Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 1. Upregulation of ALDH1A3 in oxGBM cells is associated with the increased expression and release of pro-angiogenic factors PAI-1 and IL-8. GBM cell lines were transduced with ALDH1A3 for overexpression (oxGBM) or with empty vector (evGBM). (A) Confirmation of up-regulation of ALDH1A3 mRNA level in oxGBM cells by RT2-PCR. (B) Confirmation of up-regulation of ALDH1A3 protein expression by Western blot. wt, wild-type cells. The uncropped blots are shown in Figure S7; (C) Angiogenesis array. The blots showed duplicated dots for 55 angiogenesis-related proteins in the media of oxU373 or evU373. 10 of 55 proteins were upregulated more than 2-fold in ox group compared to ev group (indicated by rectangle). They are: (1) Ang-1, (2) artemin, (3) TF, (4) ET-1, (5) GM-CSF, (6) IL-8, (7) PDGF-AA, (8) PAI-1, (9) PEDF, and (10) uPA. (D) Semi-quantification of the dots representing PAI-1 and IL-8. (E) Immunofluorescence staining of GBM cells. U373 (left panel) and LN229 (right panel). Co-localization of ALDH1A3 with PAI-1 and IL-8 was observed in oxGBM cells, whereas no immunoreactivity of PAI-1 and IL-8 was detected in evGBM cells. (F) mRNA expression of PAI-1 and IL-8 in transduced U373 cells and the effect of inhibitors. Tiplaxtinin (Tip, 30 µM) and reparixin (Rep, 1 µM) are the specific inhibitors of PAI-1 and IL-8 receptors CXCR1/2, respectively. (G) Detection of PAI-1 and potential signaling proteins by Western blot. IOD: optical density. The uncropped blots are shown in Figure S8. **, p < 0.01; ***, p < 0.001, compared with ev. ##, p < 0.01; ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation, Western Blot, Staining

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 2. Overexpression of ALDH1A3 in GBM cells activated endothelial angiogenesis in indirect co-culture with endothelial cells (ECs), which was reversed by treatment with respective inhibitors of PAI-1 or IL-8 receptors CXCR1/2. Indirect co-culture was performed by culture of HBMECs in a conditioned medium (CM) containing the media derived from evGBMs or oxGBMs and ECGM in a ratio of 1:1. PAI-1 inhibitor tiplaxtinin (Tip, 30 µM) and CXCR1/2 inhibitor reparixin (Rep, 1 µM) or vehicle DMSO (0.1%) was added to CM, followed by EC behavior study. All data were reproduced in three independent experiments. (A) Proliferation assay in HBMEC and HUVEC. Indirect co-culture of HBMECs and HUVECs with CM derived from oxU373 and oxLN229 stimulated EC proliferation, which was completely reversed by the treatment of tiplaxtinin, not by reparixin. (B) Scratch assay in HBMEC. Left panel: images were acquired 24 h after scratching. Scale bar: 200 µm. Right panel: quantitative analysis. Culture of HBMECs with CM derived from oxU373 or oxLN229 (oxCM) significantly promoted HBMEC migration, which was reversed by the treatment of tiplaxtinin and reparixin, respectively. (C) Transwell invasion assay in HBMEC. Left panel: Representative images of invaded cells were acquired after 24 h of incubation. Scale bar: 100 µm. Right panel: quantitative analysis. Culture of HBMECs with oxCM accelerated HBMEC invasion. This effect was significantly inhibited by the treatment of reparixin but not by tiplaxtinin. (D) Tube formation assay in HBMEC. Left panel: representative images of tube formation. Scale bar: 200 µm. Right panel: quantitative analysis of branching points per field. Tube formation in HBMECs was stimulated by incubation with oxCM, which was completely diminished by both inhibitors. (E) Sprouting assay in HBMEC. Left panel: representative images of sprouting in HBMECs after 24 h of co-culture. Scale bar: 100 µm. A pronounced increase in sprouting was observed in HBMECs cultured in oxCM. Tiplaxtinin and reparixin suppressed the sprouting effect resulting from oxCM. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with evCM. #, p < 0.05; ##, p < 0.01 and ###, p < 0.001, compared with oxCM.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Over Expression, Co-Culture Assay, Derivative Assay, Proliferation Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Incubation, Tube Formation Assay, Cell Culture

Journal: Cancers

Article Title: Novel Function of Cancer Stem Cell Marker ALDH1A3 in Glioblastoma: Pro-Angiogenesis through Paracrine PAI-1 and IL-8.

doi: 10.3390/cancers15174422

Figure Lengend Snippet: Figure 4. oxGBM-derived culture media stimulated angiogenesis on CAM, which was fully rescued by the treatment of tiplaxtinin and reparixin. CAM was incubated with the culture media derived from evU373 or oxU373 cells with or without tiplaxtinin (Tip, 30 µM) or reparixin (Rep, 1 µM) or DMSO (as a vehicle control, 0.1%) for 72 h. (A) Microscopy view of the vasculature structure on CAM. More enriched microvessel network was clearly visible in the ox group, which was significantly reduced in tiplaxtinin- and reparixin-treated CAMs. The images were acquired using a stereo microscope on ED13 (scale bar: 1 mm). Stem vessel (arrow); branched microvessel network (arrow heads). (B) Histological features of CAM after hematoxylin-eosin (H&E) staining. The CAM consists of the chorionic epithelium layer (ChE), allantoic epithelium (AE) layer, and the mesenchymal (MES) layer (arrows). Microvessel (arrowheads) density was much higher in the MES layer of ox section compared to ev section, which was clearly reduced in tiplaxtinin- and reparixin-treated sections. Scale bar: 50 µm. (C) Quantitative analysis of branching point of vessels based on microscopy images. The number of branching points and microvessels was counted by the ImageJ software (v1.1.53t) in 3 fields/CAM (n = 10 CAM/group). (D) Quantitative analysis of microvessel numbers based on H&E-stained CAM sections. Microvessel number was counted manually on H&E-stained CAMs. 10 fields/section (n = 6 sections/group). ***, p < 0.001, compared with ev. ###, p < 0.001, compared with ox.

Article Snippet: To evaluate the potential angiogenic factors released from oxGBMs, an array was carried out using a human angiogenesis array kit (cat# ARY007; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Incubation, Control, Microscopy, Staining, Software

Journal: Cell stem cell

Article Title: M 6 A demethylase ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in acute myeloid leukemia

doi: 10.1016/j.stem.2020.04.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lineage Cell Depletion Kit , Miltenyi Biotec , Cat#130-090-858.

Techniques: Recombinant, Control, Virus, Magnetic Beads, Reverse Transcription, Proliferation Assay, Purification, Real-time Polymerase Chain Reaction, Software

Journal: Cells

Article Title: Bone Marrow-Derived IL-1Ra Increases TNF Levels Poststroke

doi: 10.3390/cells10040956

Figure Lengend Snippet: Effect of bone marrow (BM) cells on ischemia-induced proteins in brain and serum. ( A ) BM cell treatment 30 min after tMCAo reduces infarct volumes (IFV) in LM–LM and Tg–LM mice as well as brain IL-1β levels (pg/mg) in Tg–LM mice 24 h after tMCAo, compared to LM tMCAo mice (Table with extracted data from ) (the experimental overview was created with BioRender.com). ( B – K ) Comparison of TNF ( C ), IL-4 ( D ), IL-10 ( E ), CXCL1 ( F ), IL-12p70 ( G ), IL-5 ( H ), IL-6 ( I ), IFNγ ( J ) and IL-2 ( K ), levels in the brain (pg/mg) and serum (pg/mL) of unlesioned controls and in LM, LM–LM, and Tg–LM mice 24 h after tMCAo. Data are presented as mean ± SEM ( n = 10–12/group). One-way ANOVA, followed by Sidak’s post hoc tests, was performed. Significance indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. N.D, not detected.

Article Snippet: Electrochemiluminescence analysis was performed on brain, serum, cells, and media using the MSD Mouse Proinflammatory V-Plex Plus Kit (IFNγ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, CXCL1, TNF; K15012C, Mesoscale Discovery), the U-Plex Kit (TNFR1; K15069L, Mesoscale), and the R-Plex kit (TNFR2; K150ZSR-2, Mesoscale Discovery) according to the manufacturer’s instructions.

Techniques: Comparison