Journal: Cancer Science

Article Title: Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

doi: 10.1111/cas.12464

Figure Lengend Snippet: Co-culture with platelets promotes platelet aggregation and the proliferation of osteosarcoma cells. (a) Platelets (4.0 × 10 7 ) prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 2% PPP and 250 μM CaCl 2 , followed by incubation with PBS (red lines) or osteosarcoma MG63 (left panel, black line) and HOS (right panel, black line) cells (1.0 × 10 8 cells). The transmission of light by the samples was measured using an aggregometer to determine the aggregation rate. (b) MG63 and HOS cells stably transfected with ZsGreen gene, MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel), respectively, were co-cultured with the indicated number of washed mouse platelets in medium containing 0.5% FBS. TENSV buffer was added to each well at the times indicated, and the fluorescence of ZsGreen was used to determine the number of viable osteosarcoma cells. The error bars indicate the mean ± standard deviation (SD) of triplicate experiments. (c) Washed mouse platelets (2.0 × 10 7 platelets) were resuspended in modified Tyrode's buffer containing 1% PPP and 200 μM CaCl 2 followed by incubation with PBS or osteosarcoma cells (2.5 × 10 4 cells) for 30 min. After centrifugation twice at 10 000 g for 10 min, the supernatant of osteosarcoma-platelet reactants or the PBS-platelet reactant was added to the cultures of MG63/ZsGreen (left panel) and HOS/ZsGreen (right panel) cells. After 24 h, TENSV buffer was added to each well and the fluorescence of ZsGreen was used to determine the relative number of viable cells. The error bars indicate the mean ± SD of triplicate experiments. ** P < 0.01 by the Student t- test.

Article Snippet: The human osteosarcoma cell lines, MG63 and HOS, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (DMEM growth medium).

Techniques: Co-Culture Assay, Modification, Incubation, Transmission Assay, Stable Transfection, Transfection, Cell Culture, Fluorescence, Standard Deviation, Centrifugation

Journal: Cancer Science

Article Title: Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

doi: 10.1111/cas.12464

Figure Lengend Snippet: Activation of the platelet derived growth factor receptor (PDGFR)-Akt signaling axis in MG63 cells co-cultured with platelets. (a, b) Platelets prepared with heparin from the whole blood of Jcl:ICR mice were resuspended in modified Tyrode's buffer containing 200 μM CaCl 2 . MG63/ZsGreen cells were co-cultured with buffer alone (MG63 + Buffer) or platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-RTK array were performed according to the manufacturer's protocol. Representative images of the probed arrays are shown (a). The signal intensity of each spot was determined using an LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of eight reference spots (within red squares) in each membrane were measured and defined as 100%. The relative intensities of duplicate spots are shown (b). (c, d) Analysis of the human phospho-kinase array using lysates prepared from MG63 cells co-cultured with platelets in modified Tyrode's buffer containing 200 μM CaCl 2 . MG63/ZsGreen cells were cultured with buffer alone (MG63 + Buffer) or with platelets (MG63 + Platelet) for 2 h. The preparation of cell lysates and incubation with the human phospho-kinase array were performed according to the manufacturer's protocol. Representative images of reacted membranes are shown (c). The signal intensity of each spot was measured using a LAS-3000 mini and quantified using Multi Gauge ver.3.0 software. The signal intensities of six reference spots (red squares) in each membrane were defined as 100%. The relative intensities of duplicate spots are shown (d). (e, f) Platelets prepared with 0.38% sodium citrate were resuspended Tyrode's buffer containing 200 μM CaCl 2 . MG63/ZsGreen (e) or HOS/ZsGreen (f) cells were incubated with buffer alone or platelets (2.0 × 10 7 /24-well) for 2 h. Platelets alone, osteosarcoma cells alone (+Buffer) or co-cultures of osteosarcoma cells with platelets (+Platelet) were lysed and immunoblotted using the antibodies to phospho-PDGFRβ, PDGFRβ, phospho-Akt (S473), Akt, or α-tubulin.

Article Snippet: The human osteosarcoma cell lines, MG63 and HOS, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (DMEM growth medium).

Techniques: Activation Assay, Derivative Assay, Cell Culture, Modification, Incubation, Software, Membrane

Journal: Cancer Science

Article Title: Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

doi: 10.1111/cas.12464

Figure Lengend Snippet: Platelet-derived growth factors (PDGFs) released on the osteosarcoma-induced platelet aggregation contribute to the activation of the PDGFR-Akt signaling axis. (a–c) Washed mouse platelets (2.0 × 10 7 platelets) were resuspended in modified Tyrode's buffer containing 1% PPP and 200 μM CaCl 2 followed by incubation with PBS or with the indicated osteosarcoma cells (2.5 × 10 4 cells) for 30 min. After centrifuging twice at 10 000 g for 10 min, the concentration of PDGF-BB in the supernatants was measured using an enzyme linked immunosorbent assay (ELISA) (a). The cultured MG63/ZsGreen (b) and HOS/ZsGreen (c) cells were treated with (+) or without (−) the supernatant of osteosarcoma-platelet aggregates in the presence of DMSO or 1 μM sunitinib. After a 30-min incubation, cells were lysed with TENSV buffer and immunoblotted with the indicated antibodies.

Article Snippet: The human osteosarcoma cell lines, MG63 and HOS, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (DMEM growth medium).

Techniques: Derivative Assay, Activation Assay, Modification, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer Science

Article Title: Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis

doi: 10.1111/cas.12464

Figure Lengend Snippet: Involvement of the platelet-derived growth factor receptor (PDGFR) -Akt signaling axis in the platelet-induced growth of osteosarcoma cells. (a, b) MG63/ZsGreen (a) or HOS/ZsGreen (b) cells in 24-well were co-cultured with platelets (2 × 10 7 in Tyrode's buffer containing 200 μM CaCl 2 ) in the presence of DMSO, the epidermal growth factor receptor (EGFR) inhibitor erlotinib (1 μM), the PDGFR inhibitor sunitinib (1 μM), or the PI3K inhibitor LY294002 (20 μM). After a 48-h incubation, cells were lysed and the fluorescence of ZsGreen was measured to determine relative cell growth. The error bars indicate the mean ± SD of triplicate experiments. ** P < 0.01 and * P < 0.05 (Student's t- test). NS, not significant.

Article Snippet: The human osteosarcoma cell lines, MG63 and HOS, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (DMEM growth medium).

Techniques: Derivative Assay, Cell Culture, Incubation, Fluorescence

Journal: iScience

Article Title: Cell non-autonomous control of autophagy and metabolism by glial cells

doi: 10.1016/j.isci.2024.109354

Figure Lengend Snippet: Expression of XBP-1s in CEPsh glia modulates peripheral lipid metabolism and ER remodeling in C elegans (A) Fluorescent light micrographs of wild-type (N2) and glial XBP-1s animals stained with BODIPY 493/503 dye and imaged at day 2 of adulthood. Scale bar, 250 μm. (B) Whole animal fluorescence intensity quantification of BODIPY 493/503 dye in day 2 adults in wild-type (gray) and glial XBP-1s animals (green) using COPAS BioSorter. p < 0.0001 (∗∗∗∗), using the non-parametric Mann-Whitney test. Plots are representative of three biological replicates, n = 362 (wild type) and n = 302 (glial XBP-1s). Results were normalized to the mean fluorescent intensity of wild-type animals. Each dot represents one animal, and the boxplot shows the median (horizontal line), the first and third quartiles (box), and the smallest and largest data points (whiskers). (C) Fluorescent light micrographs of wild-type and glial XBP-1s animals of intestinal lipid droplets ( dhs-3 p:: dhs-3 ::GFP) imaged at day 2 of adulthood. Scale bar, 250 μm. (D) Whole animal fluorescence intensity quantification of intestinal dhs-3 p:: dhs-3 ::GFP lipid droplet marker in wild-type (gray) and glial XBP-1s animals (green) using COPAS BioSorter. p < 0.0001 (∗∗∗∗), using the non-parametric Mann-Whitney test. Plots are representative of three biological replicates, n = 254 (wild type) and n = 532 (glial XBP-1s). Results were normalized to the mean fluorescent intensity of wild-type animals transgenic for dhs-3 p:: dhs-3 ::GFP. (E) Representative Airyscan micrographs of dhs-3 p:: dhs-3 ::GFP labeled lipid droplets from anterior intestines of day 2 wild-type and glial XBP-1s adult animals. Arrowheads point at lipid droplets: scale bar, 10 μm. (F) Quantification of intestinal dhs-3 p:: dhs-3 ::GFP labeled lipid droplets in wild-type (gray) and glial XBP-1s (green) animals. Lipid droplets were counted from three independent replicates using ImageJ and expressed as density. Density was determined by dividing the number of lipid droplets by the total area expressing dhs-3 p:: dhs-3 ::GFP in each respective image. N = 15 (wild type) and N = 22 (glial XBP-1s). p < 0.0001 (∗∗∗∗), using the non-parametric Mann-Whitney test. (G) Representative Airyscan micrographs of lmp-1 ::GFP labeled lysosomes in the anterior intestine of day 2 adults in both wild-type and glial XBP-1s animals. Arrowheads point at lysosomes: scale bar, 10 μm. (H) Quantification of intestinal lmp-1 ::GFP labeled lysosomes in wild-type (gray) and glial XBP-1s (green) animals. Lysosomes were counted from three independent replicates using ImageJ and expressed as density. Density was determined by dividing the number of lysosomes by the total area that expressed lmp-1 ::GFP in each respective image. N = 15 (wild type) and N = 16 (glial XBP-1s). p < 0.0001 (∗∗∗∗), using the non-parametric Mann-Whitney test. (I) Representative confocal micrographs of intestinal ER morphology ( vha-6p ::ERss:mRuby:HDEL, ERss = hsp- 4 ER signal sequence), wild-type and glial XBP-1s animals were imaged at day 2 of adulthood. Arrowheads mark ER puncta. Scale bar, 10 μm. Scale bar of inset, 5 μm. (J) Electron micrographs of intestine from wild-type and glial XBP-1s animals at day 2 of adulthood. Imaging was replicated in triplicate, for a total of 15–20 animals being imaged per condition. Arrowheads mark rough endoplasmic reticulum. Scale bar, 1μm. Scale bar of inset, 0.2 μm.

Article Snippet: Images of the anterior 30% of the intestine were taken using a 63× objective/1.4 Plan Apochromat objective and standard GFP filter (ZEISS filter set 46 HE).

Techniques: Expressing, Staining, Fluorescence, MANN-WHITNEY, Marker, Transgenic Assay, Labeling, Sequencing, Imaging

Journal: iScience

Article Title: Cell non-autonomous control of autophagy and metabolism by glial cells

doi: 10.1016/j.isci.2024.109354

Figure Lengend Snippet: Glial XBP-1s cell non-autonomously activates intestinal HLH-30 which transcriptionally regulates genes involved in lipid catabolism and lysosomal biogenesis (A) Fluorescent light micrographs of wild-type and glial XBP-1s animals transgenic for fluorescently tagged HLH-30 (HLH-30:GFP) grown on OP50 and imaged at day 2 of adulthood. HLH-30 translocates to the nucleus in glial XBP-1s animals. The inset shows posterior intestine. Scale bar, 250μm. Scale bar of inset, 100μm. (B) Schematic showing how HLH-30:GFP nuclear localization was scored. Categories include no nuclear enrichment (gray), weak nuclear enrichment (green), medium nuclear enrichment (blue), and strong nuclear enrichment (purple). Yellow circle indicates nucleolus, which transcription factors are excluded from. (C) Representative Airyscan micrographs of posterior intestinal cells of wild-type and glial XBP-1s animals transgenic for HLH-30:GFP grown on OP50 and imaged at day 2 of adulthood. Colored inset squares are representative of the scoring categories from (B). Scale bar, 10μm. Intestinal cells are outlined with a dashed gray line, intestinal nuclei are outlined with a dashed white line and intestinal nucleoli are outlined with a dashed yellow line. (D) Nuclear translocation of HLH-30:GFP in anterior intestinal cells of day 2 adult wild-type and glial XBP-1s animals with and without the loss-of-function unc-31 mutant, unc-31(e928) , N > 40 animals per condition from three independent replicates. Statistics done by Chi-squared test for independence with adjusted residual and Bonferroni correction, statistics shown in Table S4 . (E) Representative fluorescent micrograph of wild-type animals transgenic for HLH-30:GFP treated with either DMSO or tunicamycin. Inset shows posterior intestine. Scale bar, 250 μM. Scale bar of inset, 100 μM. Image is representative of two independent replicates, in n > 20 no animals showed nuclear localization. (F) Volcano plot demonstrating magnitude (log2[fold change]) and significance (-log10[adjusted p value]) of changes in gene expression from whole-animal RNA sequencing of glial XBP-1s versus wild-type animals at day 2 of adulthood. Differentially expressed genes (DEGs) shown in red (upregulated, adjusted p value <0.05 and log2[fold change] > 0.5, n = 86) and blue (downregulated, adjusted p value<0.05 and log2[fold change] < 0.5, n = 22). Labeled genes and DEGs with dark red (upregulated DEGs) and dark blue (down-regulated DEGs) dots correspond to HLH-30 targets which are labeled with their corresponding gene. (G) Comparison of log2(fold change) of the 86 upregulated DEGs in glial XBP-1s animals compared to wild-type animals (top) compared to the log2(fold change) of these genes in glial XBP-1s animals with a loss-of-function hlh-30 mutation, hlh-30(tm1978), compared to glial XBP-1s animals alone (bottom). log2(fold change) is color-coded via a heatmap from warm (upregulated) to cool (downregulated). Green dots above the heatmap represent HLH-30 target genes. Statistics shown in Table S5 .

Article Snippet: Images of the anterior 30% of the intestine were taken using a 63× objective/1.4 Plan Apochromat objective and standard GFP filter (ZEISS filter set 46 HE).

Techniques: Transgenic Assay, Translocation Assay, Mutagenesis, Expressing, RNA Sequencing Assay, Labeling, Comparison

Journal: iScience

Article Title: Cell non-autonomous control of autophagy and metabolism by glial cells

doi: 10.1016/j.isci.2024.109354

Figure Lengend Snippet: Glial XBP-1s cell non-autonomously activates peripheral autophagy, which is required for lifespan extension and reduced lipid levels (A) Representative Airyscan micrographs from intestines of day 2 and day 5 wild-type and glial XBP-1s transgenic animals expressing the tandem autophagy reporter mCherry::GFP::lgg-1 (green, GFP, magenta, mCherry). White arrowheads indicate autophagosomes, and yellow arrowheads indicate autolysosomes. Scale bar, 10 μm. (B and C) Quantification of mCherry puncta co-localized with GFP (autophagosomes [AP]) (B) or containing mCherry alone (autolysosome [AL]) (C) in the intestine of wild-type and glial XBP-1s transgenic animals at day 2 and day 5 of adulthood. Data are from three independent experiments, each with ≥ 5 animals. Statistics by Kruskal-Wallis with Dunn’s multiple comparison test, p < 0.0001 (∗∗∗∗). (D) Representative Airyscan micrographs of dhs-3 p:: dhs-3 ::GFP labeled lipid droplets from the intestine of day 2 wild-type and glial XBP-1s adult animals grown on HT115 E. coli expressing control or hlh-30 RNAi. Scale bar, 10 μm. Scale bar of inset, 5 μm. (E) Changes in lipid droplet density of wild-type and glial XBP-1s animals grown on control or hlh-30 RNAi from (D). Boxplot shows median, whiskers are minimum to maximum values, each dot is representative of one animal. Statistics by Kruskal-Wallis with Dunn’s multiple comparison test, p < 0.001 (∗∗∗), p > 0.05 (ns). N > 15, and from three independent replicates. (F) Representative fluorescent micrographs of age-dependent accumulation of transgenic polyQ 44 -YFP aggregates in wild-type or glial XBP-1s animals grown on control or hlh-30 RNAi. Scale bar, 1 mm. Scale bar of inset, 200 μm. (G) Quantification of age-dependent polyQ 44 aggregates from (F), grouped into animals with 0 (gray), 1–3 (green), 4–9 (blue), or >10 (purple) aggregates. N > 100 per condition. Data are representative from three independent replicates. Statistics done by Chi-squared test for independence with adjusted residual and Bonferroni correction, statistics shown in Table S4 . (H and I) Survival of wild-type and glial XBP-1s animals on control, bec-1 (D) or atg-18 RNAi (E) at 20°C. Lifespan is representative of three independent replicates. See Table S1 for lifespan statistics. (J) Representative Airyscan micrographs of transgenic dhs-3 p:: dhs-3 ::GFP labeled lipid droplets from the intestine of day 2 wild-type and glial XBP-1s adults grown on control or bec-1 RNAi. Scale bar, 10 μm. Scale bar of inset, 5 μm. (K) Changes in lipid droplet density of wild-type and glial XBP-1s animals grown on control or bec-1 RNAi from (J). Boxplot shows median, whiskers are minimum to maximum values. Statistics by Kruskal-Wallis with Dunn’s multiple comparison test, p < 0.001 (∗∗∗). N > 15 per condition from three independent replicates.

Article Snippet: Images of the anterior 30% of the intestine were taken using a 63× objective/1.4 Plan Apochromat objective and standard GFP filter (ZEISS filter set 46 HE).

Techniques: Transgenic Assay, Expressing, Comparison, Labeling

Journal: iScience

Article Title: Cell non-autonomous control of autophagy and metabolism by glial cells

doi: 10.1016/j.isci.2024.109354

Figure Lengend Snippet: Macroautophagy is required for changes in intestinal ER morphology and lipid depletion in glial XBP-1s animals (A) Representative Airyscan micrographs from intestine of day 2 wild-type and glial XBP-1s animals transgenic for the ER marker vha-6p ::ERss:mRuby:HDEL, animals were grown on control or the corresponding RNAi. ER puncta is denoted by arrowheads. (B) Quantification of ER puncta from (A). Density was determined by counting the number of ER puncta per area of intestine expressing vha-6p ::ERss:mRuby:HDEL. N > 15 per condition from three independent replicates. Statistics by Kruskal-Wallis with Dunn’s multiple comparison test, p < 0.001 (∗∗∗), p < 0.0001 (∗∗∗∗). (C) Representative Airyscan micrographs from intestines of day 2 wild-type and glial XBP-1s animals transgenic for the ER marker vha-6p ::ERss:mRuby:HDEL (pseudo-colored in magenta) and stained with Lysotracker Blue DND-22 (pseudo-colored in green). Arrowheads mark colocalization. Scale bar, 10 μm. Scale bar of inset, 5 μm. (D) Representative Airyscan micrographs from intestine of day 2 wild-type and glial XBP-1s animals transgenic for the ER marker vha-6p ::ERss:mRuby:HDEL (magenta) and autophagosome marker LGG-1:GFP (green). Arrowheads mark colocalization. Scale bar, 10 μm. Scale bar of inset, 5 μm. (E) Quantification of density of ER puncta and lysotracker colocalization. Statistics by Mann-Whitney test, p < 0.0001 (∗∗∗∗). N > 15 per condition from three independent replicates. (F) Quantification of density of ER puncta and autophagosome colocalization. Statistics by Mann-Whitney test, p < 0.0001 (∗∗∗∗). N > 15 per condition from three independent replicates. (G) Electron micrographs of intestine from wild-type and glial XBP-1s animals at day 2 of adulthood grown on either empty vector control or bec-1 RNAi. Imaging was replicated in triplicate for a total of 15–20 animals being imaged per condition. Arrowheads mark rough endoplasmic reticulum. Scale bar, 1μm. Scale bar of inset, 0.2 μm.

Article Snippet: Images of the anterior 30% of the intestine were taken using a 63× objective/1.4 Plan Apochromat objective and standard GFP filter (ZEISS filter set 46 HE).

Techniques: Transgenic Assay, Marker, Expressing, Comparison, Staining, MANN-WHITNEY, Plasmid Preparation, Imaging

Journal: iScience

Article Title: Cell non-autonomous control of autophagy and metabolism by glial cells

doi: 10.1016/j.isci.2024.109354

Figure Lengend Snippet:

Article Snippet: Images of the anterior 30% of the intestine were taken using a 63× objective/1.4 Plan Apochromat objective and standard GFP filter (ZEISS filter set 46 HE).

Techniques: Virus, Recombinant, Electron Microscopy, Gel Extraction, Purification, Software, Microscopy