Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 activates the NF-κB pathway and regulates the transcription of SASP. (A) Western blot assay was used to detect the expression levels of p-IκBα, IκBα, p-p65, and p65. (B) Immunoprecipitation was performed to assess the co-precipitation of CDK8 and p65 in C28/I2 cells treated with or without CDK8-IN-6. (C) Immunofluorescence co-localization was conducted to examine the co-localization of p65 and CDK8 in C28/I2 cells treated with IL-1β for 24 h. (D) C28/I2 cells were transfected with si-CDK8 or pCMV3-CDK8, followed by IL-1β treatment for 24 h, and then subjected to western blot analysis of nuclear and cytoplasmic proteins. (E-F) Dual-luciferase analysis demonstrated NF-κB's regulatory role in SASP transcriptional activation (E) and confirmed the direct binding sites of p65 in the promoters of the IL-6, IL-8, and MMP-13 genes (F), the statistical results indicating the significance of the differences between the truncated promoter groups and the full-length group. (G) Nucleic acid electrophoresis and (H) ChIP-qPCR further investigated the influence of CDK8 on the binding of p65 to SASP promoter region sites in C28/I2 cells. (I) Western blot assay was used to detect the expression levels of Rpb1 CTD, p-Rpb1 CTD (Ser2), and p-Rpb1 CTD (Ser5). Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Article Snippet: The primary antibodies used are listed as follows: Aggrecan (HuaBio, Cat. ET1704-57), COL2A1 (Abcam, Cat. ab34712), SOX9 (HuaBio, Cat. et1611-56), MMP3 (Abcam, Cat. ab52915), MMP13 (Abcam, Cat. ab39012), CDK8 (Proteintech, Cat. 22067–1-AP), p65 (CST, Cat. 8242), p-p65 (Affinity, Cat. AF2006), IκBα (CST, Cat. 4814), p-IκBα (CST, Cat. 2859), Rpb1 CTD (CST, Cat. 2629), p-Rpb1 CTD(ser 2)(CST, Cat. 13499), p-Rpb1 CTD(ser 5)(CST, Cat. 13523), Cleaved Caspase-1(CST, Cat. 89332), Cleaved-IL-1β (CST, Cat. 63124), Caspase1 (HuaBio, Cat. ET1608-69), NLRP3 (Abcam, Cat. ab214185), ASC (Proteintech, Cat. 10500–1-AP), FLAG (HuaBio, Cat. 0912–1), β-Actin (Fudebio, Cat. FD0060), Histone H3 (Proteintech, Cat. 68345–1-Ig), IgG (Proteintech, Cat. 30000–0-AP).

Techniques: Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Transfection, Luciferase, Activation Assay, Binding Assay, Nucleic Acid Electrophoresis, ChIP-qPCR, Control

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 and NF-κB are cooperatively recruited to SASP promoters, leading to elongation phosphorylation of the Rpb1 CTD. In C28/I2 cells transfected with si-NC or si-CDK8, with or without IL-1β treatment for one hour, (A–E) ChIP analysis shows the effects of CDK8 knockdown and IL-1β treatment on the binding of p65 (A), CDK8 (B), Rpb1 CTD (C), Rpb1 CTD phosphorylated at Ser5 (D), and Rpb1 CTD phosphorylated at Ser2 (E) to three SASP genes and a housekeeping gene. The gene diagrams are displayed at the top.

Article Snippet: The primary antibodies used are listed as follows: Aggrecan (HuaBio, Cat. ET1704-57), COL2A1 (Abcam, Cat. ab34712), SOX9 (HuaBio, Cat. et1611-56), MMP3 (Abcam, Cat. ab52915), MMP13 (Abcam, Cat. ab39012), CDK8 (Proteintech, Cat. 22067–1-AP), p65 (CST, Cat. 8242), p-p65 (Affinity, Cat. AF2006), IκBα (CST, Cat. 4814), p-IκBα (CST, Cat. 2859), Rpb1 CTD (CST, Cat. 2629), p-Rpb1 CTD(ser 2)(CST, Cat. 13499), p-Rpb1 CTD(ser 5)(CST, Cat. 13523), Cleaved Caspase-1(CST, Cat. 89332), Cleaved-IL-1β (CST, Cat. 63124), Caspase1 (HuaBio, Cat. ET1608-69), NLRP3 (Abcam, Cat. ab214185), ASC (Proteintech, Cat. 10500–1-AP), FLAG (HuaBio, Cat. 0912–1), β-Actin (Fudebio, Cat. FD0060), Histone H3 (Proteintech, Cat. 68345–1-Ig), IgG (Proteintech, Cat. 30000–0-AP).

Techniques: Phospho-proteomics, Transfection, Knockdown, Binding Assay

Journal: Nature Communications

Article Title: RNF20 links the DNA damage response and metabolic rewiring in lung cancer through HIF1α

doi: 10.1038/s41467-025-60223-4

Figure Lengend Snippet: a Schematic representation of the method for calculating Pol II pausing index (PI). b Genome tracks of merged Pol II RNA-Seq reads of control and Rnf20 + /- MLE12 cells at genes involved in glycolysis. c Log2 of the PI at genes upregulated, unchanged or downregulated upon Rnf20 LOF ( n = 3). d, e Venn diagram showing an overlap of genes with decreased PI and upregulation in Rnf20 + /- compared to control MLE12 cells ( d ) and GO analysis of the overlapping genes ( e ). f Log2 of the PI at HIF-1 signaling pathway (left) or EMT genes (right) in control and Rnf20 + /- MLE12 cells ( n = 3). g Ratio of the Pol II enrichment at the TSS and TTS of Scl2a1 , Eno1 and Pdk1 in control ( n = 3) and Rnf20 + /- lungs ( n = 5) determined by Pol II Chip-qPCR. TSS, Transcription Start Site; TTS, Transcription Termination Site. h Venn diagram showing the overlap of genes with decreased PI in Rnf20 + /- compared to control MLE12 cells and HIF1α-bound genes in A549 lung AD cells (left) and GO analysis of the overlap (right). i, j Genome tracks of merged Pol II ChIP-Seq reads of control, Rnf20 + /- and Rnf20 + /- MLE12 cells stably expressing shRNA against Hif1a ( i ) and PI at HIF1α-bound genes in lung A549 AD cells ( j , n = 2). k Chip-qPCR for RNA Pol II CTD-pSer2 (left) and CTD-pSer5 (right) at Scl2a1 , Ldha and Eno1 gene body in control, Rnf20 + /- MLE12 cells, or Rnf20 + /- MLE12 cells after shRNA mediated Hif1a silencing ( n = 3). Statistical analysis in ( c, f, g ) was performed using a two-tailed Student’s t-test. Multiple comparisons were performed using one-way ANOVA with Šídák’s ( j, k ) multiple comparisons test. Boxplots in ( c, f, j ) represent the minimum, 25 th percentile (Q1), median, 75 th percentile (Q3), and maximum of the Log2 PI in control, Rnf20 + /- MLE12 cells, or Rnf20 + /- MLE12 cells after shRNA-mediated Hif1a silencing. GO analysis in e and h was performed using Metascape (Version 3.5). Data are shown as mean ± SEM. ns, non-significant. ‘n’ indicates biological replicates.

Article Snippet: The nuclei were washed with cold PBS and centrifuged at 2500 g , 4 °C for 5 min. Chromatin was sheared using a Covaris Ultrasonicator (10% duty factor, 200 cycles, 75 A) for 6 min and the samples were clarified at 16,000 g for 15 min. Next, the extracts were pre-cleaned with 75 μl protein G beads for 2 h at 4 °C, then incubated with 0.5 μg Pol II antibody (14958S, Cell Signaling), 1 μg Pol II p-Ser5 (ab5131, Abcam), 1 μg Pol II p-Ser2 (13499 s, Cell Signaling), 1 μg H2Bub1 (5546S, Cell Signaling) or 1 μg H3K4me3 (ab8580, Abcam) antibodies overnight at 4 °C, followed by binding to 75 μl BSA-coated protein G beads.

Techniques: RNA Sequencing, Control, ChIP-qPCR, ChIP-sequencing, Stable Transfection, Expressing, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: RNF20 links the DNA damage response and metabolic rewiring in lung cancer through HIF1α

doi: 10.1038/s41467-025-60223-4

Figure Lengend Snippet: a Immunohistochemistry of tissue samples from different types and grades of lung tumors from a tissue microarray stained with HIF1α, ENO1 and LDHA antibodies. Scale bars, 100 µm. b Pearson correlation (r) of the relative staining intensity (H-Score) of RNF20 and HIF1α (top panels), RNF20 and ENO1 (middle panels), and RNF20 and LDHA (bottom panels) in AD and SCLC patients, with a two-sided p-value computed using the t-distribution. n = 70 for AD; n = 22 for SCLC. c Schematic model of the function of RNF20 in lung SCLC and AD development. PNEC pulmonary neuroendocrine cell, AT1 alveolar type 1 cell, AT2 alveolar type 2 cell, RNAPII RNA polymerase II. The figure was created in BioRender .

Article Snippet: The nuclei were washed with cold PBS and centrifuged at 2500 g , 4 °C for 5 min. Chromatin was sheared using a Covaris Ultrasonicator (10% duty factor, 200 cycles, 75 A) for 6 min and the samples were clarified at 16,000 g for 15 min. Next, the extracts were pre-cleaned with 75 μl protein G beads for 2 h at 4 °C, then incubated with 0.5 μg Pol II antibody (14958S, Cell Signaling), 1 μg Pol II p-Ser5 (ab5131, Abcam), 1 μg Pol II p-Ser2 (13499 s, Cell Signaling), 1 μg H2Bub1 (5546S, Cell Signaling) or 1 μg H3K4me3 (ab8580, Abcam) antibodies overnight at 4 °C, followed by binding to 75 μl BSA-coated protein G beads.

Techniques: Immunohistochemistry, Microarray, Staining