Journal: Cell death & disease

Article Title: Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells.

doi: 10.1038/s41419-021-04154-0

Figure Lengend Snippet: Fig. 8 Effects of S63845 and imatinib administered as single drug or in combination on the colony forming capacity of primary human CD34+ stem/progenitor cells obtained from the peripheral blood of untreated CML patients in chronic phase or from the bone marrow of healthy donors. Relative colony forming capacity of CD34+ stem/progenitor cells obtained from the peripheral blood of untreated CML patients in chronic phase (left panel; n = 6 for all S63845 concentrations) or from the bone marrow (BM) of healthy donors (right panel; n = 4 for all S63845 concentrations) in the absence or presence of 1 μM imatinib (IM) and/or the indicated concentrations of S63845. Data represent mean with range derived from four (CD34+ normal BM cells) to six (CD34+ CML cells) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Patient no. 1: ⚫; patient no. 2: ◼; patient no. 5: ◆; patient no. 6: ○; patient no. 8: △; patient no. 9: ▽; healthy donor no. 1 ⚫; donor no. 2: ◼; donor no. 3: ▲; donor no. 4: ▼.

Article Snippet: Primary human normal bone marrow CD34+ hematopoietic stem/ progenitor cells were obtained from American Type Culture Collection (Cat. No.: PCS-800-012) or from Lonza (Cat. No.: 2M-101), and kept in culture for 48 h in StemSpan SFEM II Medium supplemented with StemSpan CD34+ Expansion Supplement and 175 nM UM171 (all from STEMCELL Technologies) before starting the colony forming assay.

Techniques: Derivative Assay

Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USCs inhibited the proliferation of PBMNCs in MLR. USC-TA+, USC-TA−, BMSCs, and SMCs were used as stimulator cells and human PBMNCs from two healthy donors were used as responder cells. One-way MLR with single donor PBMNCs (donor A or B) as responder cells and two-way MLR with aliquot half dose of two different donors’ PBMNCs (donor A and B) as responder cells were detected in the same multi-well plate. Cultures were incubated at 37 °C for 5 days in 5% CO2 and 100% humidity, the cells were checked with inverted microscope on the 5th day before further experiment. There were different cell densities of PBMNCs be noticed in different wells for 5 days mix-culture, Scale bar =50 µm (A). The proliferation of PBMNCs was assessed with the BrdU cell proliferation colorimetric ELISA. Newly synthesized BrdU-DNA was quantified using a scanning multi-well spectrophotometer (B). *, P<0.05, post-hoc paired-comparisons. MLR, mixed lymphocyte reaction; BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells; SMC + PBMNC A, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor A; SMC + PBMNC B, one-way mixed lymphocyte reaction as human smooth muscle cells mixed with human peripheral blood mononuclear cells from donor B; SMC + PBMNC A + B, two-way mixed lymphocyte reaction as human smooth muscle cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; BMSC + PBMNC A, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor A; BMSC + PBMNC B, one-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with human peripheral blood mononuclear cells from donor B; BMSC + PBMNC A + B, two-way mixed lymphocyte reaction as human bone marrow mesenchymal stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B; USC + PBMNC A, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor A; USC + PBMNC B, one-way mixed lymphocyte reaction as human urine derived stem cells mixed with human peripheral blood mononuclear cells from donor B; USC + PBMNC A + B, two-way mixed lymphocyte reaction as human urine derived stem cells mixed with aliquot half dose of two different donors’ human peripheral blood mononuclear cells from donor A and donor B.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Incubation, Inverted Microscopy, Enzyme-linked Immunosorbent Assay, Synthesized, Spectrophotometry, Derivative Assay, Activity Assay

Journal: Translational Andrology and Urology

Article Title: Functional characterization of the immunomodulatory properties of human urine-derived stem cells

doi: 10.21037/tau-21-506

Figure Lengend Snippet: USC showed a characteristic cytokine release profile. The supernatants from USC-TA+, USC-TA−, and BMSCs cultured alone or co-cultured with PBMNCs (direct mixed culture with PBMNCs, or indirect mixed culture with PBMNCs in transwell insert for 48 h) (A) were assessed for their relative levels of cytokines and chemokines by using the human cytokine array panel A, as described in “Methods” (B). A: human cytokine array panel template; B: PBMNCs background; C: USC-TA+ culture alone; D: USC-TA− culture alone; E: BMSCs culture alone; F: USC-TA+ direct mix culture with PBMNCs; G: USC-TA− direct mix culture with PBMNCs; H: BMSCs direct mix culture with PBMNCs; I: USC-TA+ in-direct mix culture with PBMNCs in transwell insert; J: USC-TA− in-direct mix culture with PBMNCs in transwell insert; K: BMSCs in-direct mix culture with PBMNCs in transwell insert: The immunoblot of cytokine expression levels were visualized and quantifed (C). *, P<0.05, comparing with BMSCs alone; †, P<0.05, comparing direct and indirect mixed culture with PBMNCs. BMSC, human bone marrow mesenchymal stem cells; SMC, human smooth muscle cells; USC TA+, human urine derived stem cells with high telomerase activity; USC TA−, human urine derived stem cells with low telomerase activity; PBMNC, human peripheral blood mononuclear cells.

Article Snippet: Human peripheral blood mononuclear cells (PBMNCs, normal human, ATCC PCS-800-011) from two different donors were purchased from ATCC (American Type Culture Collection, MD, USA).

Techniques: Cell Culture, Western Blot, Expressing, Derivative Assay, Activity Assay

Journal: Developmental biology

Article Title: Strains matter: Success of murine in vitro spermatogenesis is dependent on genetic background.

doi: 10.1016/j.ydbio.2019.08.007

Figure Lengend Snippet: Fig. 4. Immunohistochemical identification of the steroidogenic function of Leydig cells by expression of CYP17A1 enzyme (brown) in mice testic- ular fragments sections throughout organ culture. A. C57BL/6 J (n ¼ 3) and B. B6D2F2 (n ¼ 4) mouse donors. Insets show isotype IgG negative controls. For both strains, CYP17A1 was expressed by Leydig cells before cultured and maintained during the entire culture period. Arrow-head: CYP17A1-positive Leydig cell. Scale bars represent 20 μm. Pictures were taken using an Olympus BX41 bright-field microscope with an Olympus DP20 color camera.

Article Snippet: Sections were subsequently incubated with 1:1000 rabbit polyclonal LIN28 (ab46020, Abcam, UK); 1:100mousemonoclonal AR (sc-7305, Santa Cruz Biotechnology, USA) or 1:800 rabbit polyclonal CYP17A1 (ab80206, Abcam, UK) primary antibody overnight at 4 C and, after washing, followed by 1 h incubation with goat-anti Mouse/Rabbit poly-HRP secondary antibody (DPVO110HRP, Immunologic, the Netherlands).

Techniques: Immunohistochemical staining, Expressing, Organ Culture, Cell Culture, Microscopy

Journal: Cancer Prevention Research

Article Title: KAVA Chalcone, Flavokawain A, Inhibits Urothelial Tumorigenesis in the UPII-SV40T Transgenic Mouse Model

doi: 10.1158/1940-6207.capr-13-0219

Figure Lengend Snippet: Figure 5. Antiproliferative and proapoptotic effects of dietary FKA in male UPII-SV40T transgenic mice. A, representative H&E staining of bladder tissues from vehicle control and dietary FKA-fed male low-copy UPII- SV40T transgenic mice. FKA feeding reduced number of high-grade papillary UCCs compared with control group. Original magnification, 100. B, immunohistochemical staining of Ki67 expression in bladder tissues. Slides were counterstained with hematoxylin and photographed using a light microscope. Original magnification, 200. Ki67-positive cells were counted in 12 fields in each group. The percentage of Ki67- positive cells was calculated and presented as mean SD (the left 2 panels). The percentages of Ki67-positive cells are significantly lower in the FKA-fed groups (n ¼ 6) than those in the vehicle control group (n ¼ 6; Student t test, P < 0.01). C, representative DAB-stained tissue specimens from vehicle control and 0.6% FKA-fed group showing brown-colored TUNEL-positive cells are depicted at 200 magnifications. Original magnification, 200. TUNEL-positive cells were counted in 12 fields in each group. The percentage of TUNEL-positive cells was calculated and presented as mean SD (the left 2 panels). The percentages of TUNEL-positive cells are significantly higher in the FKA-fed groups (n ¼ 6) than those in the vehicle control group (n ¼ 6; Student t test, P < 0.01).

Article Snippet: The sections were then incubated with mouse monoclonal anti-Ki67 antibody (1:800; Abcam), anti-survivin (1:200; Cell Signaling), anti-DR5 (1:100; Abcam), and anti-p27/Kip (1:100; BD) for 1 hour at 37 C in ahumidity chamber.Negative controls were treated only with PBS under identical conditions.

Techniques: Transgenic Assay, Staining, Control, Immunohistochemical staining, Expressing, Light Microscopy, TUNEL Assay

Journal: Development (Cambridge, England)

Article Title: Acid sphingomyelinase is a gatekeeper of placental labyrinthine architecture and function

doi: 10.1242/dev.204425

Figure Lengend Snippet: Increased autophagy in Smpd1 −/− placentas. (A) Densiometric analysis (left) of phospho-TFEB (p-TFEB) and total TFEB protein expression in gestational day (d) 15.5 wild-type and Smpd1 −/− placentas, normalized to β-actin. P-TFEB protein levels are significantly decreased in Smpd1 −/− placentas ( P <0.05). Although slightly elevated in wild-type placenta, there was no significant change in total TFEB levels. (Right) A TFEB immunoblot of wild-type (+/+) and Smpd1- deficient (−/−) d13.5, d15.5 and d17.5 placentas. (B) (Top) Immunofluorescence analysis of TFEB localization examined by confocal microscopy in d15.5 wild-type and Smpd1 −/− placentas at 40× magnification. TFEB (red) localizes to the cytoplasm of wild-type placental labyrinth and to the nucleus of labyrinth trophoblast in the Smpd1 −/− placental labyrinth. Lysosomes immunostained by LAMP1 (green) and nuclei are stained with DAPI (blue). Scale bars: 5 μm. (Bottom) Quantitation of TFEB localization in sinusoidal and syncytial cells. Smpd1 −/− placentas have a higher percentage of labyrinth trophoblasts, which have nuclear TFEB staining, compared to wild type ( P <0.05). (C) A phospho-mTOR (Ser2448) and a mTOR immunoblot (right) of wild-type and Smpd1 −/− d15.5 placentas, and densiometric analysis (left) of p-mTOR and mTOR protein expression normalized to β-actin. pmTOR protein level is significantly increased in Smpd1 −/− placentas. (D) A Phospho-p70S6K (Thr389) and p70S6K immunoblot (right) and densiometric analysis (left). All data are mean±s.e.m. per placenta obtained from at least two different dams. Different letters represent statistical significance ( P <0.05). For details of the statistical test(s) used, see the Materials and Methods.

Article Snippet: Primary antibodies used for western blotting were anti-β-actin, goat polyclonal (I-19) (Santa Cruz Biotechnology,sc-1616; 1:500), anti-LAMP-1 (DSHB, 1D4B; 1:1000), anti-TFEB (Bethyl, A303-673A; 1:2000), anti-mTOR Ser2448 (Cell Signaling, 2971; 1:800), anti-mTOR (Cell Signaling, 2972; 1:1000), anti-p62 (PROGEN Biotechnik, GP62-C; 1:1000), anti-LC3 (MBL, PM036; 1:1000), anti-ATP6V1B2 (Proteintech, 15097-1-AP; 1:1000), anti-Cathepsin D (Bio-Vision, 3191R-100; 1:2000), anti-DDDK-HRP (Proteintech, HRP-66008; 1:15,000), anti-p70 S6 kinase Thr389 (Cell Signaling, 9205; 1:800) and anti-p70 S6 kinase (Cell Signaling, 9202; 1:1000).

Techniques: Expressing, Western Blot, Immunofluorescence, Confocal Microscopy, Staining, Quantitation Assay