Journal: bioRxiv

Article Title: The Hippo/YAP Pathway Mediates the De-differentiation of Corneal Epithelial Cells into Functional Limbal Epithelial Stem Cells In Vivo

doi: 10.1101/2024.06.11.596348

Figure Lengend Snippet: ( A ) Immunostaining of YAP in frozen sections of normal cornea and LESCs-ablation cornea at indicated days after the limbal epithelium removal. The limbus was shown. ( B ) 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 the mean fluorescence intensity (MFI) of Cx43 in limbal basal cells were quantified. ( C ) 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 of YAP in limbal basal cells and the percentage of Ki67 + limbal basal cells were quantified. ( D ) 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 after the LESCs ablation. Data are the mean ± SD, n=5 biological replicates; statistical analysis were performed by unpaired ( B ) or paired ( C ) one-way ANOVA with Tukey’s test. Scale bars, 20 um.

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-p-YAP-Ser127 monoclonal antibody (Abcam, ab76252; 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: Immunostaining, Fluorescence

Journal: Nature Communications

Article Title: Multiplexed and reproducible high content screening of live and fixed cells using Dye Drop

doi: 10.1038/s41467-022-34536-7

Figure Lengend Snippet: a Relative cell viability in OptiPrep™ fixed cells as compared to live cell microscopy following 24 h treatments with increasing concentrations of dinaciclib, paclitaxel, staurosporine, and vincristine in MCF 10A-H2B-mCherry cells. Error bars represent the standard error of the mean of eight technical replicates from one representative biological replicate. b Deep Dye Drop protocol steps: EdU and LDR dye are added in 10% OptiPrep™ followed by fixation with 4% formaldehyde in 20% Optiprep™. Cells are then permeabilized with 0.5% Triton X-100 in 10% OptiPrep, and the EdU is labeled with a fluorescent dye azide via Click chemistry in 20% OptiPrep™. The contents of the well are aspirated, cells are blocked, and then stained with a conjugated antibody against phospho-histone H3 (pH3) in 10% OptiPrep™. One well of a multi-well plate is depicted. c Schematic and representative image of cells stained with the Deep Dye Drop protocol. The image shown is an example of typical of Deep Dye Drop staining as performed in 58 breast cancer cell lines in this work. Nuclei are stained with Hoechst (gray-scale), dead cells are stained with LIVE/DEAD red (blue), S-phase cells are labeled with EdU (red) and M-phase cells are stained with phospho-histone H3 (green). Scale bar is 100 µm. d Thresholds set to classify dead cells shown on a distribution of LDR intensity values and e to identify cells in M-phase shown on a distribution of pH3 intensity values. f Scatter plot of EdU intensity versus DNA content. The red dotted lines represent gating applied to assign cells to the sub G1, G1, G2, beyond G2, and S-phases of the cell cycle (see online “Methods”). g DNA content in BT20 cells treated for 72 h with inhibitors targeting CHK1 (1 µM LY2606368), CDK1/2 (3.16 µM BMS-265246) and PLK4 (0.316 µM CFI-400495) and untreated controls. All cells from a single well in a 384-well plate are shown per condition. h The difference in GR values calculated from Deep Dye Drop and conventional assays with respect to the GR value from the Deep Dye Drop assay. The gray bars represent the 90% confidence intervals for GR values from Deep Dye Drop experiments performed in biological triplicate. Source data are provided as a Source Data file.

Article Snippet: Next, an Odyssey blocking buffer solution containing 10% OptiPrepTM (final concentration of 6% iodixanol) and 1:2000 Alexa 488-conjugated anti-phospho-histone H3 (S10) antibody (pH3) (clone D2C8, catalog number 3465, lot number 14, Cell Signaling Technology, Danvers, MA) to label M-phase cells and 2 μg/ml Hoechst 33342 to stain all nuclei, was dropped onto the cells and incubated overnight at 4 °C.

Techniques: Microscopy, Labeling, Staining

Journal: Nature Communications

Article Title: Multiplexed and reproducible high content screening of live and fixed cells using Dye Drop

doi: 10.1038/s41467-022-34536-7

Figure Lengend Snippet: a MCF7 cells stained with phospho-pRb and b actin untreated and after 72 h in 1 µM palbociclib; effects of increasing concentrations of palbociclib on the fraction of phospho-pRb positive MCF7 cells, or b on cell size as detected with actin staining after 72 h. c MCF 10 A cells stained with 53BP1 untreated and after 72 h in 1 µM etoposide; induction of DNA damage by increasing concentrations of etoposide in MCF 10 A cells as detected with 53BP1 staining after 72 h. d MCF7 cells stained with cytochrome C untreated and after 72 h in 0.1 µM actinomycin D; effect of increasing concentrations of actinomycin D on release of cytochrome C from the mitochondria in MCF7 cells after 72 h, performed in duplicate. Nuclei are stained with Hoechst (gray-scale), and EdU (red) in all images. Error bars represent the standard deviation of the mean of four replicates. Scale bars are 50 µm. e Schematic and representative image from three cell lines in a 384-well plate stained with the addition of a fifth channel to the Deep Dye Drop assay. Cells are stained with Hoechst (gray-scale), LDR (blue, 1), EdU (red, 2), pH3 (purple, 3) and 53BP1 (green, 4). Scale bar is 50 µm. Source data are provided as a Source Data file.

Article Snippet: Next, an Odyssey blocking buffer solution containing 10% OptiPrepTM (final concentration of 6% iodixanol) and 1:2000 Alexa 488-conjugated anti-phospho-histone H3 (S10) antibody (pH3) (clone D2C8, catalog number 3465, lot number 14, Cell Signaling Technology, Danvers, MA) to label M-phase cells and 2 μg/ml Hoechst 33342 to stain all nuclei, was dropped onto the cells and incubated overnight at 4 °C.

Techniques: Staining, Standard Deviation

Journal: Nature Communications

Article Title: Multiplexed and reproducible high content screening of live and fixed cells using Dye Drop

doi: 10.1038/s41467-022-34536-7

Figure Lengend Snippet: a Schematic and representative image from technical triplicates of multiplexing Deep Dye Drop assays with cyclic immunofluorescence: the Hoechst (gray-scale), EdU (red), pH3 (green), beta-catenin (cyan), phospho-pRb (blue), and p21 (yellow) signals are displayed, and contrast was adjusted for visualization purposes only. Scale bar is 100 µm. b UMAP representation of MCF7 and MCF 10 A cells treated with BMS-265246 (1 µM, 10 µM), ribociclib (10 µM) or DMSO stained with Deep Dye Drop and cyclic immunofluorescence. c The number of MCF7 cells in S-phase following treatment with increasing concentrations of ribociclib, d palbociclib, and e abemaciclib after 6, 24, 48, and 72 h. Data are presented as mean +/− standard deviation of four technical replicates. EdU versus DNA content scatter plots show the single cell cell-cycle distributions from one well at 1 µM doses of each drug at the time points indicated, the percentage of cells in S-phase (average from four technical replicates) is indicated on each plot. The scatter plots show all cells in a single, representative well of a 384-well plate for each condition. f Illustration of possible patterns of the emergence of resistant cells. A, clonal, genetic resistance or B, non-clonal, non-genetic adaptation followed by representative images from biological duplicates performed in technical quadruplicate of MCF7 cells treated with 1 µM palbociclib for 24 h or 72 h. Nuclei are visualized with Hoechst in white and EdU positive cells are shown in red, scale bars are 100 µm. Source data are provided as a Source Data file and on Synapse.

Article Snippet: Next, an Odyssey blocking buffer solution containing 10% OptiPrepTM (final concentration of 6% iodixanol) and 1:2000 Alexa 488-conjugated anti-phospho-histone H3 (S10) antibody (pH3) (clone D2C8, catalog number 3465, lot number 14, Cell Signaling Technology, Danvers, MA) to label M-phase cells and 2 μg/ml Hoechst 33342 to stain all nuclei, was dropped onto the cells and incubated overnight at 4 °C.

Techniques: Multiplexing, Immunofluorescence, Staining, Standard Deviation

Journal: Investigative ophthalmology & visual science

Article Title: Loss of citron kinase affects a subset of progenitor cells that alters late but not early neurogenesis in the developing rat retina.

doi: 10.1167/iovs.14-15272

Figure Lengend Snippet: FIGURE 2. Number of progenitor cells in WT and KO retinae is comparable at E12. Immunohistochemistry on retinal sections obtained from embryonic day E12 embryos where the mother was pulsed with EdU 1 hour before harvest. (A, D) Retinal progenitor cells positive for Ki67 (green) and PH3 (red) in WT (A) and KO (D). (B, E) Retinal progenitor cells positive for EdU (magenta) in WT (B) and KO (E). DAPI (blue) marks all nuclei. (C, F) Merged image showing RPCs positive for Ki67 (green) and EdU (magenta) in WT (C) and KO (F). DAPI (blue) marks all nuclei. Inset shows the higher magnification image of the boxed region in the apical end of the ONBL where the solid arrow points to an RPC that is EdUþ and Ki67þ. (G) Quantification of S-phase RPCs (EdUþcells) as a percentage of all RPCs (Ki67þcells) in WT (blue, n¼6) and KO (red, n¼9). (H) Quantification of M-phase RPCs (PH3þ cells) as a percentage of all RPCs (Ki67þ cells) in WT (blue, n ¼ 6) and KO (red, n ¼ 9).

Article Snippet: Primary antibody ([product No. 40.2D6, mouse anti-Islet1, 1:300; Developmental Studies Hybridoma Bank, Iowa, IA, USA]; [product No. 556003, mouse anti-Ki67; BD Biosciences, San Jose, CA, USA]; [product No. IHC-00061, rabbit anti–Phospho Histone H3 (PH3), 1:300; Bethyl Laboratories, Inc., Montgomery, TX, USA]; [product No. PRB-278P, rabbit anti-Pax6, 1:300; Covance, Dedham, MA, USA]; [product No. MABN15, mouse anti-rhodopsin, clone 4D2, 1:300; Millipore, Billerica, MA, USA]; and [product No. AB5405, rabbit anti-red/green opsin, 1:300; Millipore]) was incubated in PBTS buffer overnight at 48C.

Techniques: Immunohistochemistry

Journal: Investigative ophthalmology & visual science

Article Title: Loss of citron kinase affects a subset of progenitor cells that alters late but not early neurogenesis in the developing rat retina.

doi: 10.1167/iovs.14-15272

Figure Lengend Snippet: FIGURE 3. Number of RPCs and RGCs in WT and KO retinae is comparable at E13. Immunohistochemistry on retinal sections obtained from E13 embryos where the mother was pulsed with EdU at E12. (A, D) Retinal progenitor cells positive for Ki67 (green) and PH3 (red) in WT (A) and KO (D). (B, E) Retinal progenitor cells positive for EdU (magenta) in WT (B) and KO (E). (C, F) Merged image showing RPCs positive for Ki67 (green) and EdU (magenta) in WT (C) and KO (F). DAPI (blue) marks all nuclei. Inset shows the higher magnification image of the boxed region in the apical end of the ONBL where the solid arrow points to an RPC that is EdUþ and Ki67þ. (G–H’’) Retinal sections showing EdUþ cells (magenta) in WT (G) and KO (H) along with IHC for Islet1 (green) in WT (G’) and KO (H’). Shown in (G’’) and (H’’) are the merged images of (G) and (G’) and

Article Snippet: Primary antibody ([product No. 40.2D6, mouse anti-Islet1, 1:300; Developmental Studies Hybridoma Bank, Iowa, IA, USA]; [product No. 556003, mouse anti-Ki67; BD Biosciences, San Jose, CA, USA]; [product No. IHC-00061, rabbit anti–Phospho Histone H3 (PH3), 1:300; Bethyl Laboratories, Inc., Montgomery, TX, USA]; [product No. PRB-278P, rabbit anti-Pax6, 1:300; Covance, Dedham, MA, USA]; [product No. MABN15, mouse anti-rhodopsin, clone 4D2, 1:300; Millipore, Billerica, MA, USA]; and [product No. AB5405, rabbit anti-red/green opsin, 1:300; Millipore]) was incubated in PBTS buffer overnight at 48C.

Techniques: Immunohistochemistry

Journal: Investigative ophthalmology & visual science

Article Title: Loss of citron kinase affects a subset of progenitor cells that alters late but not early neurogenesis in the developing rat retina.

doi: 10.1167/iovs.14-15272

Figure Lengend Snippet: FIGURE 4. Number of RGCs in WT and KO retinae is comparable at E14 but the number of RPCs in S-phase are fewer in the KO retina. Immunohistochemistry on retinal sections obtained from E14 embryos where the mother was pulsed with EdU at E12. (A, D) Retinal progenitor cells positive for Ki67 (green) and PH3 (red) in WT (A) and KO (D). (B, E) Retinal progenitor cells positive for EdU (magenta) in WT (B) and KO (E). (C, F) Merged image showing RPCs positive for Ki67 (green) and EdU (magenta) in WT (C) and KO (F). DAPI (blue) marks all nuclei. Inset shows the higher magnification image of the boxed region in the apical end of the ONBL where the solid arrow points to an RPC that is EdUþ and Ki67þ. (G–H’’) Retinal sections showing EdUþ cells (magenta) in WT (G) and KO (H) along with IHC for Islet1 (green) in WT (G’) and KO (H’). Shown in (G’’) and (H’’) are the merged images of (G) and (G’) and (H) and (H’), respectively. Inset shows the higher magnification image of the boxed region in the GCL where the solid arrow points to an RGC that is EdUþ and Islet1þ. DAPI (blue) marks all nuclei. (I) Quantification of S-phase RPCs (EdUþ cells) as a percentage of all RPCs (Ki67þ cells) in WT (blue, n ¼ 3) and KO (red, n ¼ 9); Student’s t-test, P ¼ 0.01. (J) Quantification of RGCs born after E12 by determining Islet1þ and EdUþ cells as a percentage of all Islet1þ cells within the GCL in WT (blue, n ¼ 6) and KO (red, n ¼ 7).

Article Snippet: Primary antibody ([product No. 40.2D6, mouse anti-Islet1, 1:300; Developmental Studies Hybridoma Bank, Iowa, IA, USA]; [product No. 556003, mouse anti-Ki67; BD Biosciences, San Jose, CA, USA]; [product No. IHC-00061, rabbit anti–Phospho Histone H3 (PH3), 1:300; Bethyl Laboratories, Inc., Montgomery, TX, USA]; [product No. PRB-278P, rabbit anti-Pax6, 1:300; Covance, Dedham, MA, USA]; [product No. MABN15, mouse anti-rhodopsin, clone 4D2, 1:300; Millipore, Billerica, MA, USA]; and [product No. AB5405, rabbit anti-red/green opsin, 1:300; Millipore]) was incubated in PBTS buffer overnight at 48C.

Techniques: Immunohistochemistry

Journal: Investigative ophthalmology & visual science

Article Title: Loss of citron kinase affects a subset of progenitor cells that alters late but not early neurogenesis in the developing rat retina.

doi: 10.1167/iovs.14-15272

Figure Lengend Snippet: FIGURE 6. Reduction in the ONBL in the CitK KO retina along with absence of bipolar cells at P2. (A–D’) Immunohistochemistry on P0 retinal section showing PH3 (green) in WT (A) and KO (A’); Islet1 (green) in WT (B) and KO (B’); Ki67 (green) in WT (C) and KO (C’); and Pax6 (green) in WT (D) and KO (D’). (E–H’) Immunohistochemistry on P2 retinal section showing PH3 (red) in WT (E) and KO (E’); Islet1 (green) in WT (F) and KO (F’); Ki67 (green) in WT (G) and KO (G’); and Pax6 (green) in WT (H) and KO (H’). DAPI (blue) marks all the nuclei.

Article Snippet: Primary antibody ([product No. 40.2D6, mouse anti-Islet1, 1:300; Developmental Studies Hybridoma Bank, Iowa, IA, USA]; [product No. 556003, mouse anti-Ki67; BD Biosciences, San Jose, CA, USA]; [product No. IHC-00061, rabbit anti–Phospho Histone H3 (PH3), 1:300; Bethyl Laboratories, Inc., Montgomery, TX, USA]; [product No. PRB-278P, rabbit anti-Pax6, 1:300; Covance, Dedham, MA, USA]; [product No. MABN15, mouse anti-rhodopsin, clone 4D2, 1:300; Millipore, Billerica, MA, USA]; and [product No. AB5405, rabbit anti-red/green opsin, 1:300; Millipore]) was incubated in PBTS buffer overnight at 48C.

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: Live dynamics of induced cell-cell fusion between mitotic and interphasic cells

doi: 10.64898/2026.01.27.700572

Figure Lengend Snippet: (A) Comparison of Mad2 localization in control prometaphase and anaphase cells and mitotic fused cells undergoing premature mitotic exit. Mad2 can be detected at some kinetochores despite the presence of nuclear envelope membranes. (B) Time lapse of cerulean-Cyclin B1 transiently transfected in LLC-PK1 cells expressing H2B-mCherry after cell-cell fusion. Cyclin B1 was not degraded during induced mitotic exit of the mitotic cell. (C) Analysis of pH3-s10 in fixed prometaphase control cells and at different time points after cell-cell fusion. At 30 minutes after cell-cell fusion there is a mixed population of cells with high (second panel) and low (third panel) pH3-s10 levels, despite the presence of nuclear envelope membranes. At 60 minutes after cell-cell fusion, most cells show very low levels of pH3-s10. Magenta squares show magnification of nuclear envelope membranes around chromosomes. (D) Quantification of pH3-s10 in telophase control cells and at several time points after cell-cell fusion. Control, n=19 cells; 20 min, n=20 cells; 30 min, n=22 cells; 40 min, n=27 cells; 50 min, n=32 cells and 60 min, n=26 cells. Note that only 50 and 60 minutes after cell-cell fusion the levels of pH3-s10 become similar to control levels. Statistics, non-parametric Mann-Whitney test. Time is h:min. Scale bar is 10μm.

Article Snippet: Primary mouse mab414 (1:1000; ab24609, Abcam), mouse anti-α-Tubulin (1:500, DM1A, sigma), mouse anti-Mad2 (1:100, sc-65492, Santa Cruz) and rabbit anti-pH3-s10 (1:200, 3377, Cell Signaling).

Techniques: Comparison, Control, Transfection, Expressing, MANN-WHITNEY