Journal: Translational Neurodegeneration

Article Title: Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

doi: 10.1186/s40035-020-00211-4

Figure Lengend Snippet: PINCH is transcriptionally regulated by MEF2A under inflammatory conditions. a Quantification of TNFα production by neurons exposed to Tat at different time points. b Quantification of PINCH mRNA levels in neurons untreated or exposed to Tat or TNFα for 48 h. c Bioinformatic analysis of lims1/pinch promoter sequence predicted a conserved putative binding site for different transcription factors (TF). Inset: Sequences show conserved binding sites for MEF2A, Cc-FOS, Cc-Jun, and AP-1. d ChIP-assay was performed in neurons untreated or exposed to Tat or TNFα. Antibodies specific for MEF2A, c-Jun, Foxd1, and HoxA9 were used to immunoprecipitate the chromatin and the fold enrichment of lims1/pinch promoter relative to the matched input control was quantified by qPCR. e Representative Western blot of lysates from neurons untreated or exposed to Tat or TNFα and probed with antibodies specific for phospho-CamkII, CamkII, phospho-P38, P38, phospho-MEF2A, MEF2A, PINCH and GAPDH. f-i Quantification of relative protein abundance of phospho-CamkII/CamkII ( f ), phospho-P38/P38 ( g ), phospho-MEF2A/MEF2A ( h ), and PINCH/GAPDH ( i ) from ( e ). j Schematic representation of the lims1/pinch promoter-luciferase constructs. The MEF2A consensus response element at − 169-175 base pairs (bp) (TATTATA) is shown in the oval. k Neurons transfected with control and lims1/pinch luciferase constructs were untreated or exposed to Tat or TNFα for 48 h and luciferase activity was measured. Data represent mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3–5 (one-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with lims1/pinch (PINCH) (NM_004987) promoter sequence (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for the luciferase assay.

Techniques: Sequencing, Binding Assay, Control, Western Blot, Quantitative Proteomics, Luciferase, Construct, Transfection, 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: 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