Journal: BMC Cancer

Article Title: Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

doi: 10.1186/s12885-017-3319-0

Figure Lengend Snippet: ATM activation is linked to γ-H2AX formation while ATR activity contributes to proliferation in A673 spheroids. a Activation of the hypoxia-responsive factor HIF1-α ( red with blue Hoechst 33,342 counter-stain) is observed in the peri-necrotic zone of A673 spheroids 570–650 μm in diameter. b Activation of ATM (phosphorylation at serine 1981 (pATM); red with blue Hoechst 33,342 counter-stain) is seen in the hypoxic, peri-necrotic zone. c The distribution of γ-H2AX ( red , with blue Hoechst 33,342 counter-stain) in spheroids maintained for 96 h in either vehicle, the ATM inhibitor KU55933, or the ATR inhibitor VE-821. Spheroids were <500 μm in diameter at the start of the experiment and vehicle-treated spheroids were approximately 700 μm in diameter at the end of 96 h. Both KU55933 and VE-821 impaired spheroid growth. d The distribution of Ki-67 ( green , with blue Hoechst 33,342 counter-stain) in spheroids maintained for 96 h in either vehicle, the ATM inhibitor KU55933, or the ATR inhibitor VE-821. e Bar graph showing percentage of γ-H2AX-positive cells per spheroid when maintained for 96 h in either vehicle, the ATM inhibitor KU55933, or the ATR inhibitor VE-821. Mean and standard deviation are shown; each bar represents at least 15 spheroids from three independent experiments. f Percentage of Ki-67-positive cells per spheroid when maintained for 96 h in either vehicle, the ATM inhibitor KU55933, or the ATR inhibitor VE-821 is shown as the mean and standard deviation; each bar represents at least 15 spheroids from three independent experiments. One way ANOVA with Dunnett’s post-test. *** P < .001, ns P > .05

Article Snippet: Human Ewing Sarcoma A673 (CRL-1598; ATCC, Manassas, VA) and LLC (CRL-1642; ATCC, Manassas, VA); Dulbecco’s Modified Eagles Medium, DMEM, (11,965,092; Thermoscientific, Waltham, MA); Fetal Bovine serum - FBS, (TMS-013-B; Millipore, Billerica, MA); Agar (J637; Amresco, Solon, OH); DMSO (67–68-5, Sigma, St. Louis, MO); Proox-p110 ProCO 2 Hypoxia Chamber (Biospherix, Parish, NY); Inflatable Glove Bag Model X-37-27 (108D; Glas-Col, Terre Haute, IN); ATM inhibitor KU55933 (S1092; Selleckchem, Houston, TX); ATR inhibitor VE-821 (S8007; Selleckchem, Houston, TX); Click-IT Plus EdU imaging kit (MP10637, Life technologies, Carlsbad, CA).

Techniques: Activation Assay, Activity Assay, Staining, Phospho-proteomics, Standard Deviation

Journal: BMC Cancer

Article Title: Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

doi: 10.1186/s12885-017-3319-0

Figure Lengend Snippet: ATM or ATR inhibition attenuates the severe hypoxia-induced increase in γ-H2AX levels in spheroids. a A673 spheroids ~400 μm in diameter were maintained for 12 h in either 20% O 2 or 1% O 2 in vehicle, 1% O2 in KU55933 or 1% O2 in VE-821. The effect of ATM inhibition (KU55933) or ATR inhibition (VE-821) on γ-H2AX distribution ( red , with blue Hoechst 33,342 counter-stain) is shown. b Percentage of γ-H2AX-positive cells in spheroids was quantitated. Bar graph shows the mean and standard deviation from >14 spheroids and at least 3 independent experiments. One way ANOVA with Dunnett’s post-test. *** P < .001, ** P < .01, ns P > .05

Article Snippet: Human Ewing Sarcoma A673 (CRL-1598; ATCC, Manassas, VA) and LLC (CRL-1642; ATCC, Manassas, VA); Dulbecco’s Modified Eagles Medium, DMEM, (11,965,092; Thermoscientific, Waltham, MA); Fetal Bovine serum - FBS, (TMS-013-B; Millipore, Billerica, MA); Agar (J637; Amresco, Solon, OH); DMSO (67–68-5, Sigma, St. Louis, MO); Proox-p110 ProCO 2 Hypoxia Chamber (Biospherix, Parish, NY); Inflatable Glove Bag Model X-37-27 (108D; Glas-Col, Terre Haute, IN); ATM inhibitor KU55933 (S1092; Selleckchem, Houston, TX); ATR inhibitor VE-821 (S8007; Selleckchem, Houston, TX); Click-IT Plus EdU imaging kit (MP10637, Life technologies, Carlsbad, CA).

Techniques: Inhibition, Staining, Standard Deviation

Journal: BMC Cancer

Article Title: Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

doi: 10.1186/s12885-017-3319-0

Figure Lengend Snippet: 1% O 2 is sufficient to induce DNA damage in A673 monolayers and inhibition of ATR promotes H2AX phosphorylation. a The effect of maintaining monolayers of A673 cells in 1% O 2 for 12 h was monitored. Immunofluorescence indicates the formation of distinct γ-H2AX foci ( red ) in A673 cells. b Western blot shows elevation of γ-H2AX levels in A673 cells maintained in 1% O 2 for 12 h. c Alkaline COMET assay shows induction of COMET tails (a direct measure of DNA damage) when A673 cells were maintained in 1% O 2 for 12 h. Tail moments were quantitated using OpenCOMET and are plotted as the mean and standard deviation. d Bar graph showing the percentage of γ-H2AX-positive A673 cells when monolayers were maintained for 12 h in either 20% O 2 or 1% O 2 in vehicle, 1% O 2 in KU55933 (ATM inhibitor), or 1% O2 in VE-821 (ATR inhibitor). Mean and standard deviation from two independent experiments are shown. e Immunofluorescence for γ-H2AX ( red ) in A673 monolayers maintained for 12 h in either 20% O 2 or 1% O 2 in vehicle, 1% O2 in KU55933 (ATM inhibitor), or 1% O2 in VE-821 (ATR inhibitor). ATR inhibition under hypoxia results in cells with either intense pan-nuclear γ-H2AX or distinct foci, while only distinct γ-H2AX foci are seen under hypoxia in the presence of either vehicle or ATM inhibitor. One way ANOVA with Tukey’s multi-comparison post-test. *** P < .001, ** P < .01, * P < .05, ns P > .05

Article Snippet: Human Ewing Sarcoma A673 (CRL-1598; ATCC, Manassas, VA) and LLC (CRL-1642; ATCC, Manassas, VA); Dulbecco’s Modified Eagles Medium, DMEM, (11,965,092; Thermoscientific, Waltham, MA); Fetal Bovine serum - FBS, (TMS-013-B; Millipore, Billerica, MA); Agar (J637; Amresco, Solon, OH); DMSO (67–68-5, Sigma, St. Louis, MO); Proox-p110 ProCO 2 Hypoxia Chamber (Biospherix, Parish, NY); Inflatable Glove Bag Model X-37-27 (108D; Glas-Col, Terre Haute, IN); ATM inhibitor KU55933 (S1092; Selleckchem, Houston, TX); ATR inhibitor VE-821 (S8007; Selleckchem, Houston, TX); Click-IT Plus EdU imaging kit (MP10637, Life technologies, Carlsbad, CA).

Techniques: Inhibition, Phospho-proteomics, Immunofluorescence, Western Blot, Alkaline Single Cell Gel Electrophoresis, Standard Deviation, Comparison