Journal: Advanced Science

Article Title: Homoisoflavanone Delays Colorectal Cancer Progression via DNA Damage‐Induced Mitochondrial Apoptosis and Parthanatos‐Like Cell Death

doi: 10.1002/advs.202511406

Figure Lengend Snippet: HIF induces DNA damage‐mediated parthanatos in CRC cells. (A) Heatmap of differentially expressed genes in HCT15 and HCT116 cells treated with or without HIF. (B) Venn diagram illustrating overlapping differentially expressed genes between HCT15 and HCT116 cells. (C) GO enrichment analysis of HIF‐regulated genes in HCT15 and HCT116 cells. (D, E) GSEA of genes modulated by HIF in HCT15 and HCT116 cells. (F) Immunofluorescence analysis of γ‐H2AX expression in HCT15 and HCT116 cells following HIF treatment. (G) Expression levels of DNA damage markers (P53, P21, γ‐H2AX, c‐PARP, and PARP). (H) Gel electrophoresis analysis of plasmid DNA treated with HIF. The values represent the mass ratio of PKF to plasmid DNA, indicating that 200 ng of plasmid DNA was incubated with 0, 1, 3, 10, 30, and 100 µg of HIF for 2 h. (I, J) Cell viability assessment of HCT15 and HCT116 cells with HIF or combined with Olaparib (Ola) treatment. (K) Genomic DNA integrity analysis via gel electrophoresis after HIF treatment. (N, O) ROS levels after HIF treatment in HCT15 and HCT116 cells. (L) Time‐course analysis of DNA damage repair‐related proteins, including RAD51 and phosphorylated ATM (p‐ATM), ATR (p‐ATR), and Chk1 (p‐Chk1) in HCT15 and HCT116 cells at 0.5, 2, and 6 h following HIF treatment. ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: The membranes were then blocked with a 5% non‐fat milk TBST (TBS containing 0.1% Tween‐20) for 1 h at room temperature and incubated at 4°C for overnight with primary antibodies against the following proteins: cleaved Caspase 3 (25128‐1‐AP, Proteintech), cleaved Caspase 8 (9496, CST), PARP (9542, CST), cleaved PARP (9541, CST), P21 (2947, CST), P53 (2527, CST), Cyclin A2 (91500, CST), Cyclin D1 (2978, CST), CDK1 (bs‐1341R, Bioss), CDK2 (bs‐10726R, Bioss), CDK7 (bs‐0569R, Bioss), AKT (4691, CST), p‐AKT (4060, CST), ERK (9102, CST), p‐ERK (9101, CST), MEK (9126, CST), p‐MEK (9154, CST), ATR (13934, CST), p‐ATR (2853, CST), Chk1 (2360, CST), p‐Chk1 (2348, CST), RAD51 (14961‐1‐AP, Proteintech), ATM (27156‐1‐AP, Proteintech), p‐ATM (5883, CST), γ‐H2AX (9718, CST), AIF (5318, CST), MIF (75038, CST), Cyt c (10993‐1‐AP, Proteintech), Lamin B (66095‐1‐Ig, Proteintech), VDAC1 (81538‐1‐RR, Proteintech) and GAPDH (60004‐1‐Ig, Proteintech).

Techniques: Immunofluorescence, Expressing, Nucleic Acid Electrophoresis, Plasmid Preparation, Incubation

Journal: PLoS Genetics

Article Title: Elevated temperature increases meiotic crossover frequency via the interfering (Type I) pathway in Arabidopsis thaliana

doi: 10.1371/journal.pgen.1007384

Figure Lengend Snippet: (A, B) γH2Ax foci counting. (A) Pollen mother cells containing chromosomes (DAPI, blue) and γH2Ax foci (red). (B) Boxplots of γH2Ax foci at 20˚C and 28˚C; mean shown as white circle. (C, D) RAD51 foci counting. (C) Pollen mother cells containing chromosomes (DAPI, blue) and RAD51 foci (red). (D) Boxplots of RAD51 foci at 20°C and 28°C; mean shown as white circle. p-values are indicated as follows: ns = p > 0.5, * = p ≤ 0.05, ** = p ≤ 0.01. Scale bars represent 5 μm.

Article Snippet: The slides were incubated overnight with primary antibodies diluted 1:200 (RAD51, γH2AX, MLH1) in blocking buffer (goat serum, AR0009, Bosterbio) at 4°C and then at 37°C for 60 min with secondary antibody (1:1000, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555, catalog # A-21428, Thermo Fisher Scientific).

Techniques:

Journal: BMC Genomics

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

doi: 10.1186/s12864-015-1425-4

Figure Lengend Snippet: Construction of the recA -inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr- neo , a lambda Pr promoter fused to the neomycin resistance gene ( neo ), and c I- spc , which contains c I encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene ( spc ). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis . cat , chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene ( tet ) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’- amyE and 3’- amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA . The closed arrowheads indicate the inducible recA derived from pX-recA.

Article Snippet: Each membrane was blocked in 5% (wt/vol) skimmed milk in TBS containing 0.05% (vol/vol) Tween 20, washed three times in TBST for 10 min each and then reacted with anti-RecA polyclonal antibody (Abnova, PAB15568) overnight at 4°C.

Techniques: Plasmid Preparation, Construct, BAC Cloning, Sequencing, Expressing, Homologous Recombination, Southern Blot, Clone Assay, Derivative Assay

Journal: BMC Genomics

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

doi: 10.1186/s12864-015-1425-4

Figure Lengend Snippet: Western blot analysis and optimization of RecA induction. (a) Western blot analysis with an anti-RecA antibody indicated the expression of RecA in the presence of xylose. Remarkably, expression of RecA in the absence of xylose was strictly repressed. BEST310/ ΔrecA , that was constructed by replacing the endogenous recA of BEST310 with tet of pCTP, was used as a negative control. (b) Schematic diagram of the cloning procedure. erm , erythromycin resistance gene; Em S , erythromycin sensitive; Em R , erythromycin resistant. (c) Numbers of erythromycin-resistant colonies under various induction times after addition of 1.0% xylose. Error bars, s.d. n = 3. (d) Numbers of erythromycin-resistant colonies under various xylose concentrations at 150 min induction. Error bars, s.d. n = 3. (e) Erythromycin-resistant recombinants were obtained only in the presence of xylose. The picture of xylose (+) is representative plate at a final xylose concentration of 1.0% at the induction time of 150 min. (f) All colonies formed on LB plates containing erythromycin were fluorescent due to their GFP gene.

Article Snippet: Each membrane was blocked in 5% (wt/vol) skimmed milk in TBS containing 0.05% (vol/vol) Tween 20, washed three times in TBST for 10 min each and then reacted with anti-RecA polyclonal antibody (Abnova, PAB15568) overnight at 4°C.

Techniques: Western Blot, Expressing, Construct, Negative Control, Cloning, Concentration Assay

Journal: BMC Genomics

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

doi: 10.1186/s12864-015-1425-4

Figure Lengend Snippet: Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. Tet S , tetracycline sensitive; Tet R , tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA . ( d and e ) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.

Article Snippet: Each membrane was blocked in 5% (wt/vol) skimmed milk in TBS containing 0.05% (vol/vol) Tween 20, washed three times in TBST for 10 min each and then reacted with anti-RecA polyclonal antibody (Abnova, PAB15568) overnight at 4°C.

Techniques: Clone Assay, Stable Transfection, Southern Blot, Marker

Journal: BMC Genomics

Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

doi: 10.1186/s12864-015-1425-4

Figure Lengend Snippet: Stable gene manipulation in the iREX by preventing the deletion. (a) Schematic diagram of the insertion of IRES - tauEGFP - c I- spc and IRES - tauLacZ into MOR42-3 and MOR42-2 , respectively, and the assumed deletion at the IRES - tau sequences. (b) The signal derived from the deletion was observed in BEST310/BAC1-GL (open arrowhead). The same result was obtained in iREX/BAC1-GL in the presence of xylose because of the induced RecA. In contrast, the signal derived from the deletion was not observed for iREX/BAC1-GL in the absence of xylose due to the strong repression of recA . The closed arrowheads indicate the signals derived from the intact inserts of BEST310/BAC1-GL and iREX/BAC1-GL. BEST310/BAC1-GL-deletion, which was screened from BEST310/BAC1-GL culture by neomycin resistance and spectinomycin sensitivity, was used as a control of deletion. The BGM vector is indicated as a closed arrow. (c) The proportions of the spectinomycin-resistant clones containing two IRES-tau sequences were 69% and 73% in the BEST310/BAC1-GL and iREX/BAC1-GL with xylose, respectively. In contrast, the proportion of the spectinomycin-resistant clones was 93% in the iREX/BAC1-GL without xylose. Error bars, s.d. n = 3.

Article Snippet: Each membrane was blocked in 5% (wt/vol) skimmed milk in TBS containing 0.05% (vol/vol) Tween 20, washed three times in TBST for 10 min each and then reacted with anti-RecA polyclonal antibody (Abnova, PAB15568) overnight at 4°C.

Techniques: Derivative Assay, Control, Plasmid Preparation, Clone Assay