Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Common Markers and Small Molecule Inhibitors in Golgi Studies

doi: 10.1007/978-1-0716-2639-9_27

Figure Lengend Snippet: Commonly used antibodies for immunoblotting of Golgi proteins

Article Snippet: Trans -Golgi , GCC88 (GCC1) , rabbit , 1:200 , Proteintech, 16271-1-AP , RRID: AB_2107197.

Techniques: Western Blot, Transduction

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Common Markers and Small Molecule Inhibitors in Golgi Studies

doi: 10.1007/978-1-0716-2639-9_27

Figure Lengend Snippet: Commonly used markers to visualize Golgi and other cellular structures by immunofluorescence microscopy

Article Snippet: Trans -Golgi , GCC88 (GCC1) , rabbit , 1:200 , Proteintech, 16271-1-AP , RRID: AB_2107197.

Techniques: Immunofluorescence

Journal: Theranostics

Article Title: FN1 mRNA 3'-UTR supersedes traditional fibronectin 1 in facilitating the invasion and metastasis of gastric cancer through the FN1 3'-UTR-let-7i-5p-THBS1 axis

doi: 10.7150/thno.82492

Figure Lengend Snippet: THBS1 , CPED1 , and AMOTL2 were regulated by FN1 3'-UTR via let-7i-5p and miR-629-5p. The oncogenic and core regulatory network using FN1 3′-UTR as the core (A). The relative expression levels of THBS1, CPED1, and AMOTL2 determined by western blotting, ImageJ software analysis, and real-time PCR in FN1 3′-UTR overexpressed and negative cells (B, C, D). The regulatory relationships between let-7i-5p and THBS1, let-7i-5p and CPED1, and miR-629-5p and AMOTL2 in the core subnetwork were validated by western blot analysis and real-time PCR (E, F, G). Schematic diagram (H) and real-time PCR results (I) of the RNA immunoprecipitation chip assay based on AGO2. The relative expression levels of THBS1, CPED1, and AMOTL2 determined by western blot analysis and ImageJ software in FN1 3′-UTR overexpressed cells, FN1 protein overexpressed cells, and negative control cells (J, K). The data are presented as a histogram of the mean ± SEM of three independent experiments in C, D, F, G, I, and K and compared using Student's t -test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, n = 3).

Article Snippet: Antibodies against THBS1 (1:1000, 18304-1-AP), CPED1 (1:1000, 20924-1-AP), AMOTL2 (1:1000, 23351-1-AP), TGFβ1 (1:1000, 21898-1-AP), TGFβ2 (1:1000, 19999-1-AP), and GAPDH (1:10000, 60004-1-Ig) were purchased from Proteintech Group (IL, USA).

Techniques: Expressing, Western Blot, Software, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation, Negative Control

Journal: Nucleic acids research

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

doi: 10.1093/nar/gkac890

Figure Lengend Snippet: Figure 1. KRCC1 promotes CHK1 activation and efficient checkpoint. (A, B) OV90 and U2OS cells were transfected with control siRNA (siCTL) or siRNA targeting KRCC1 (siKRCC1) for 72 h. Cells were stained with DAPI and anti-RPA2 antibody and visualized by fluorescence microscopy (scale bar, 20 m). Percentage of cells with >10 foci is quantitated. The data are from manual scoring of ∼200 cells per condition and from three experiments ± SDs. (C) Lysates from above transfected siCTL and siKRCC1 OV90 and U2OS cells were analyzed by immunoblotting for CHK1-mediated DDR markers. (D) Immunoblotting for CHK1-mediated DDR markers after 72 h KRCC1 silencing in the presence or absence of CPT (1 M for 1 h). (E) KRCC1 interaction with CHK1 and 14-3-3 was evaluated using co-immunoprecipitation in EV or HA-tagged KRCC1 (HA-KRCC1) overexpressed cells treated with or without CPT (3 M, 2 h). (F) CHK1 interaction with KRCC1 was evaluated using co-immunoprecipitation in EV or Halo-tagged KRCC1 (Halo-KRCC1) overexpressed cells treated with CPT (3 M, 2 h) in the presence or absence of ATR inhibitor (ATRi, 5 M, 4 h) and quantification of immunoprecipitated Halo-KRCC1 by densitometry analysis using NIH ImageJ and normalized to their respective CHK1 levels and compared to CPT treatment only, which was set to 1. Experiments were repeated three times. Data represent mean ± SD. Marker (M) and IgG lanes are shown. (G) OV90 cells transfected with EV or HA-KRCC1 were treated with CPT (1 M for 1 h) and released for up to 180 min. Cells were collected at the indicated time points and processed for immunoblotting. The black right-pointing triangle indicates KRCC1. Short exposure (SE) and long exposure (LE) blots for KRCC1 are shown. The right panel depicts quantification of pCHK1-S296 by densitometry analysis using NIH ImageJ, normalized to their respective CHK1 levels and compared to the no treatment control group (NT), which was set to 1. (H) Proposed model of CHK1 activation. Following DNA damage and ATR-mediated phosphorylation of CHK1 at S345, CHK1 associates directly or indirectly with KRCC1 and 14-3-3. We posit that this association induces a conformational change in CHK1 to favor autophosphorylation at S296 and enhance kinase activity toward CDC25A.

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: Activation Assay, Transfection, Control, Staining, Fluorescence, Microscopy, Western Blot, Immunoprecipitation, Marker, Phospho-proteomics, Activity Assay

Journal: Nucleic acids research

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

doi: 10.1093/nar/gkac890

Figure Lengend Snippet: Figure 2. KRCC1 inhibition suppresses HRR. (A) Schematic of functional HRR assay. (B) Live cell images of DR-GFP U2OS cells transfected with I-SceI endonuclease in the presence or absence of KRCC1 siRNA. (C) GFP-positive cells from live imaging were counted and compared to siCTL, which was set to 1. The data are from manual scoring of ∼300 cells per condition per experiment and from three experiments ± SDs. (D, E) OV90 and U2OS cells transfected with control siRNA (siCTL) or siRNA targeting KRCC1 (siKRCC1) were treated with CPT (1 M for 1 h) and released for 2 h. Cells were stained with DAPI and anti-RAD51 antibody and visualized by fluorescence microscopy. If a cell had >10 RAD51 foci, it was counted as positive and this was set to 100%. Similarly, in the KRCC1 silenced CPT treated group, RAD51 foci positive cells were counted and expressed relative to the control. Experiments were repeated independently three times and 150 cells were counted per group per experiment. Data represent mean ± SD. (F) Experimental design of panels (G) and (H). OV90 cells transfected with control or KRCC1 siRNA and treated with CPT (1 M for 1 h) were released for 8 h and collected at 2 h intervals. (G, H) Quantitation of H2AX and RAD51. Cells with >10 foci were scored as positive. The data are from manual scoring of ∼200 cells per condition per experiment and from three experiments ± SDs.

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: Inhibition, Functional Assay, Transfection, Imaging, Control, Staining, Fluorescence, Microscopy, Quantitation Assay

Journal: Nucleic acids research

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

doi: 10.1093/nar/gkac890

Figure Lengend Snippet: Figure 3. Silencing KRCC1 results in delayed S-phase progression and accumulation of cells at the late S phase. Asynchronous HeLa and U2OS cells transfected with control or KRCC1 siRNA and labeled with 20 M EdU for 15 min. DNA synthesis, DNA content and cell cycle distribution were assessed by flow cytometry. (A) Experimental images of the EdU/PI distribution of control and KRCC1 silenced cells. (B) Percentage of cells at the late S– G2 boundary was calculated as a fraction of % EdU-positive cells in the small gate over % total EdU-positive cells. (C) HeLa cells transfected with control or KRCC1 siRNA or treated with CHK1i (AZD7762) or CDC7 inhibitor (CDC7i, TAK-931) were labeled with EdU and subjected to flow cytometry. (D) HeLa cells transfected with control or KRCC1 siRNA or treated with CDC7i (TAK-931) were G1–S synchronized by double thymidine block and released for 12 h. The cells were collected at the indicated time points and analyzed by flow cytometry. (E) Immunoblotting of indicated proteins in control, KRCC1 depleted, CHK1 inhibited or CDC7 inhibited cells. SE and LE blots for pMCM2-S40/41 are shown.

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, DNA Synthesis, Flow Cytometry, Blocking Assay, Western Blot

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: Nucleic acids research

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

doi: 10.1093/nar/gkac890

Figure Lengend Snippet: Figure 5. Model of the role of KRCC1 in genome maintenance. Following replication stress and DNA damage, ATR phosphorylates CHK1 at S345. KRCC1 then associates directly or indirectly with CHK1 and 14-3-3 to promote autophosphorylation of CHK1 at S296 and facilitate kinase activity toward CDC25A. CDC25A is then targeted for proteasomal degradation to induce a CHK1-mediated checkpoint. Failure to fully activate CHK1 may result in reduced HRR. We speculate that the KRCC1–CDC7 axis may promote CDC7-mediated events and overall replication integrity, disruption of which leads to replication stress. Overall, replication defects and failure to fully activate checkpoint may result in premature mitotic entry and subsequent apoptosis.

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: Activity Assay, Disruption

Journal: PeerJ

Article Title: Porphyrobacter mercurialis sp. nov., isolated from a stadium seat and emended description of the genus Porphyrobacter

doi: 10.7717/peerj.1400

Figure Lengend Snippet: Phenotypic comparison of Coronado T and other members of the Porphyrobacter genus.

Article Snippet: On the basis of phenotypic and phylogenetic data presented in this study, strain Coronado T represents a novel species in the Porphyrobacter genus for which the name Porphyrobacter mercurialis sp. nov. is proposed; the type strain is Coronado T (=DSMZ 29971, =LMG 28700).

Techniques:

Journal: Clinical and Translational Medicine

Article Title: HSF4/COIL complex‐dependent R‐loop mediates ultraviolet‐induced inflammatory skin injury

doi: 10.1002/ctm2.1336

Figure Lengend Snippet: HSF4 is a key transcription factor regulating ultraviolet (UV)‐induced inflammation and forms an HSF4–COIL complex with COIL under UV induction. (A) Strategies for studying human skin databases. (B) Enrichment analysis of differentially expressed genes induced by ultraviolet radiation showed that 340 genes were upregulated (red) and 500 genes were downregulated (blue). (C) Pathway enrichment analysis of genes whose expression was upregulated after UV induction showed that a large number of genes were enriched in immune‐inflammation‐related pathways after UV induction. (D) The transcription factors that mainly regulate the upregulation of gene expression were analyzed by landscape in silico deletion analysis (LISA). (E) The downstream genes regulated by the top three transcription factors were marked by bioinformatics. (F) The mRNA expression of intracellular HSF4 in the experimental group and UV‐treated group. (G) Detection of HSF4 protein expression levels in cells before and after UV induction by Western blot. (H) The location and expression of HSF4 in the cells of the control group and UV‐treated group were detected by immunofluorescence, and the intensity of the HSF4 fluorescence signal in the nucleus of the control group and UV‐treated group was quantified. (I) Quantification of HSF4 fluorescence signal intensity in nuclei from control and UV‐treated groups. (J) GST‐pulldown assay with HSF4 protein in NIH‐3T3 cells after UV stimulation. The peptides were identified in the same way as above with this combined sequence database. (K) Venn diagram of HSF4 pull‐down and LISA analysis results. (L) Co‐immunoprecipitation (Co‐IP) assay protein–protein interactions were analyzed by Co‐IP experiments. The interaction test of HSF4 and COIL was performed on NIH‐3T3 cells before and after UV induction. (M) Immunofluorescence (IF) analysis of the colocalization of HSF4 and COIL before and after UV induction: HSF4, green; COIL, yellow. (N) The performed proximity ligation assay (PLA) experiments demonstrated enhanced HSF4‐COIL intranuclear interaction after UV induction. Error bars represent mean ± s.d.; * p ≤ 0.05; ** p ≤ .01; *** p ≤ .0005.

Article Snippet: Protein products were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight at 4°C with primary antibodies against HSF4 (Affinity), COIL, (Proteintech), LIMS1 (Affinity), ATG7 (Affinity), TFPI (Affinity), H2AX (Zenbio) and GAPDH (Affinity) and then for 1 h at room temperature in horseradish peroxidase conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Beyotime).

Techniques: Expressing, Gene Expression, In Silico, Western Blot, Control, Immunofluorescence, Fluorescence, GST Pulldown Assay, Sequencing, Co-Immunoprecipitation Assay, Protein-Protein interactions, Proximity Ligation Assay

Journal: Clinical and Translational Medicine

Article Title: HSF4/COIL complex‐dependent R‐loop mediates ultraviolet‐induced inflammatory skin injury

doi: 10.1002/ctm2.1336

Figure Lengend Snippet: HSF4–COIL complex activates the transcription and expression of genes related to inflammation and senescence (A) The downstream genes regulated by COIL and HSF4 after ultraviolet (UV) stimulation and the gene Venn with differential messenger RNA (mRNA) expression under UV stimulation showed all coregulated genes. (B) Quantitative polymerase chain reaction (qPCR) revealed differences in the expression of co‐regulated genes after UV stimulation. (C) Chromatin immunoprecipitation (ChIP)–qPCR analysis of HSF4 and COIL mRNA expression levels of Atg7, Tfpi and Lims1 before and after UV induction. (D) ChIP–reChIP verified the co‐regulation of HSF4 and COIL on Atg7, Tfpi and Lims1. (E) The luciferase reporter assay detected that the transcription factor HSF4 enhanced the transcriptional activation of Atg7, Tfpi and Lims1 before and after UV treatment. (F) Differences in Atg7, Tfpi and Lims1 mRNA expression before and after UV induction. (G) Differential expression of Atg7, Tfpi and Lims1 protein before and after UV induction. Error bars represent mean ± s.d.; * p ≤ 0.05; ** p ≤ .01; *** p ≤ .0005.

Article Snippet: Protein products were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight at 4°C with primary antibodies against HSF4 (Affinity), COIL, (Proteintech), LIMS1 (Affinity), ATG7 (Affinity), TFPI (Affinity), H2AX (Zenbio) and GAPDH (Affinity) and then for 1 h at room temperature in horseradish peroxidase conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Beyotime).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, ChIP-qPCR, Luciferase, Reporter Assay, Activation Assay, Quantitative Proteomics

Journal: Clinical and Translational Medicine

Article Title: HSF4/COIL complex‐dependent R‐loop mediates ultraviolet‐induced inflammatory skin injury

doi: 10.1002/ctm2.1336

Figure Lengend Snippet: R‐loop is involved in ultraviolet (UV) response mediated by HSF4–COIL. (A) The distribution statistics of HSF4 and COIL UV‐induced/non‐UV‐induced CUT&RUN‐seq binding peak positions. (B) CUT&RUN ‐seq showed enhanced binding of HSF4 and COIL to Atg7, Tfpi and Lims1 after UV induction. (C) Chromatin immunoprecipitation (ChIP)‐reChIP showed enhanced binding of Atg7, Tfpi and Lims1 to HSF4 and COIL in the promoter regions (R1, R2 and R3) after UV induction. (D) RNA content of HSF4, COIL and ChIP samples treated with different nucleases. (E) The immunofluorescence image shows that the content of the R‐loop increases after UV stimulation, and RNaseH can specifically degrade the R‐loop. Statistical charts make statistics on the fluorescence signal of the R‐loop. (F) DRIP‐qPCR showed that R‐loop enhanced the regulation of Atg7, Tfpi and Lims1 after UV induction. (G) CUT&RUN‐seq showed the colocalization of Atg7, Tfpi, Lims1 and R‐loop peaks under UV induction. (H) DRIP‐qPCR showed that R‐loop mediates the expression of Atg7, Tfpi and Lims1 after UV induction.Error bars represent mean ± s.d.; * p ≤ .05; ** p ≤ .01; *** p ≤ .0005.

Article Snippet: Protein products were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight at 4°C with primary antibodies against HSF4 (Affinity), COIL, (Proteintech), LIMS1 (Affinity), ATG7 (Affinity), TFPI (Affinity), H2AX (Zenbio) and GAPDH (Affinity) and then for 1 h at room temperature in horseradish peroxidase conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Beyotime).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Immunofluorescence, Fluorescence, Expressing

Journal: Clinical and Translational Medicine

Article Title: HSF4/COIL complex‐dependent R‐loop mediates ultraviolet‐induced inflammatory skin injury

doi: 10.1002/ctm2.1336

Figure Lengend Snippet: Ultraviolet (UV)‐induced R‐loop pathological increase is dependent on the HSF4–COIL complex. (A) Immunofluorescence showing the effects of overexpression/knockout HSF4 and COIL on R‐loop expression before and after UV stimulation. Statistical graphs are used to perform statistics on the R‐loop fluorescence signal. (B) Proximity ligation assay (PLA) experiments showed colocalization analysis of R‐loops after the overexpression/knockdown of HSF4 and COIL, proving that COIL is a key protein that binds to R‐loops. Statistical plots count the fluorescent signals generated by colocalization. (C) DRIP–qPCR demonstrated the regulation of Atg7, Tfpi and Lims1 by R‐loops. (D) The luciferase reporter gene showed the transcriptional activities of Atg7, Tfpi and Lims1 under different R‐loop conditions. (E, F) Messenger RNA (mRNA) and protein expression levels of Atg7, Tfpi and Lims1 under different R‐loop conditions. Error bars represent mean ± s.d.; * p ≤ .05; ** p ≤ .01; *** p ≤ .0005.

Article Snippet: Protein products were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight at 4°C with primary antibodies against HSF4 (Affinity), COIL, (Proteintech), LIMS1 (Affinity), ATG7 (Affinity), TFPI (Affinity), H2AX (Zenbio) and GAPDH (Affinity) and then for 1 h at room temperature in horseradish peroxidase conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Beyotime).

Techniques: Immunofluorescence, Over Expression, Knock-Out, Expressing, Fluorescence, Proximity Ligation Assay, Knockdown, Generated, Luciferase

Journal: Clinical and Translational Medicine

Article Title: HSF4/COIL complex‐dependent R‐loop mediates ultraviolet‐induced inflammatory skin injury

doi: 10.1002/ctm2.1336

Figure Lengend Snippet: HSF4‐COIL complex and R‐Loop promoted skin inflammation and ageing under UV conditions. (A, B) Effect of knockout or overexpression of Atg7 (A) and Tfpi (B) on the expression of inflammatory factors. (C) Effect of knocking out or overexpressing Lims1 on the expression of age‐related markers. Error bars represent mean ± s.d.; * p < .05; ** p < .01; *** p < .0005. (D) qPCR shows the effects of inflammatory factors and senescence factors before and after UV induction, and after simultaneous knockout/overexpression of HSF4, COIL and RNaseH. (E–G) HE staining showed the changes of epidermal thickness before and after UV induction, and immunohistochemistry showed the expression of inflammatory factors and aging factors in the skin after UV induction. (H) IHC showed that the expression of HSF4, COIL, and R‐loop in the tissues after UV induction changed, accompanied by epidermal thickening. (I) The score of immunohistochemistry showed that the expression levels of HSF4, COIL and R‐loop increased after UV induction, and the score of skin thickness proved that UV induction leads to skin thickening. (J) The expression of melanin content before and after UV induction and after knockout/overexpression of HSF4 and COIL. (K) The tyrosinase activity before and after UV induction, and after knockout/overexpression of HSF4 and COIL. Error bars represent mean ± s.d.; * p ≤ .05; ** p ≤ .01; *** p ≤ .0005.

Article Snippet: Protein products were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight at 4°C with primary antibodies against HSF4 (Affinity), COIL, (Proteintech), LIMS1 (Affinity), ATG7 (Affinity), TFPI (Affinity), H2AX (Zenbio) and GAPDH (Affinity) and then for 1 h at room temperature in horseradish peroxidase conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Beyotime).

Techniques: Knock-Out, Over Expression, Expressing, Staining, Immunohistochemistry, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: HSF4/COIL complex‐dependent R‐loop mediates ultraviolet‐induced inflammatory skin injury

doi: 10.1002/ctm2.1336

Figure Lengend Snippet: Nucleotide analogue N6‐(2‐hydroxyethyl)‐adenosine (HEA) can reduce the presence of R‐loop. (A) The docking energies predicted by the docking conformation of various nucleoside analogue drugs combined with R‐loop were ranked, and the docking results were evaluated. (B) Structure display of COIL binding to R‐loop, and structure display of COIL binding to N6‐(2‐hydroxyethyl)‐adenosine (HEA). (C, D) Proximity ligation assay (PLA) experiments verified that HEA would attenuate the binding of COIL to the R‐loop, and the statistical graph was the number of co‐localized fluorescent signals in the nucleus. (E) qPCR showed that high and low doses of HEA reduced the expression of downstream inflammatory factors. (F, G) Immunofluorescence test showed that after UV induction γ‐ The increased expression of H2AX indicates an increase in DNA damage. (H) Cck8 showed that the activity of cells treated with HEA‐H/L was significantly higher than that of cells treated with UV. (I–K) Immunohistochemistry of mouse back skin showed that Atg7, Tfpi and Lims1 increased pathologically after UV irradiation, and the expressions of Atg7, Tfpi and Lims1 decreased after smearing HEA on the back of the UV model mice. (L) Immunofluorescent techniques on fixed skin tissue showed that HEA reduced R‐loop expression at the tissue level. (M) Statistics of the skin thickness of the mice after UV irradiation and HEA treatment, the immunohistochemical score (H‐score) was then calculated. And the statistics of the S9.6 fluorescence signal in the immunofluorescence nucleus on the tissue. (N, O) Mouse skin IHC showing the effect of HEA treatment on the expression of inflammatory factors. Statistical graphs are immunohistochemical scores. (P) Proposed model for the role of HSF4‐COIL complex and R‐loop in inflammatory injury caused by UV radiation. Error bars represent mean ± s.d.; * p ≤ .05; ** p ≤ .01; *** p ≤ .0005.

Article Snippet: Protein products were separated by electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes and incubated overnight at 4°C with primary antibodies against HSF4 (Affinity), COIL, (Proteintech), LIMS1 (Affinity), ATG7 (Affinity), TFPI (Affinity), H2AX (Zenbio) and GAPDH (Affinity) and then for 1 h at room temperature in horseradish peroxidase conjugated goat anti‐rabbit or goat anti‐mouse IgG secondary antibodies (Beyotime).

Techniques: Binding Assay, Proximity Ligation Assay, Expressing, Immunofluorescence, Activity Assay, Immunohistochemistry, Irradiation, Immunohistochemical staining, Fluorescence