Journal: Drug Design, Development and Therapy

Article Title: Targeted Degradation of Histone Deacetylase 8 Using Proteolysis Targeting Chimeras Technology: A Promising Approach for Glioblastoma Treatment

doi: 10.2147/DDDT.S555228

Figure Lengend Snippet: HDAC8 PROTAC (1) downregulated Cdk1, Cdk2, Cdk4, Cdk6, and Cyclin B1 in glioblastoma cells. Representative Western blots and bar graphs of HDAC8, Cdks, and cyclins compared to GAPDH after 72-h treatment with the HDAC8 PROTAC (1) , HDAC8 inhibitor (2) , and pomalidomide (3) at 10 μM. Data are represented as the mean ± S.D. of five independent experiments. (** p < 0.01, *** p < 0.001).

Article Snippet: Rabbit polyclonal Cyclin B1 antibody , Cell Signaling Technology , CS#4138 , 1:1000.

Techniques: Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Targeted Degradation of Histone Deacetylase 8 Using Proteolysis Targeting Chimeras Technology: A Promising Approach for Glioblastoma Treatment

doi: 10.2147/DDDT.S555228

Figure Lengend Snippet: Proposed mechanism of HDAC8 PROTAC (1) in U-87 MG glioblastoma cells. HDAC8 degradation mediated by HDAC8 PROTAC (1) selectively caused cytotoxic toward glioblastoma cells and inhibited glioblastoma cell growth by reducing Cdk1, Cdk2, Cdk4, Cdk6, and cyclin B1 leading to S-phase arrest in glioblastoma cells. In addition, the HDAC8 PROTAC (1) also activates IRE1α observed by increasing of BiP and XBP1s level. Then, the activated IRE1α/XBP1s signal activates JNK as shown by the increase of phosphorylated JNK. Next, phosphorylated JNK induces CHOP which downregulates Bcl-2 protein leading to apoptosis in U87-MG cells. Downward red arrow refers to downregulate of protein level, while upward red arrow refers to upregulate of protein level. This figure was created in BioRender. Chotitumnavee, (J) (2025) https://BioRender.com/j5c7j5q .

Article Snippet: Rabbit polyclonal Cyclin B1 antibody , Cell Signaling Technology , CS#4138 , 1:1000.

Techniques:

Journal: bioRxiv

Article Title: Identification and inhibition of the Cyclin D Rb-docking interface that drives cell division

doi: 10.64898/2026.01.14.699544

Figure Lengend Snippet: A. Lentiviral integration constructs containing doxycycline-inducible shRNA targeting the 3’ UTR of CCND1 and doxycycline-inducible CCND1 cDNA fused to a FLAG affinity tag sequence. Constructs were integrated into RPE-1 cells. B. Immunoblot analysis of lysates from RPE-1 cells containing the constructs indicated in (A) that were treated with doxycycline for 48 hours to knockdown the endogenous cyclin D1 and express the indicated CCND1 mutant. WT denotes wild-type cyclin D1, A2’-1 and A2’-2 denote two different sets of alanine mutations in cyclin D1’s A2’ helix, and HP denotes mutation of the hydrophobic patch. C. Flow cytometry analysis of cell cycle phase distribution using EdU incorporation and DAPI staining after 48 hours of doxycycline treatment as in B. D. Cell size analysis using a Coulter counter of the indicated RPE-1 cell lines after 48 hours of doxycycline treatment as in B. E. Characteristic images of the indicated RPE-1 cell lines growing on soft agar in media containing doxycycline. F. Quantification of colony area for soft agar growth assays in E.

Article Snippet: Membranes were incubated with the following antibodies: Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (Cell Signaling Technology #8516), Phospho-Rb (Ser780) (D59B7) Rabbit mAb (Cell Signaling Technology #8180), Rb (4H1) Mouse mAb (Cell Signaling Technology #9309), Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma-Aldrich T9026), and Rabbit polyclonal cyclin D1 Antibody H-295 (Santa Cruz Biotechnology sc-753).

Techniques: Construct, shRNA, Sequencing, Western Blot, Knockdown, Mutagenesis, Flow Cytometry, Staining

Journal: The EMBO Journal

Article Title: Dual pathways via CENP-C and Mis18C recruit HJURP for CENP-A deposition into vertebrate centromeres

doi: 10.1038/s44318-025-00674-z

Figure Lengend Snippet: ( A ) Immunoblot analysis to detect HJURP with an anti-HJURP antibody in Tet-based HJURP conditional knockout (cKO) cells expressing EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) at the indicated time points after Tet addition. α-tubulin was probed as a loading control. Molecular weight markers (kDa) are shown on the left. ( B ) Flow cytometry analysis of HJURP-cKO cells expressing EGFP-fused HJURP (WT) or HJURP 2E (2E) at 4, 5, and 6 days after Tet addition. G1, S, G2/M, Sub-G1, and apoptotic phases are shown. ( C ) Quantification of cell populations of HJURP-cKO cells expressing EGFP-fused HJURP (WT) or HJURP 2E (2E) at 4, 5, and 6 days after Tet addition as determined by flow cytometry analysis. ( D ) Co-IP experiments using cytoplasmic extracts with an anti-GFP antibody in Tet-based HJURP conditional knockout cells expressing either EGFP-HJURP or EGFP-HJURP 2E (V117E/I120E) as a bait. Tetracycline was added to the culture medium 24 h before extraction to replace endogenous HJURP expression with EGFP-fused HJURP variants. Immunoblot analyses to detect HJURP and CENP-A in either EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) expressing cells. The co-immunoprecipitated CENP-A amounts in WT cells were comparable to those in cells expressing EGFP-HJURP 2E , indicating that the 2E mutation does not affect HJURP-CENP-A binding. Each assay was performed twice. Molecular weight markers (kDa) are shown on the left. The workflow of the cytoplasmic extract preparation and immunoprecipitation is shown on the left. ( E ) Immunoblot analysis to detect HJURP, CENP-C, and KNL2 with anti-HJURP, anti-CENP-C, and anti-KNL2 antibodies, respectively, in AID-based CENP-C or KNL2 knockout cells with Tet-based HJURP-cKO background. Cells expressing either EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) were analyzed in the presence (+) or absence (−) of IAA (5 h) or Tet (24 h) as indicated. α-tubulin was probed as a loading control. Molecular weight markers (kDa) are shown on the left. ( F ) EGFP-HJURP localization in DT40 cells at various cell-cycle stages stained with an anti-Cyclin B2 antibody. EGFP-HJURP signals are classified as high, medium, or low intensity, corresponding to different cell-cycle stages. In Cyclin B2-negative interphase cells (early G1 phase), EGFP-HJURP signals are high. In contrast, EGFP-HJURP signals were weak (medium and low) in Cyclin B2-positive G1 cells and they were undetectable in Cyclin B2-positive S and G2 phases. DNA was stained with DAPI. Scale bar, 10 μm. ( G ) Left: Spike-in normalized ChIP-seq profiles around the centromere of the chicken chromosome 5 (Cen5) with an anti-HJURP antibody in AID-based CENP-C or KNL2 knockout cells expressing EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) in either the absence or the presence of IAA. Right: Quantification of normalized mapped-read ratios on the Cen5 region by Spike-in ChIP-seq for each condition. Each assay was performed three times. Results are presented as the mean with individual experimental values shown as dots. For the ChIP-seq profiles, the representative data sets are shown.

Article Snippet: Cyclin B2 Rabbit polyclonal antibody , Proteintech , Cat#: 21644-1-AP; RRID: AB_10755304.

Techniques: Western Blot, Knock-Out, Expressing, Control, Molecular Weight, Flow Cytometry, Co-Immunoprecipitation Assay, Extraction, Immunoprecipitation, Mutagenesis, Binding Assay, Staining, ChIP-sequencing

Journal: The EMBO Journal

Article Title: Dual pathways via CENP-C and Mis18C recruit HJURP for CENP-A deposition into vertebrate centromeres

doi: 10.1038/s44318-025-00674-z

Figure Lengend Snippet: ( A ) Experimental scheme to analyze the significance of the interaction between CENP-C and HJURP in cells. EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) expressing AID-based CENP-C or KNL2 knockout cells with HJURP-cKO background were generated. Tetracycline was added to the culture medium 24 h before fixation to replace wild-type HJURP expression with EGFP-fused HJURP variants. At 5 h after IAA addition, CENP-C or KNL2 were undetectable in the respective knockout cells as shown in Fig. . In CENP-C On or Off cells expressing EGFP-HJURP 2E , the Mis18C pathway is expected to be still active, while in KNL2 Off cells expressing EGFP-HJURP 2E , both Mis18C and CENP-C pathways are expected to be inactive. ( B ) Localization of EGFP-tagged HJURP (WT) or HJURP 2E (2E) in AID-based CENP-C or KNL2 knockout cells with HJURP-cKO background as described in ( A ). Cyclin B2-negative cells were identified as early G1 cells. Cyclin B2 staining profiles with HJURP localization are shown in Fig. . CENP-T was used as a centromere marker. DNA was stained with DAPI. Scale bar, 10 μm. ( C ) Relative intensities of EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) in AID-based CENP-C or KNL2 knockout cell lines in the absence or presence of IAA. Each assay was conducted twice ( n = 15 cells per condition), and results are presented as median relative signal intensities (bold dotted-line) with first and third quartiles (dotted-lines) using violin plots. ( D ) Experimental scheme for quantitative ChIP-seq with an anti-HJURP antibody to evaluate HJURP levels at G1 centromeres in AID-based CENP-C or KNL2 knockout cell lines expressing EGFP-HJURP (WT) or EGFP-HJURP 2E . Cells were treated with tetracycline for 24 h before fixation to replace endogenous HJURP expression with the EGFP-fused HJURP variants. Cells were then cultured with Nocodazole in the absence or presence of IAA for 8 h, with Reversine added 0.5 h before fixation. Fixed G1-accumulated cells were subjected to Spike-in ChIP-seq. ( E ) Spike-in normalized ChIP-seq profiles around the centromere of the chicken chromosome Z (CenZ) with an anti-HJURP antibody in AID-based CENP-C or KNL2 knockout cells expressing EGFP-HJURP (WT) or EGFP-HJURP 2E (2E) in either the absence or the presence of IAA as described in ( D ). ( F ) Quantification of normalized mapped-read ratios on the CenZ region by spike-in ChIP-seq experiments shown in ( E ). Each assay was performed three times. Results are presented as the mean with individual experimental values shown as dots. For the ChIP-seq profiles, a representative data set is shown in ( E ). .

Article Snippet: Cyclin B2 Rabbit polyclonal antibody , Proteintech , Cat#: 21644-1-AP; RRID: AB_10755304.

Techniques: Expressing, Knock-Out, Generated, Staining, Marker, ChIP-sequencing, Cell Culture

Journal: The EMBO Journal

Article Title: Dual pathways via CENP-C and Mis18C recruit HJURP for CENP-A deposition into vertebrate centromeres

doi: 10.1038/s44318-025-00674-z

Figure Lengend Snippet: ( A ) Amino acid sequence of the short repeats of HJURP from chicken, human, and mouse. Putative key residues for the Mis18α/β binding are annotated on the top. ( B ) An AF3 model for the interaction between chicken HJURP (aa 493–523) and the Mis18α/β complex containing two Mis18α molecules (aa 160–204) and one Mis18β molecule (aa 160–213). In the magnified view, the residues involved in the putative interface are shown as sticks. ( C ) An AF3 model showing the interaction between chicken HJURP (aa 546–576) and the Mis18α/β complex. ( D ) GST pull-down assay using lysate from bacterial cells expressing GST-ggHJURP 480-580 and His 6 -ggMis18α 38-204 :MBP-ggMis18β 45-213 . Molecular weight markers (kDa) are shown on the left. ( E ) Competitive GST pull-down assay for the ggHJURP 480-580 binding to the Mis18α/β complex and the CENP-C C-terminal region. The concentration of the purified ggCENP-C 677-864 in the reaction was 3.1 µM (+) or 6.2 µM (++). Molecular weight markers (kDa) are shown on the left. ( F ) Schematic representation of the experimental scheme to analyze the significance of the interaction between Mis18C and HJURP in cells. AID-based CENP-C knockout cells with a Tet-inducible HJURP-cKO background expressing Halo-HJURP (WT) or Halo-HJURP 8K (8K) were used. Tet was added to the culture medium 24 h before fixation to replace wild-type HJURP expression with Halo-fused HJURP variants. In CENP-C off cells, the Mis18C pathway alone could be examined in cells expressing Halo-HJURP or Halo-HJURP 8K . ( G ) Immunoblot analysis of HJURP, KNL2, and CENP-C using anti-HJURP, anti-KNL2, and anti-CENP-C antibodies, respectively, in AID-based CENP-C knockout cells with Tet-based HJURP-cKO background expressing Halo-HJURP (WT) or Halo-HJURP 8K (8K) in the presence (+) or absence (−) of IAA (5 h). Signal intensities of CENP-C or CENP-C AID on the blot were measured with normalization to α-tubulin intensity in each blot. Wild-type cells (Cl18) were also analyzed. α-tubulin was probed as a loading control. Molecular weight markers (kDa) are shown on the left. ( H ) Localization of Halo-tagged HJURP (WT) or HJURP 8K (8 K) in AID-based CENP-C knockout cells with a HJURP-cKO background as described in ( F ). Cyclin B2-negative cells were identified as early G1 cells and marked with yellow dotted lines. CENP-T was used as a centromere marker. DNA was stained with DAPI. Scale bar, 10 μm. ( I ) Relative intensities of Halo-HJURP (WT) or Halo-HJURP 8K (8K) in AID-based CENP-C knockout cell lines in the absence or presence of IAA. Data are presented as median relative signal intensities (bold line) with first and third quartiles (dotted lines) using violin plots.

Article Snippet: Cyclin B2 Rabbit polyclonal antibody , Proteintech , Cat#: 21644-1-AP; RRID: AB_10755304.

Techniques: Sequencing, Binding Assay, Pull Down Assay, Expressing, Molecular Weight, Concentration Assay, Purification, Knock-Out, Western Blot, Control, Marker, Staining