Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: Cr(VI) induces ALDH1A1 expression through KLF4. (A) CrT cells transfected with siRNAs (50 nM) targeting KLF4, DACH1, ABCB5, MERTK, SOX2 or EGF for 72 h and were lysed for immunoblot analyses with the indicated antibodies. (B) CrT cells transfected with or without EGF siRNA (50 nM, 72 h) were lysed for ELISA analyses for detecting secreted EGF levels in the culturing media. (C) CrT cells transfected with siRNAs (50 nM) targeting KLF4, DACH1, ABCB5, MERTK, SOX2 or EGF for 72 h and were lysed for qRT‐PCR analysis of ALDH1A1 mRNA expression levels. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (D) ALDH1A1 High and ALDH1A1 Low CrT cells were lysed for immunoblot analyses with the indicated antibodies. (E) ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4 for 72 h were lysed for immunoblot analysis with the indicated antibodies. ALDH1A1 Low CrT cells transfected with or without Flag‐ALDH1A1 were lysed for immunoblot analyses with the indicated antibodies. (F) ALDH1A1 Low CrT cells transfected with or without KLF4 siRNA (50 nM) for 72 h were lysed for immunoblot analysis with the indicated antibodies. ALDH1A1 High CrT cells transfected with or without ALDH1A1 siRNA were lysed for immunoblot analyses with the indicated antibodies. (G) Schematic image represents the KLF4 binding sequence within the ALDH1A1 transcriptional regulation region. (H) Luciferase reporter assays were performed in BEAS‐2B and CrT cells transfected with pGL‐3.0 vector containing ALDH1A1 WT or mutant promoter. Data represent the mean ± SD of triplicate experiments. ** p < .001. (I) CrT cells with or without KLF4 depletion and BEAS‐2B cells with or without expression of Flag‐KLF4 were transfected with a luciferase reporter gene under the control of the ALDH1A1 promoter for 24 h. Luciferase reporter assays were performed. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (J) BEAS‐2B cells, CrT cells, and CrT/TICs were used for ChIP‐qPCR analysis of the ALDH1A1 promoter with the indicated antibody. Data are presented as the mean ± SD of triplicate experiments. * P < .01, ** P < .001. (K) CrT cells with or without KLF4 depletion were used for the detection of ALDH1A1 activity by flow cytometry. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (L) Tumoursphere formation assays using ALDH1A1 High CrT cells transfected with or without KLF4 siRNA. (M) In vitro limiting dilution assays on ALDH1A1 High CrT cells transfected with or without KLF4 siRNA. ** p < .001. (N) In vitro limiting dilution assays on ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4. ** p < .001. (O) Tumoursphere formation assays using ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Flow Cytometry, In Vitro

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: CrT/TIC‐secreted EGF activates EGFR signalling and promotes LUSC cell growth. (A) HCC95 and H226 cells incubated with a conditioned medium or co‐cultured with the indicated cells were lysed for immunoblot analysis with the indicated antibodies; HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (B) HCC95 and H226 cells incubated with CrT/TIC‐derived conditioned medium for 12 h in the presence or the absence of human recombinant truncated EGF or EGF L26G were lysed for immunoblot analysis with the indicated antibodies; HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (C) HCC95 and H226 cells incubated with CrT/TIC‐derived conditioned medium for 12 h in the presence or the absence of EGF‐neutralising antibodies were lysed for immunoblot analyses with the indicated antibodies. (D) HCC95 and H226 cells co‐cultured with CrT/TICs with or without ALDH1A1 depletion were lysed for immunoblot analyses with the indicated antibodies. (E) HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (F) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without A37 were lysed for immunoblot analyses with the indicated antibodies. (G) HCC95 and H226 cells incubated with conditional medium derived from ALDH1A1 Low CrT or ALDH1A1 High CrT were lysed for immunoblot analyses with the indicated antibodies. (H) Growth curves of HCC95 and H226 cells cultured with BEAS‐2B‐, CrT‐, and CrT/TIC‐derived conditioned medium. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (I) Growth curves of HCC95 and H226 cells cultured with CrT/TIC‐derived conditioned medium pretreated with truncated EGF or EGF L26G. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (J) Growth curves for the HCC95 and H226 cells cultured with CrT/TICs‐derived conditional medium pretreated with or without anti‐EGF antibody. Data represent the mean ± SD of triplicate experiments. ** p < .001. (K) Growth curves of HCC95 and H226 cells cultured with the indicated conditioned medium derived from CrT/TICs with or without ALDH1A1 depletion. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (L) Growth curves of HCC95 and H226 cells cultured with the indicated conditioned medium derived from CrT/TICs with or without A37 treatment. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (M) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without U0126 were lysed for immunoblot analyses with the indicated antibodies. (N) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without PD98 were lysed for immunoblot analyses with the indicated antibodies. (O) Growth curves of HCC95 and H226 cells with or without U0126 treatment cultured with the indicated conditioned medium derived from CrT/TICs. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (P) Growth curves of HCC95 and H226 cells with or without PD98 treatment cultured with the indicated conditioned medium derived from CrT/TICs. Data are presented as the mean ± SD of triplicate experiments. ** P < .001.

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Incubation, Cell Culture, Western Blot, Transfection, Derivative Assay, Recombinant

Journal: International Journal of Stem Cells

Article Title: Immunomodulatory Effect of Epidermal Growth Factor Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells on Atopic Dermatitis

doi: 10.15283/ijsc21173

Figure Lengend Snippet: List of abbreviation

Article Snippet: On the day of transfection, 200 pmol of control siRNA (siCTL; Santa Cruz Biotechnology, USA) or human EGF-targeting siRNA (siEGF; Santa Cruz Biotech-nology) was mixed with 10 μl Lipofectamine 3000 in 1 ml Opti-MEM medium (Invitrogen).

Techniques: Activation Assay, Modification, Recombinant, Real-time Polymerase Chain Reaction, Small Interfering RNA, Negative Control, Positive Control

Journal: Clinical and Translational Medicine

Article Title: Mechanisms of interactions between lung‐origin telocytes and mesenchymal stem cells to treat experimental acute lung injury

doi: 10.1002/ctm2.231

Figure Lengend Snippet: Potential roles of OPN in the interaction between TCs and MSCs. MSCs or TCs were pre‐activated with LPS or vehicle and then cultured alone or together (A). The principle of the present study is to evaluate potential effects of TCs on MSCs through TCs‐produced OPN as paracrine ways, MSCs‐produced OPN as autocrine ways, or external OPN using monoclonal antibody against OPN (B). The influence of inflammation in mRNA and protein expression of OPN was evaluated in MSCs (C) or TCs (D) 4, 8, 24, and 48 h after LPS stimulation at different doses. Effects of secreted OPN on MSCs migration was assessed (E) in MSCs preactivated by LPS (aM), TCs preactivated by LPS (aT), and both co‐culture by the mono‐antibody against OPN (Anti‐OPN) or nonspecific antibody (IgG) at different doses. * and # stand for P values < .05, as compared with aM group and aM+aT without IgG or Anti‐OPN, respectively. We screened and selected the high efficacy of siRNA against OPN genes (siRNA‐OPN) to inhibit OPN mRNA and protein expression in MSCs (F1) or TCs (F2) after LPS stimulation. * and ## stands for P values < .05, as compared with cells with vehicle and cells stimulated by LPS and treated with siRNA carrier (Carrier), respectively. We further evaluated the migration capacity of co‐cultured MSCs and TCs without pre‐activation (M+T), MSCs OPN‐ preactivated by LPS (aM‐OPN), TCs OPN‐ preactivated by LPS (aT‐OPN), MSCs preactivated by LPS (aM), or TCs preactivated by LPS (aT) 24 h after LPS challenge (G). * and # stand for P values < .05, as compared with as compared with vehicle and animals treated with vehicle and aM+aT, respectively. The dynamic proliferation (H1) and movement (H2) of MSCs were measured by Cell‐IQ in groups of aM+aT, aM+aT‐Carrier, aM‐OPN+aT, aM+aT‐OPN, aM‐OPN+aT‐OPN, or M+T

Article Snippet: MSCs or TCs were plated into 12 well plate 24 hrs and then transfected with RNA interference OPN siRNA (sc‐36130, Santa Cruz Co. Ltd, USA), EGF siRNA (sc‐39417, Santa Cruz Co. Ltd), and the fluorescein‐conjugated control siRNAs (sc‐36869, Santa Cruz Co. Ltd) using lipofectamine 2000 (Invitrogen, CA).

Techniques: Cell Culture, Produced, Expressing, Migration, Co-Culture Assay, Activation Assay

Journal: Clinical and Translational Medicine

Article Title: Mechanisms of interactions between lung‐origin telocytes and mesenchymal stem cells to treat experimental acute lung injury

doi: 10.1002/ctm2.231

Figure Lengend Snippet: Roles of EGF‐EFGR axis in the interaction between TCs and MSCs. The expression of EGF mRNA and protein in MSCs (A) or TCs (B) were measured 0, 4, 8, 24, and 48 h after activated with LPS at different doses, while LPS‐induced mRNA and protein expressions of EGF in MSCs (C) or TCs (D) were blocked with siRNA‐EGF. * and ** stand for P values < .05, as compared to cells with vehicle, respectively. Effects of EGF from MSCs or TCs in the migration capacity of MSCs were evaluated (E) in capacity of the following groups: MSCs preactivated by LPS(aM), co‐cultured MSCs preactivated by LPS (aM) and TCs preactivated by LPS (aT), MSCs EGF‐ preactivated by LPS (aM‐EGF), TCs EGF‐ preactivated by LPS (aT‐EGF), MSCs preactivated by LPS (aM), or TCs preactivated by LPS (aT) 24 h after LPS challenge and co‐cultured MSCs preactivated by LPS (aM) and TCs preactivated by LPS without LPS challenge (aT‐Sham). * and # stand for P values < .05, as compared with as compared with groups aM and aM+aT, respectively. Dynamic effects of EGF originated from MSCs‐TCs contacts were investigated in the alive cell‐monitoring system (F1), including dynamic proliferation (F2) and movement (F3) of MSCs in the coculture of aM+aT, aM+aT with carrier, aM+aT‐EGF, am‐EGF+aT, aM‐EGF+aT‐EGF, or MSCs and TCs without pre‐activation (M+T). The role of EGFR in MSCs‐TCs interaction was evaluated by imaging alive cell behaviors (G1) and dynamic proliferation (G2) and movement (G3) in the co‐culture of aM+aT with vehicle, aM+aT with IgG, aM+aT with AG1478 at different doses, or M+T. Potential mechanisms of the EGF‐EFGR axis regulation in the MSCs‐TCs interaction are proposed (H)

Article Snippet: MSCs or TCs were plated into 12 well plate 24 hrs and then transfected with RNA interference OPN siRNA (sc‐36130, Santa Cruz Co. Ltd, USA), EGF siRNA (sc‐39417, Santa Cruz Co. Ltd), and the fluorescein‐conjugated control siRNAs (sc‐36869, Santa Cruz Co. Ltd) using lipofectamine 2000 (Invitrogen, CA).

Techniques: Expressing, Migration, Cell Culture, Activation Assay, Imaging, Co-Culture Assay