Journal: Investigative Ophthalmology & Visual Science

Article Title: De-Differentiation of Corneal Epithelial Cells Into Functional Limbal Epithelial Stem Cells After the Ablation of Innate Stem Cells

doi: 10.1167/iovs.65.13.32

Figure Lengend Snippet: The role of Hippo/YAP pathway in the de-differentiation of CECs in vivo. ( A ) Immunostaining of YAP in frozen sections of normal cornea and LESCs-ablation cornea at indicated days after the limbal epithelial removal. The limbus was shown. ( B , C ) MFI and N/C ratio of YAP in limbal basal cells were quantified after the limbal epithelial removal. ( D – F ) The administration of vehicle or VTP or TRULI on cornea for 4 days after the LESCs ablation, and expressions of ApoE and Cx43 were examined by immunostaining at 6 days. The percentage of ApoE + LESCs and MFI of Cx43 in limbal basal cells were quantified. ( G – J ) The frozen-section YAP, Ki67, and pH3 (indicators of cell proliferation) immunostaining against cornea of scratched limbus either with or without NaOH application at 10 days after the LESCs ablation. Arrows point to CECs with nuclear YAP. The MFI and N/C ratio of YAP in limbal basal cells and the percentage of Ki67 + limbal basal cells were quantified. ( K – M ) Corneas with scratched limbus and localized NaOH application were administrated with VTP for 4 days. The frozen-section ApoE and Cx43 immunostaining against cornea of scratched limbus either with or without NaOH application at 6 days. The percentage of ApoE + LESCs and MFI of Cx43 in limbal basal cells were quantified. ( N ) Comparison of MFI of Cx43 in limbal basal cells of corneas that were administrated with vehicle or VTP at 6 days after LESCs ablation and NaOH treatment. Data are the mean ± SD, n = 5 biological replicates ( B , E , F , H , J , L – N ) or 10 cells ( C , I ); vehicle = 39.76 ± 7.15, VTP = 66.25 ± 9.51, and TRULI = 3.46 ± 5.23 ( E ); vehicle = 22.52 ± 3.47, VTP = 14.21 ± 4.49, and TRULI = 35.73 ± 17.42 ( F ); limbus (S) = 23.28 ± 11.11, central cornea = 51.03 ± 11.28, and limbus (S+NaOH) = 53.27 ± 13.03 ( H ); limbus (S) = 55.63 ± 23.16, central cornea = 72.10 ± 20.68, and limbus (S+NaOH) = 69.72 ± 25.14 ( I ); limbus (S) = 8.92 ± 2.68, central cornea = 27.37 ± 4.66, and limbus (S+NaOH) = 51.20 ± 10.66 ( J ); limbus (S) = 43.21 ± 6.65, and limbus (S+NaOH) = 0 ± 0 ( L ); limbus (S) = 21.67 ± 4.09, and limbus (S+NaOH) = 44.51 ± 4.23 ( M ); vehicle = 45.40 ± 6.28 and VTP = 44.51 ± 4.23 ( N ); statistical analysis were performed by unpaired ( E , F ) or paired ( H – J ) 1-way ANOVA with Tukey's test or unpaired Student's t -test ( L – N ). Scale bars = 20 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-CK12 monoclonal antibody (Abcam, ab185627; 1:400), rabbit anti-CK7 monoclonal antibody (Abcam, ab181598; 1:400), rabbit anti-connexin 43 (Cx43) monoclonal antibody (CST, #3512; 1:80), rabbit anti-ApoE monoclonal antibody (Abcam, ab183596; 1:400), rabbit anti-CK14 monoclonal antibody (Abcam, ab119695; 1:200), rabbit anti-deltaN-p63 polyclonal antibody (BioLegend, 619002; 1:400), rabbit anti-p75NTR monoclonal antibody (CST, #8238; 1:800), rabbit anti-CD63 polyclonal antibody (Bioworld, BS72936; 1:400), rabbit anti-TSPAN7 polyclonal antibody (Proteintech, 18695-1-AP; 1:100), rabbit anti-IFITM3 monoclonal antibody (CST, #59212; 1:100), mouse anti-ATF3 monoclonal antibody (Santa Cruz, sc-518032; 1:100), rabbit anti-YAP monoclonal antibody (CST, #14074; 1:100), rabbit anti-active-YAP (non-phosphorylated YAP) monoclonal antibody (Abcam, ab205270; 1:200), rabbit anti-Ki67 monoclonal antibody (CST, #9129; 1:100), rabbit anti-p-Histone H3-Ser10 (pH3) monoclonal antibody (CST, #53348; 1:100), and mouse anti-CK15 monoclonal antibody (Santa Cruz, sc-47697; 1:200).

Techniques: In Vivo, Immunostaining, Comparison

Journal: Cancer Research

Article Title: MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia

doi: 10.1158/0008-5472.CAN-21-0061

Figure Lengend Snippet: TH1579 kills AML cells by causing G 2 –M arrest, increases ROS, genomic 8oxodG, and DNA damage. A, A total of 24 hours treatment of TH1579 induced cell death mostly from G 2 –M. n = 3. B, TH1579 causes G 2 –M arrest in HL60 cells. Mean ± SEM, n = 3 . C, TH1579 increases number of mitotically arrested cells. After 24 hours treatment, cells were stained with Histone H3-pS10 antibodies and DAPI. H3pS10-positive cells with chromatin condensation were scored as mitotic cells. Data shown as percentage of total cells. Mean ± SEM from two to four independent experiments. ***, P < 0.001, one-way ANOVA, Tukey multiple comparison. D, TH1579 induces ROS levels in HL60 cells. ROS was measured using ROS indicator CM-H2DCF. After 18 hours of treatment, it was analyzed using FACS and normalized to control (DMSO)-treated cells. Mean ± SEM from four independent experiments. *, P < 0.05, one-way ANOVA, Tukey multiple comparison. E, Quantitative data of modified comet assay from HL60 cells treated with MTH1 inhibitor for 24 hours showing an increase in tail moment after addition of OGG1 enzyme in DNA of HL60 cells. Data shown as mean ± SEM from n = 2 independent experiments. F, TH1579 (400 nmol/L, 18 hours treatment) significantly elevates ROS in NB4 and A3 cells, while no ROS induction was observed in the resistant KBM3 cells. Data normalized to control (DMSO)-treated cells. Mean ± SEM from four independent experiments. *, P < 0.05 compared with control-treated cells in NB4, A3, KBM3, respectively. Student t test. G, NAC treatment partly rescues TH1579-induced toxicity. HL60, NB4, and A3 cells treated with 2.5 mmol/L NAC and TH1579 at concentrations as mentioned in the figure for 72 hours. Mean ± SEM; n = 3 independent experiments. *, P < 0.05, Student t test. (full curves in Supplementary Fig. S1D–F). H, Quantification of Western blot analysis from three independent treatment experiments following 24-hour TH1579 treatment in HL60 cells showing increased expression of DNA damage, mitotic, and apoptotic markers. Mean ± SEM. **, P <0.01; ****, P < 0.0001. Two-way ANOVA, Tukey multiple comparison (representative Western blot analysis in Supplementary Fig. S2A). I, A typical example of Western blot analysis following 18-hour TH1579 treatment in HL60, NB4, and KBM3 cells, showing elevated levels of DNA damage in the TH1579-sensitive NB4 and HL60, but no DNA damage response in resistant KBM3 cells. J, Dose-dependent increase in percentage of dead cells measured by FACS analysis of the subG 1 population in HL60 cells. Data shown as mean ± SEM from three to four experiments.

Article Snippet: Following antibodies were used: rabbit anti-53BP1 (A300-272A, Bethyl Laboratories), mouse anti beta-Actin (ab6276, Abcam), rabbit anti-cyclin B1 (sc-594, Santa Cruz Biotechnology), rabbit anti-GAPDH (sc-25778, Santa Cruz Biotechnology), mouse anti-H2AX phospho S139 (05-636, Millipore), mouse anti-Histone H3 phospho-S10 (pH3; ab14955, Abcam), rabbit anti-pH3 (ab5176, Abcam), goat anti-MAD2 (sc-6329, Santa Cruz Biotechnology), rabbit anti-MTH1 (NB100-109, Novus Biologicals), and rabbit anti-cleaved PARP (#9541, Cell Signaling Technology).

Techniques: Staining, Comparison, Control, Modification, Single Cell Gel Electrophoresis, Western Blot, Expressing

Journal: Nature aging

Article Title: Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids.

doi: 10.1038/s43587-020-00001-7

Figure Lengend Snippet: Fig. 4 | Sestrin is important for maintenance of ISCs in response to DR. a,b, Gut stem cell activity of wDah and Sesn3F6 flies under DR. a, Representative images from 20-day-old flies. Actively dividing stem cells are indicated by arrows. b, Quantification of pH3-positive cells in Sesn3F6 females under DR. n = 25 guts (wDah, Sesn3F6, 2.0×; Sesn3F6, 1.0×), n = 24 guts (wDah, 1.0×). c,d, Gut turnover rates on Sestrin knockdown (KD). c, Representative images after 7 d induction. d, Quantification of gut turnover rates in Sestrin knockdown flies under DR. n = 14 guts (wDah, 2.0×, 1.0×), n = 16 guts (Sesn KD, 2.0×), n = 17 guts (Sesn KD, 1.0×). e,f, Gut stem cell activity of SesnR407A mutant flies under DR conditions. e, Representative images from 20-day-old flies. f, Quantification of pH3-positive cells in SesnR407A mutants under DR. n = 25 guts (Sesnwt, 2.0×, 1.0×), n = 24 guts (SesnR407A, 2.0×, 1.0×). g,h, Gut turnover rates in SesnR407A mutant flies under DR. g, Representative images after 7 d induction. h, Quantification of gut turnover rates in SesnR407A mutants under DR. n = 14 guts (Sesnwt, 2.0×; SesnR407A, 1.0×), n = 15 guts (Sesnwt, 1.0×; SesnR407A, 2.0×). i,j, Quantification of gut turnover rates in SesnR407A mutants on addition of all amino acids (i) and addition of methionine and BCAAs (MBC) (j). n = 13 guts (i), n = 16 guts (j). Scale bars, 50 μm. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (b,d,f,h–j). Outliers are shown as open circles. Interaction between diet and genotype was significant in b,d,f and h–j: two-way ANOVA, P = 0.02 (b), P = 0.01 (d), P = 0.02 (f), P = 0.01 (h), P < 0.0001 (i), P = 0.047 (j). Statistics in b,d,f and h–j: two-way ANOVA followed by Bonferroni’s post-hoc test, P values were adjusted for multiple comparisons.

Article Snippet: Lee Anti pH3 (S10) rabbit polyclonal, Cell signaling, Cat# 9701 Anti Myc (9B11) mouse monoclonal, Cell signaling, Cat# 2276 Anti Flag M2 mouse monoclonal, Sigma, Cat# F1804 Anti HA rabbit polyclonal, Sigma, Cat# H6908 Anti pS6K (T398) rabbit polyclonal, Cell signaling, Cat# 9209 Anti S6K rabbit polyclonal, home-made Anti pAMPKα (T172) (40H9) rabbit monoclonal, Cell signaling, Cat# 2535 Anti pAKT (S505) rabbit polyclonal, Cell signaling, Cat# 4054 Anti AKT rabbit polyclonal, Cell signaling, Cat# 9272 Anti Tubulin (DM1A) mouse monoclonal, Sigma, Cat# T9026 Goat anti-rabbit IgG IRDye 680RD, LI-COR, Cat# 926-68071 Goat anti-mouse IgG IRDye 800CW, LI-COR, Cat# 926-32210 Goat anti-rabbit IgG (H+L), Alexa Fluor 594, Thermo Fisher Scientific, Cat# A11012 Validation Anti Sestrin rabbit polyclonal, home-made, gift from J.H.

Techniques: Activity Assay, Knockdown, Mutagenesis, Whisker Assay

Journal: Nature aging

Article Title: Sestrin is a key regulator of stem cell function and lifespan in response to dietary amino acids.

doi: 10.1038/s43587-020-00001-7

Figure Lengend Snippet: Fig. 5 | Sestrin overexpression in gut stem cells improves gut homeostasis. a,b, Ubiquitous overexpression of Sestrin reduced gut stem cell activity. a, Representative images from 30-day-old flies without (RU486−) or with (RU486+) induction. Actively dividing stem cells are indicated by arrows. Scale bar, 50 μm. b, Quantification of pH3-positive cells on days 10, 30 and 50. n = 20 guts (RU486−), n = 22 guts (RU486+), day 10; n = 23 guts (RU486−, RU486+), day 30; n = 24 guts (RU486−, RU486+), day 50. Generalized linear modelling analysis confirmed that Sestrin overexpression significantly reduced the increase in age-dependent stem cell activity (P = 0.012). c, Overexpression of Sestrin in enterocytes (5966GS) did not affect pH3-positive cell numbers. Day 30: n = 22 guts (RU486−), n = 21 guts (RU486+); day 46: n = 22 guts (RU486−), n = 20 guts (RU486+). d, In contrast, overexpression of Sestrin in gut stem cells (5961GS) significantly reduced stem cell activity. Day 30: n = 24 guts (RU486−), n = 25 guts (RU486+); day 46: n = 22 guts (RU486−), n = 23 guts (RU486+). e, Sestrin overexpression (OE) prolonged the G1 phase in gut stem cells. Fly-Fucci and Sestrin were driven by the esg-Gal4 driver. Sestrin overexpression significantly changed the cell cycle distribution of gut stem cells. n = 416 cells, wDah; n = 382 cells, Sesn OE. Chi-square test. f–h, Sestrin overexpression in gut stem cells decreased gut dysplasia (f,g) and the proportion of Smurf flies (h). f, Representative gut images from 50-day-old flies. DNA (DAPI) in blue. The gut epithelium is indicated by dashed lines. Scale bar, 20 μm. g, The difference in gut dysplasia was significant. n = 13 guts. h, Flies were 60 days old. n = 15 vials. Median, 25th and 75th percentiles, and Tukey whiskers are indicated in box-and-whisker plots (b–d,g,h). Outliers are shown as open circles. Statistics in b–d: two-tailed, Mann–Whitney test; statistics in g and h: unpaired, two-tailed t-test.

Article Snippet: Lee Anti pH3 (S10) rabbit polyclonal, Cell signaling, Cat# 9701 Anti Myc (9B11) mouse monoclonal, Cell signaling, Cat# 2276 Anti Flag M2 mouse monoclonal, Sigma, Cat# F1804 Anti HA rabbit polyclonal, Sigma, Cat# H6908 Anti pS6K (T398) rabbit polyclonal, Cell signaling, Cat# 9209 Anti S6K rabbit polyclonal, home-made Anti pAMPKα (T172) (40H9) rabbit monoclonal, Cell signaling, Cat# 2535 Anti pAKT (S505) rabbit polyclonal, Cell signaling, Cat# 4054 Anti AKT rabbit polyclonal, Cell signaling, Cat# 9272 Anti Tubulin (DM1A) mouse monoclonal, Sigma, Cat# T9026 Goat anti-rabbit IgG IRDye 680RD, LI-COR, Cat# 926-68071 Goat anti-mouse IgG IRDye 800CW, LI-COR, Cat# 926-32210 Goat anti-rabbit IgG (H+L), Alexa Fluor 594, Thermo Fisher Scientific, Cat# A11012 Validation Anti Sestrin rabbit polyclonal, home-made, gift from J.H.

Techniques: Over Expression, Activity Assay, Whisker Assay, Two Tailed Test, MANN-WHITNEY