Journal: Stem Cell Reviews

Article Title: Human Menstrual Blood-Derived Stromal Cells Promote Recovery of Premature Ovarian Insufficiency Via Regulating the ECM-Dependent FAK/AKT Signaling

doi: 10.1007/s12015-018-9867-0

Figure Lengend Snippet: Physiological changes to evaluate ovarian status. a Serum hormone levels assessed by ELISA kits in each group for measuring FSH (left), E2 (middle) and AMH (right), n = 6 (data were represented as mean ± SEM, * P < 0.05, ** P < 0.01). b Representative images of parturition in each group after mating with male mice. c Records of live births after CTX injection followed by MenSCs transplantation, n = 6 (data were represented as mean ± SEM, ** P < 0.01). d Apoptosis evaluation using Hoechst kit. Yellow arrows pointed to apoptotic cells with fragmented or condensed nuclei of apoptotic cells. Original magnification, ×100. e Apoptosis evaluation performed by TUNEL assay. White arrows pointed to FITC-labeled apoptotic cells. Original magnification, ×100. f Relative mRNA expression by q-RT PCR analysis for AMH, DDX4 and VEGFA controlled to GAPDH with fold change measured by 2 -ΔΔCT , n = 6. g Relative expression at protein levels of AMH, DDX4 and VEGFA (upper panel) and quantitative analysis (lower panel)

Article Snippet: TUNEL assay was performed using TUNEL FITC Apoptosis Detection Kit (Vazyme, Nanjing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Transplantation Assay, TUNEL Assay, Labeling, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Bioengineering & Translational Medicine

Article Title: Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

doi: 10.1002/btm2.10538

Figure Lengend Snippet: RANTES/IL‐15‐secreting GBM tumor cells enhance T‐cell trafficking and expansion. (a) RANTES and IL‐15 were detected in culture supernatant collected from transduced U87 tumor cells. (b) U87 or U87 RANTES‐IL‐15 were seeded and cultured for 24 or 48 h. Transwell inserts were added and CSPG4‐CAR‐T or control T cells were seeded therein. T cells were allowed to migrate through inserts for 5 h. Afterward, inserts were removed and media was collected for flow cytometry. (c) Migration of CSPG4‐CAR‐T or control T cells toward U87 or U87 RANTES‐IL‐15 cultured for 24 or 48 h was evaluated by flow cytometry ( n = 3). (d) NSG mice were implanted i.c. with U87, U87 RANTES‐IL‐15 , or PBS in the right hemisphere of the brain. Five days later, mice were infused i.v. with CSPG4‐CAR‐T. Mice were sacrificed 1, 4, or 7 days following CSPG4‐CAR‐T infusion. Brains were harvested, bisected along the interhemispheric fissure, and processed for flow cytometry ( n = 3). (e) CSPG4‐CAR‐T concentrations in single cell suspensions generated from bisected mouse brains (contralateral hemisphere vs tumor hemisphere) were analyzed by flow cytometry. Schematic(s) created with BioRender.com . * p < 0.05, ** p < 0.01, *** p < 0.001. CAR‐T, chimeric antigen receptor‐modified T cell; CSPG4‐CAR‐T, CAR‐T cells targeting chondroitin sulfate proteoglycan 4 antigen; GBM, glioblastoma; IL‐15, interuekin 15; NSG, NOD.Cg‐ Prkdc scid Il2rg tm1Wjl /SzJ.

Article Snippet: The U87 glioma cell line and human telomerase reverse transcriptase (hTERT)‐immortalized normal human fibroblasts (NHF1) were obtained from the American Type Culture Collection and as previously described.

Techniques: Cell Culture, Control, Flow Cytometry, Migration, Generated, Modification

Journal: Bioengineering & Translational Medicine

Article Title: Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

doi: 10.1002/btm2.10538

Figure Lengend Snippet: Development and characterization of hiNeuroS RANTES‐IL‐15 . (a) Schematic representation of lentivirus encoding human RANTES and IL‐15. (b) Human fibroblasts were transduced with LV‐RANTES‐IL‐15‐GFP, LV‐Sox2, and LV‐rtTA in succession. Reprogramming and primary hiNeuroS formation were initiated by switching to NSC growth media supplemented with growth factors and tetracycline. Three rounds of cell cluster selection were performed by serial seeding on laminin‐coated plates. After each round of selection, nonadhering cells were collected and expanded to yield secondary hiNeuroS. (c) Fluorescence imaging of hiNeuroS RANTES‐IL‐15 confirms successful LV‐RANTES‐IL‐15‐GFP transduction. Scale bar represents 100 μm. (d) RANTES and IL‐15 were detected in culture supernatant collected from transduced hiNeuroS. (e) Migration of CSPG4‐CAR‐T toward U87 ± NS‐CM or control media using a transwell migration assay was evaluated by flow cytometry ( n = 3). (f) CSPG4‐CAR‐T counts after 3 days in co‐culture with U87 ± NS‐CM ( n = 3). (g) Anti‐tumor activity of CSPG4‐CAR‐T after 3 days in co‐culture with U87 ± NS‐CM was assessed by bioluminescence ( n = 8–10). Schematic(s) created with BioRender.com . * p < 0.05, ** p < 0.01, *** p < 0.001. CAR‐T, chimeric antigen receptor‐modified T cell; CSPG4‐CAR‐T, CAR‐T cells targeting chondroitin sulfate proteoglycan 4 antigen; NSC, neural stem cell.

Article Snippet: The U87 glioma cell line and human telomerase reverse transcriptase (hTERT)‐immortalized normal human fibroblasts (NHF1) were obtained from the American Type Culture Collection and as previously described.

Techniques: Transduction, Selection, Fluorescence, Imaging, Migration, Control, Transwell Migration Assay, Flow Cytometry, Co-Culture Assay, Activity Assay, Modification

Journal: Bioengineering & Translational Medicine

Article Title: Utilizing induced neural stem cell‐based delivery of a cytokine cocktail to enhance chimeric antigen receptor‐modified T‐cell therapy for brain cancer

doi: 10.1002/btm2.10538

Figure Lengend Snippet: Combination hiNeuroS RANTES‐IL‐15 + CAR‐T therapy increases CAR‐T infiltration and eradicates non‐invasive GBM. (a) Schematic representation of study design. NSG mice were implanted i.c. with FLuc + U87 ± hiNeuroS RANTES‐IL‐15 in the right hemisphere of the brain. Four days later, mice were infused i.v. with CSPG4‐CAR‐T or PBS ( n = 5–7). (b) Representative bioluminescent images depict U87 tumor growth. (c) Luminescence was quantified by calculating total flux within a region of interest centered on the head, then normalizing to Day 0 values. Dotted line signifies CAR‐T infusion. (d) Kaplan–Meier survival curves. (e) AUC was calculated for each normalized tumor growth curve. (f) CAR‐T tumor infiltration (brown) was assessed by immunohistochemistry. Schematic(s) created with BioRender.com . *** p < 0.001. CAR‐T, chimeric antigen receptor‐modified T cell; CSPG4‐CAR‐T, CAR‐T cells targeting chondroitin sulfate proteoglycan 4 antigen; GBM, glioblastoma; NSG, NOD.Cg‐ Prkdc scid Il2rg tm1Wjl /SzJ.

Article Snippet: The U87 glioma cell line and human telomerase reverse transcriptase (hTERT)‐immortalized normal human fibroblasts (NHF1) were obtained from the American Type Culture Collection and as previously described.

Techniques: Immunohistochemistry, Modification

Journal: Carcinogenesis

Article Title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

doi: 10.1093/carcin/bgn230

Figure Lengend Snippet: ( A ) Kaplan–Meier plot on cumulative survival of patients with ovarian cancer with high and low Gli1 expression showed that high Gli1 expression was associated with poor prognosis. ( B ) Representative mRNA expression of Shh, Patched, Smo and Gli1 in various ovarian tissues detected by RT–PCR. (N1 and N2, normal tissues; B1, benign tumor; B2, borderline tumor; C1–C6, cancers; Ptch, Patched.) Elevated expression of Shh mRNA was observed in cancer samples, whereas sustained expression of Patched mRNA and heterogeneous expression of Gli1 mRNA was present in ovarian cancers. ( C ) Results of RT–PCR for Shh, Patched, Smo and Gli1 and western blotting for Patched and Gli1 in immortalized normal ovarian epithelial cell lines (HOSE 6-3, HOSE 11-12) and ovarian cancer cell lines (OVCA420, OVCA429, OVCA433, OVCAR3 and SKOV3). Sustained expression of Patched and variable expression of Gli1 mRNA and protein was observed in all seven cell lines (Ptch, Patched). ( D ) Mesothelial marker calretinin (Cal) and mesenchymal marker vimentin (Vim) were expressed in normal cell lines, whereas epithelial marker E-cadherin (E-Cad) and cytokeratin 7 (CK 7) were present in cancer cell lines. Snail expression was observed in all the normal and cancer cell lines.

Article Snippet: Two human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker

Journal: Carcinogenesis

Article Title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

doi: 10.1093/carcin/bgn230

Figure Lengend Snippet: Ectopic overexpression of Gli1 promoted proliferation, migration and invasion of ovarian cancer cells. ( A ) Transient transfection of pcDNA3.1-HisB-hGli1 promotes cancer cell proliferation as assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Error bars, SD. ( B ) Increased cell number was found to migrate through Matrigel-coated chamber after transfection of pcDNA3.1-HisB-hGli1 (OVCAR3 cells were shown). Bar chart below showed an increase in percentage of cells migrating through Matrigel-coated chamber in Gli1-transfected cells when compared with empty vector-transfected cells and untransfected (untreated) cells (* P < 0.01; ** P < 0.05). (U, untransfected cells; V, vector; G, pcDNA3.1-HisB-hGil) Error bars, SD. ( C ) Wound-healing assay shows increased cell migration ability of SKOV3 with ectopic overexpression of Gli1. ( D ) Increased expression of Bcl-2, total form of caspases-3, 7, 9 and PARP as well as β1-integrin was present in Gli1-transfected cancer cells. (Casp-3, 7, 9, caspases-3, 7, 9, respectively; U, untransfected; V, empty vector; G, pcDNA3. 1-HisB-hGli1.)

Article Snippet: Two human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Wound Healing Assay, Expressing

Journal: Carcinogenesis

Article Title: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation

doi: 10.1093/carcin/bgn230

Figure Lengend Snippet: Ectopic Gli1 overexpression induced increased expression of MT1-MMP and VEGF mRNA as well as epithelial differentiation markers in ovarian cancer cells. (V, empty vector; His-Gli1 and G, pcDNA3.1-HisB-hGli1.) ( A ) Real-time PCR showed increased expression of MT1-MMP in OVCAR3 and VEGF in all cancer cells. Error bars, SD. ( B ) Increased expression of E-cadherin (E-Cad) and vimentin (Vim) was present in cancer cells after transfection of pcDNA3.1-HisB-hGli1 (V, empty vector, G, pcDNA3.1-HisB-hGli1). ( C ) E-cadherin mRNA expression was increased with ectopic overexpression of Gli1 in SKOV3 detected by real-time PCR. Error bars, SD. ( D ) Dual luciferase assay confirmed upregulation of E-cadherin expression at transcriptional level in SKOV3 cells after transfection (* P < 0.05). Error bars, SD.

Article Snippet: Two human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Luciferase

Journal: PLoS ONE

Article Title: The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation

doi: 10.1371/journal.pone.0204792

Figure Lengend Snippet: HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Multiple clones were established for each cell line. Addition of the tetracycline analog, doxycycline (dox), for 24 hours relieves TR repression and induces BNIP3 expression as detected by immunofluorescence (A), Western blot (B) and RT-PCR. In (A), nuclei are visualized by Hoescht dye (blue) and BNIP3 is seen in the cytoplasm (green fluorescence).

Article Snippet: MEF cells (obtained from Animal Care Services, University of Manitoba) and HEK293 cells (obtained from ATCC) were cultured in DMEM media (Thermofisher) consisting of 4mM L-glutamine, 4mM sodium pyruvate, 4.5 microg/mL glucose and supplemented with either 10% fetal bovine serum (MEFs) or 5% fetal bovine serum (HEK293), and 100 units/ml penicillin/streptomycin.

Techniques: Stable Transfection, Clone Assay, Expressing, Immunofluorescence, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fluorescence

Journal: PLoS ONE

Article Title: The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation

doi: 10.1371/journal.pone.0204792

Figure Lengend Snippet: HEK293 cells were engineered to stably co-express the Tet Repressor protein (TR) and tetracycline-inducible BNIP3 (TO-BNIP3) or beta-galactosidase (TO-β-gal) as described in the Materials & Methods. Cells were untreated or treated with doxycycline (1 microg/mL) to induce gene expression. (A) Reactive oxygen species (ROS) production was increased upon BNIP3 (but not β-gal) induction. ROS were detected using the dye CM-H2DCFDA, which is oxidized to green fluorescent DCF (dichlorofluorescein) by hydrogen peroxide. (B) Autophagy was also increased upon BNIP3 (but not beta-gal) induction, as determined by GFP-LC3 localization. TO-cells were transiently transfected with GFP-LC3. 24 hours after transfection, cells were untreated or treated with doxycycline for an additional 24 hours. (C) Cell death was not induced upon BNIP3 or β-gal induction, as determined by the membrane permeability assay. (D) Cell growth was severely restricted upon BNIP3 (but not beta-gal) induction in HEK293 cells. For each cell type, 1.5x105 cells were seeded in one well of a 6-well culture dish, with or without doxycycline (gene induction). Three days later, the number of adherent cells in each well was determined using a Beckman Coulter Counter. The fold-increase represents the ratio of cells on day three compared to the number of cells originally seeded. Results represent the average of three independent experiments; error bars represent standard deviation. *p < .01.

Article Snippet: MEF cells (obtained from Animal Care Services, University of Manitoba) and HEK293 cells (obtained from ATCC) were cultured in DMEM media (Thermofisher) consisting of 4mM L-glutamine, 4mM sodium pyruvate, 4.5 microg/mL glucose and supplemented with either 10% fetal bovine serum (MEFs) or 5% fetal bovine serum (HEK293), and 100 units/ml penicillin/streptomycin.

Techniques: Stable Transfection, Gene Expression, Transfection, Membrane, Permeability, Standard Deviation

Journal: PLoS ONE

Article Title: The BH3 only Bcl-2 family member BNIP3 regulates cellular proliferation

doi: 10.1371/journal.pone.0204792

Figure Lengend Snippet: (A) HEK293 cells were transfected with either an empty control vector or nuclear BNIP3 expression vector. Total lysates from transfected cells were western blotted for BNIP3. The blot was stripped and reprobed with GAPDH as loading control. The results shown are representative of three independent experiments (three replicates each). (B) Proliferation of transfected cells was measured using the Click iT EdU cell proliferation assay. The results are an average of three independent experiments (three replicates each). The error bars show standard error of mean. Student’s t-test was used to calculate statistical significance (*p<0.0005). Results shown are representative of three independent experiments.

Article Snippet: MEF cells (obtained from Animal Care Services, University of Manitoba) and HEK293 cells (obtained from ATCC) were cultured in DMEM media (Thermofisher) consisting of 4mM L-glutamine, 4mM sodium pyruvate, 4.5 microg/mL glucose and supplemented with either 10% fetal bovine serum (MEFs) or 5% fetal bovine serum (HEK293), and 100 units/ml penicillin/streptomycin.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Western Blot, Proliferation Assay

Journal: Molecular Medicine Reports

Article Title: lncRNA FLVCR1-AS1 drives colorectal cancer progression via modulation of the miR-381/RAP2A axis

doi: 10.3892/mmr.2020.11778

Figure Lengend Snippet: FLVCR1-AS1 expression is upregulated in CRC. (A) RT-qPCR results show that FLVCR1-AS1 expression is higher in CRC tissues compared with normal tissues (n=26). (B) RT-qPCR was used to measure the expression of FLVCR1-AS1 in CRC cell lines (Caco-2, SW480, LoVo and SW1116) and the NCM460 normal colonic epithelial cell line. (C) RT-qPCR results showing the level of FLVCR1-AS1 in CRC tissues of different TNM stages. *P<0.05. (D) Kaplan-Meier analysis reveals the association of high FLVCR1-AS1 expression with poor overall survival in patients with CRC. FLVCR1-AS1, feline leukemia virus subgroup C receptor 1 antisense RNA 1; CRC, colorectal cancer; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Four human CRC cell lines, namely Caco-2, SW480, LoVo and SW1116, the NCM460 normal colonic epithelial cell line and 293T cells were purchased from the American Type Culture Collection.

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

Journal:

Article Title: Antitumor Activity and Prolonged Expression from a TRAIL-Expressing Adenoviral Vector

doi:

Figure Lengend Snippet: Induction of apoptosis in tumor cells by Ad.TRAIL-GFP vector. (A) Structure of Ad.TRAIL-GFP and Ad.GFP. The adenoviral sequences E1 and E3 are deleted. Ad.TRAIL-GFP vector expresses both TRAIL and GFP. (B) Western blot analysis of Ad.GFP-or Ad.TRAIL-GFP-transduced IMR90 cells or U87 glioma cells using an anti-TRAIL antibody (top), an anti-PARP antibody (middle), an antitubulin antibody (bottom), respectively. (C) Apoptotic index of cells (U87, T98, HUVEC, IMR90, NHA [MOI 100], and TA3 [MOI 50]) infected with Ad.TRAIL-GFP or Ad.GFP. Statistical significance between treatment was evaluated by Student's t test (asterisk: P<0.01). (D) Expression of four TRAIL receptors in a panel of cell lines, as determined by semiquantitative RT-PCR.

Article Snippet: The human umbilical vein endothelial cells (HUVECs), U87MG, T98 (human glioblastoma cells), 293 (human embryonic kidney cells), Colo-205 (human colon carcinoma), Ramos (Burkitt lymphoma), and IMR90 (human primary fibroblast cells) were purchased from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Plasmid Preparation, Western Blot, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction