Journal: Molecular carcinogenesis

Article Title: Regulation of SELENOF translation by eIF4a3: Possible role in prostate cancer progression.

doi: 10.1002/mc.23616

Figure Lengend Snippet: FIGURE 4 eIF4a3 binds to the 3′UTR of SELENOF mRNA. (A) PC3 cells were transiently transfected with eIF4a3 siRNA, scrambled RNA, eIF4a3 expression vector, or empty vector for 3 days. Cell lysates were collected, electrophoresed, and probed with SELENOF, eIF4a3, and β‐ tubulin antibodies. The band intensities of SELENOF were normalized to corresponding tubulin values and plotted in (ii). The data is presented as the SD with an n = 3. (B) RNA‐IP was performed on PC3 cell lysates. (i) eIF4a3 and the bound SELENOF mRNA were coimmunoprecipitated using mouse anti‐eIF4a3 IgG or mouse isotype control IgG as a control for the specificity of the pull‐down. The protein‐bound mRNA in the precipitate was isolated, reverse‐transcribed, and PCR‐amplified using SELENOF‐specific primers (Supporting Information: Table S1). (ii) Western blot of immunoprecipitated protein with eIF4a3 specific antibodies showing recovery of SELENOF mRNA only when anti‐eIF4a3 antibodies and not control IgG was used. (C) Same as in (B) except the cells were transfected with the SECIS‐like or SECIS containing reporter constructs shown in Figure 2 as indicated, and PCR amplification was performed using one primer complementary to sequences in the reporter construct to amplify sequences from the transfected constructs and endogenous SELENOF mRNA. PC3 cells were transiently transfected with pMirGlo vectors with SECIS‐like and SECIS element inserts for 72 h. RNA‐IP was performed with the transfected cell lysates with mouse anti‐ eIF4a3 IgG and mouse isotype control IgG. The eIF4a3‐bound mRNA was isolated, PCR amplified using construct‐specific primers as mentioned in the methods section, electrophoresed in agarose gels, and visualized.

Article Snippet: Antibodies against SELENOF (Abcam Cat#2118, RRID:AB_10972510) were used at 1:2000, GAPDH (Cell Signaling Technologies, Cat#: 2118, RRID:AB_561053) at 1:10000, β‐actin (Abcam Cat#AB8226, RRID:AB_306371) at 1:10,000, eIF4a3 (Santa Cruz Biotechnology Cat# sc‐365549, RRID:AB_108472215)at 1:1000, α‐tubulin (Cell Signaling Technology Cat#2146, RRID:AB2210545) 1:10,000 dilutions respectively.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Isolation, Reverse Transcription, Amplification, Western Blot, Immunoprecipitation, Construct

Journal: Brain and behavior

Article Title: KEAP1-NRF2/HO-1 Pathway Promotes Ferroptosis and Neuronal Injury in Schizophrenia.

doi: 10.1002/brb3.70311

Figure Lengend Snippet: FIGURE 3 Dysregulation of ferroptosis pathway in cINs derived from hiPSCs of SCZ patients. (A–C) Immunofluorescence and Western blot showed expression of β-tubulin and SOX6 in cINs derived from SCZ patients (n = 3). (D–E) qRT-PCR and Western blot indicated a significant decrease in KEAP1 expression in SCZ-derived cINs (n = 3). **p < 0.01 compared to normal group.

Article Snippet: Proteins acquired after lysis of cells were carefully separated on SDS-PAGE and then shifted onto nitrocellulose membranes that were incubated with primary antibodies specific to β-tubulin (Cell signaling#2146, 1:900), SOX6 (Proteintech, Cat. #14010-1-AP, 1:25), NRF2 (Proteintech#80593-1, 1:50), HO-1 (Novus Bio# NBP197507, 1:75), GAPDH (Santa Cruz#sc32233, 1:20), GPX4 (Thermo Fischer#MA5-32827, 1:30), and KEAP1 (Cell signaling#8047).

Techniques: Derivative Assay, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: Sub-lethal oxidative stress induces two waves of γH2AX, 53BP1 foci and phosphorylated p53 (Ser15) in synchronized L6 cells. L6 cells were synchronized using thymidine before being treated with 50 μM H 2 O 2 for 1 h and cell cycle analysis, γH2AX foci, 53BP1 foci, p53 expression and p53 phosphorylation were assessed. ( A ) Line plots capturing the percentage of cells in each cell cycle phase at the indicated time points. ( B ) Representative nucleus showing γH2AX foci in control and H 2 O 2 -treated cells. ( C ) Quantification of the average number of γH2AX foci per nuclei from the analysis of at least 30 to 40 nuclei. ( D ) Representative nucleus showing 53BP1 foci in control and H 2 O 2 -treated cells. ( E ) Quantification of the average number of 53BP1 foci per nuclei from the analysis of at least 30–40 nuclei. ( F ) Kinetics of phosphorylated p53 (ser15) and total p53 in control and 50 μM H 2 O 2 -treated cells. Plots shown in (C) and (E) are representative of at least two independent experiments and error bars represent ± SD of number of foci per nucleus counted within the experiment.

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Cell Cycle Assay, Expressing, Phospho-proteomics, Control

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: ATM and ATR are activated after endogenous DNA damage induction. Cells were synchronized using thymidine before being treated with 50 μM H 2 O 2 for 1 h. ( A ) Western blot analysis of the levels of γH2AX and phosphorylated p53 (ser15) at 6, 9 and 12 h following synchronized cells exposure to 50 μM H 2 O 2 and the inhibition of ATM using KU55933 3 h after 50 μM H 2 O 2 treatment. ( B ) Western blot analysis of γH2AX and phosphorylated p53 (ser15) after inhibition of ATR using AZ20 at the time of second wave of DNA damage (6–12 h). AZ20 was added 2 h before the collection of cells at the indicated time points.

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Western Blot, Inhibition

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: Replicative stress induced by sub-lethal oxidative stress leads to the formation of DSB following replicative fork collapse. Cells were synchronized using thymidine before being treated with 50 μM H 2 O 2 for 1 h. ( A ) Western blot showing the levels of RPA32 at the chromatin level at indicated time. ( B ) Densitometry plots of the chromatin level of RPA32 at 6, 10 and 24 h from (A). ( C ) Immunofluorescence images showing the levels of RPA32 foci in control and H 2 O 2 -treated cells in representative nucleus at indicated time points. ( D ) Co-localization analysis of 53BP1 and RPA32 foci. ( E ) Western blot of γH2AX and phosphorylated p53 (ser15) at 9 h, after addition of 10 μM aphidicolin (DNA polα inhibitor) 1 h after 50 μM H 2 O 2 treatment.

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Western Blot, Immunofluorescence, Control

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: Exposure of L6 cells to 50 μM exogenous H 2 O 2 induced senescence-associated persistent foci/DNA-SCARS. Cells were synchronized in late G1 using thymidine before being treated with 50 μM H 2 O 2 for 1 h and the appearance of senescence-associated persistent foci/DNA-SCARS assessed. ( A ) γH2AX and 53BP1 foci after 24, 48 and 72 h post-H 2 O 2 treatment. ( B ) Immunofluorescence images showing the co-localization of PML and γH2AX 96 h after exposure to H 2 O 2 . ( C ) Total p53, Rad51 and γH2AX protein expression levels in a chromatin extract 72 h after cells exposure to 50 μM H 2 O 2 . Total histone H3 is used as a loading control. ( D ) Rad51 and γH2AX foci in control and 50 μM H 2 O 2 -treated cells at indicated time points. Representative immunofluorescence images of Rad51 and γH2AX foci in a nucleus are shown. Line plots showing the average of Rad51 foci ( E ) and γH2AX foci ( F ) from at least 30–40 nuclei at indicated time points are also shown. Error bars represent ± SD of number of foci per nucleus counted within the experiment.

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Immunofluorescence, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: Endogenous DNA damage induced p53-dependent senescence growth arrest. Cells were synchronized with thymidine before being treated with 50 μM H 2 O 2 for 1 h. ( A ) NMA analysis of the nuclei of untreated and 50 μM H 2 O 2 -treated synchronized cells at 72 h in CoSi and Sip53-transfected cells. Nuclei types and its corresponding phases are described as below. N—normal (interphase), S—small (mitosis), SR—small regular (apoptosis), SI—small irregular (mitotic abnormality), LR—large regular (senescence), LI—large irregular (mitotic catastrophe) and I—irregular (mitotic catastrophe). ( B ) Phase contrast images showing the cell morphology of untreated and 50 μM H 2 O 2 -treated cells in CoSi and Sip53-transfected cells 48 h following cells exposure to the oxidant. ( C ) Cells were stained with crystal violet to quantify adherent cell density of untreated and 50 μM H 2 O 2 -treated cells at 48 h upon silencing with CoSi and Sip53. Values are normalized to the day of trigger. The data shown in (C) represent the mean of at least three independent experiments and the error represents ± SD. P -value (one-tailed, Student’s t -test, ** P -value < 0.01, ns—not significant) of the comparison groups are indicated in the figures.

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Transfection, Staining, One-tailed Test, Comparison

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: p53 transcriptionally repressed Rad51 and Lamin B1. Cells were synchronized using thymidine before being treated with 50 μM H 2 O 2 for 1 h. ( A ) Western blot showing Rad51expression in CoSi and Sip53-transfected cells. ( B ) Rad51 protein expression at 24 and 48 h following cells exposure to 50 μM H 2 O 2 . ( C ) Rad51 mRNA expression in CoSi and Sip53-transfected cells 24 and 48 h after cells exposure to 50 μM H 2 O 2 . Data shown are the representative of four independent experiments and the error represents ± SD. ( D ) Western blot showing Lamin B1 expression in CoSi and Sip53-transfected cells 24 and 48 h following cells exposure to 50 μM H 2 O 2 . ( E ) Lamin B1 mRNA expression at 24 and 48 h upon silencing p53. Data shown are the representative of four independent experiments and the error represents ± SD. ( F ) Western blot showing Lamin B1 expression at indicated time points in 50 μM H 2 O 2 -treated cells. ( G ) Depletion of Lamin B1 in H 2 O 2 -treated cells at indicated time points, shown by immunofluorescence. White boxed nucleus are magnified to show the intensity of Lamin B1 at various time points indicated (right column). The Lamin B1 fluorescence intensity along the diameter of the nucleus is also quantified. Note the gradual decrease of Lamin B1 fluorescence intensity in 50 μM H 2 O 2 -treated cells (right bottom column).

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Western Blot, Transfection, Expressing, Immunofluorescence, Fluorescence

Journal: Nucleic Acids Research

Article Title: Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress

doi: 10.1093/nar/gkx684

Figure Lengend Snippet: Proposed model of the induction of cellular senescence by a sub-lethal oxidative stress. We propose that the acquisition of cellular senescence following cells’ exposure to a sub-lethal oxidative stress occurs in three phases. Phase 1 (1–3 h): upon exposure of cells to a sub-lethal oxidative stress, variety of complex DNA ends including oxidized deoxyriboses, DSBs, SSBs and OCDLs are generated. Phase 2 (4–12 h): despite the presence of unrepaired DNA, cells progress to S phase. Upon the progression to S phase, presence of unrepaired DNA damage induces replication stress. The inability to resolve the stress results in the stalling of the replication fork, replication fork collapse leading to the formation of DSB. At the same time activation of p53 inhibits the transcription of the HR repair protein Rad51 and the nuclear protein Lamin B1. Phase 3 (12–72 h): repression of Rad51 by active p53 is critical for the formation of DNA-SCARS. In addition, reciprocally, DNA-SCARS formation is critical to maintain the low level of p53 activation necessary to maintain cell cycle arrest. On the other hand, aggregation of DSB into micronuclei like structures or the formation of chromatin budding initiated by the unresolved replication stress associated with the p53-dependent repression of Lamin B1 facilitates the translocation of chromatin via micronuclei like structures to the cytoplasm. Furthermore, similar to the p53–Rad51 feedback loop, a feedback mechanism between p53 and Lamin B1 is critical to maintain growth arrest. Following the establishment of the p53–Rad51 and p53–Lamin B1 feedback loops, cells will then enter a deep senescent state, characterized by growth arrest, an increase in SA-β-Gal activity, activation of a p16-RB axis, establishment of DNA-SCARS, increase in HMGA2 expression as a marker of SAHF, formation of CCF-like micronuclei and secretion of IL-6.

Article Snippet: The primary antibodies used: rabbit-γH2AX (Ser139) (#2577), rabbit-p-p53 (Ser15) (#9284), rabbit-total p53 (#9282) rabbit- β-tubulin (#2146) and horseradish peroxidase (HRP)-conjugated goat anti-rat secondary antibody (#7077) were purchased from Cell Signaling.

Techniques: Generated, Activation Assay, Translocation Assay, Activity Assay, Expressing, Marker