Journal: Nucleic acids research

Article Title: KRCC1, a modulator of the DNA damage response.

doi: 10.1093/nar/gkac890

Figure Lengend Snippet: Figure 4. KRCC1 depletion results in premature mitotic entry. (A, B) HeLa and U2OS cells transfected with control or KRCC1 siRNA and labeled with 20 M EdU for 15 min. Cells were stained with DAPI and then immunofluorescence was performed for EdU and pH3-S10. (C) Percentage of pH3-S10 positive cells. (D) Percentage of EdU and pH3-S10 dual positive cells. The data shown are from three independent experiments ± SDs. (E) HeLa cells were transfected with control or KRCC1 siRNA or treated with CDC7i, TAK-931 (300 nM, 24 h). The cells were pulse labeled with EdU for 15 min, fixed and processed for immunofluorescence for EdU to denote S-phase cells and 53BP1 to label under-replicated DNA sequestered in 53BP1 nuclear bodies (53BP1-NBs). (F) Percentage of G1 cells with 53BP1-NBs. Experiments were repeated independently at least three times. Data represent mean ± SD; ordinary one-way ANOVA was performed for statistical analysis and asterisks indicate significance. (G) HeLa cells were labeled with EdU for 30 min, and then cells were fixed and proteins binding to EdU-labeled DNA captured by the Dm-ChP technique as described in the ‘Materials and Methods’ section.

Article Snippet: The following primary antibodies were used: pCHK1-S345 (2348), pCHK1S296 (2349), H2AX (2577), CHK1 (2360), HA-tag (3724), pH3-S10 (3377) and pan 14-3-3 (8312) from Cell Signaling Technology (Danvers, MA, USA); KRCC1 (16916-1- AP) from Proteintech (Rosemont, IL, USA); CDC25A (sc7389) and CDC7 (sc-56275) from Santa Cruz Biotechnol- ogy (Dallas, TX, USA); pRPA-S33 (A300-246A), AND1 (A301-141A), PSF3 (A304-124A), CHK1 (A300-298A) and pMCM2-S40/41 (A300-788A) from Bethyl Laboratories (Montgomery, TX, USA); DBF4 (ab124707) and pCyclinB1-S126 (ab55184) from Abcam; anti-Halo-tag (G921A) from Promega; RPA32 (MABE285) from EMD Millipore; RAD51 (NB100-148) from Novus Biological; 53BP1 (88439) from Cell Signaling Technology; and - tubulin and -actin from Sigma–Aldrich.

Techniques: Transfection, Control, Labeling, Staining, Immunofluorescence, Binding Assay

Journal: Frontiers in immunology

Article Title: Transformation of primary murine peritoneal mast cells by constitutive KIT activation is accompanied by loss of Cdkn2a/Arf expression.

doi: 10.3389/fimmu.2023.1154416

Figure Lengend Snippet: FIGURE 7 PMC-306 cells are characterized by enhanced cell cycle activity and sporadic DNA damage. The cell cycle of primary WT PMCs was compared to PMC-306 cells. (A) Cells were stained with an antibody directed against MKI67 (AL700-A). Left: Quantification of MKI67+ cells (% of single cells, n=3). Right: Representative FACS-plots of MKI67+ cells (% of single cells, n=3). (B) Quantification of the mean fluorescence intensity (MFI, n=3) of MKI67 in single cells (left) and a representative histogram showing MKI67 intensities in primary PMCs and PMC-306 cells. (C) Cells were stained with an antibody directed against phosphorylated histone H3 (pH3, AL647-A). Left: Quantification of pH3 cells (% of single cells, n=3). Right: Representative FACS-plots of pH3+ cells (% of single cells). (D) Cells were stained with an antibody directed against phosphorylated Histone H2Ax (pH2Ax, PerCP- Cy5.5-A). Left: Quantification of pH2ax cells (% of single cells, n=3). Right: Representative FACS-plots of pH2Ax+ cells (% of single cells). Data are expressed as mean +SD. Unpaired, two-tailed Student’s t test with Welsh’s correction. *p<0.05, **p<0.01.

Article Snippet: Intracellular co-staining of cells was performed using a mix of fluorophore-labelled antibodies against pH2Ax-PerCP-Cy5.5 (1:100, #564718, S139, BD Biosciences) together with pH3-AL647 (1:100, #3458, S10, Cell Signaling Technology) or MKi67-AL700 (1:100, #56-5698-82, Invitrogen Thermo Fisher Scientific) diluted in FACS-blocking buffer (mixture of 0.66% human/rabbit/mouse serum, Sigma-Aldrich, and 1% Bovine Serum Albumin, Sigma-Aldrich in PBS) for 30 minutes at 4°C.

Techniques: Activity Assay, Staining, Two Tailed Test

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: 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