-NA), rabbit anti–phospho-SMAD2 (Cell Signaling Technologies, 138D4), goat anti–IL-33 (R&D Systems, AF3626), rabbit anti-NRF2 (MBL International, PM069), rat anti-CD45 (BD Biosciences, 30-F11), rat anti-CD11b (BD Biosciences, M1/70), rat anti-CD3 (BD Biosciences, 17A2), rat anti-CD8a (eBioscience, 4SM15), rat anti-CD4 (eBioscience, 4SM95), rat Ly6G (BioLegend, HK1.4), rat anti-F4/80 (Bio-Rad, Cl:A3-1), Armenian hamster anti-FcεRIα (BioLegend, MAR-1), rat anti-integrin α6 (BD Biosciences, GoH3), mouse anti–α-tubulin (Sigma-Aldrich, DM1A), rabbit anti–keratin 14 (Abcam, EPR17350 ), rabbit anti–keratin 10 (Abcam, EP1607IHCY), goat anti-CD31 (R&D Systems, AF3628), goat antitryptase/MCP-6 (R&D Systems, AF3736), goat antihuman IL-33 (R&D Systems, AF3025), rabbit anti-CD206 (Abcam, ab64693), rabbit anti–E-cadherin (Cell Signaling Technologies, 24E10), rabbit anti SIP1/ZEB2 (Bethyl, IHC-00691), and rabbit anti–V5-tag (Abcam, ab9116).

Techniques: Immunolabeling, shRNA, Staining, Flow Cytometry, Expressing, Activity Assay, Whisker 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: Oxidative Medicine and Cellular Longevity

Article Title: Novel Antioxidant Tripeptide “ACQ” Can Prevent UV-Induced Cell Death and Preserve the Number of Epidermal Stem Cells

doi: 10.1155/2015/359740

Figure Lengend Snippet: Decreased DCF staining and increased p63 positive cells in ACQ fed mice. (a) After UVB irradiation, control mice showed strong DCF staining but ACQ fed mice showed weak DCF staining (dashed line means basement membrane; scale bar is 50 μ M). (b) Confocal microscopic examination showed that there is strong signal of p63 and integrin α 6 in ACQ fed mice compared to control mice. There was nonspecific staining of integrin α 6 at the upper dermis (green; p63 staining, red; integrin α 6 staining, ×200, scale bar is 50 μ M).

Article Snippet: Antibodies that recognize p63 (sc-8343) and integrin α 6 (sc-6597) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, Irradiation, Control, Membrane

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Novel Antioxidant Tripeptide “ACQ” Can Prevent UV-Induced Cell Death and Preserve the Number of Epidermal Stem Cells

doi: 10.1155/2015/359740

Figure Lengend Snippet: Confocal microscopic examination of skin equivalents. (a) The number of p63 positive cells increased significantly in response to treatment with ACQ compared to the control. In addition, the expression of α 6 integrin was also significantly increased but nonspecific staining was observed at the upper epidermis (×200, scale bar is 50 μ M).

Article Snippet: Antibodies that recognize p63 (sc-8343) and integrin α 6 (sc-6597) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Expressing, Staining