Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 1. Structural model of the interaction between pUL97 and cyclin H. (A) Predicted binding site of pUL97(231-280) (orange) superimposed with the experimental cyclin H–CDK7–MAT1 complex structure (gray, cyan, and dark blue). This model suggests that pUL97(231-280) uses the same binding pocket as MAT1 for targeting the cyclin H–CDK7 complex. (B) Model of a ternary pUL97–cyclin H–CDK7 complex, in which pUL97 is attached to cyclin H exclusively through IF2 formed by the 231-280 sequence stretch. The pUL97 kinase domain (residues 329-634, marked in red) is connected to the complex by a nonstructured, flexible linker (residues 281-328, indicated as dark orange connecting line). (C) Model of a pUL97–cyclin H complex, in which pUL97 interacts with cyclin H both through IF2, pUL97(231-280) (orange), and the globular kinase domain IF1, pUL97(329-634) (red), thereby displacing CDK7.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Binding Assay, Sequencing

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 3. Viral protein substrate phosphorylation by CDK7. HFFs were infected with HCMV AD169-GFP at MOI of 1 and lysed 3 d.p.i. (A) Viral proteins were coimmunoprecipitated with cellular kinase CDK7 (lanes 3-10) and exposed to the non-radioactive IVKA reaction. Specific phosphorylation signals are marked to indicate single (◦) or continuous (arrows in respective colors) bands. Autophosphorylation signals of viral kinase pUL97 served as a positive control (lane 2). Du plicates of each immunoprecipitate were treated with 5 µM of CDK7-inhibitor LDC4297 shortly before the IVKA reaction (lanes 4, 6, 8, 10). (B) An IVKA reaction conducted in parallel without ATPγS served as a negative control. Successful IP (C) and reliable expression levels (D) of all proteins of interest were demonstrated by Wb analysis. (E) Phosphorylation signals from IVKA reactions shown in (A) were quantitated three times with varying background parameters, resulting in triplicate determinations of each sample, and mean values ± SD were then normalized to IP control.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, Positive Control, Negative Control, Expressing, Control

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 6. Human CDK7 does not increase pUL97 in vitro kinase activity. (A) 293T cells were transiently transfected with plasmids encoding pUL97-Flag. Cells were lysed 2 d post-transfection, and pUL97-Flag and CDK7 were immu noprecipitated using the indicated antibodies; a mouse Fc fragment served as a negative control. Dynabeads-bound proteins were eluted in 150 µl enzyme buffer. 25 µl of eluted Dynabeads were denatured in 25 µl 2x loading buffer and subjected to SDS-PAGE and Wb to confirm successful precipitation of pUL97- Flag and CDK7. (B) Samples derived from immunoprecipitation of CDK7 plus pUL97-Flag, as well as Fc control samples, were subjected to a qSox-IVKA (using the pUL97-specific sensor peptide AQT0258). Optionally, 0.01 µM of CDK7-specific inhibitor LDC4297 (corresponds to the mean antiviral EC50 value) or equal amounts of DMSO were added to the reactions. Background activity determined with the Fc control was subtracted from values of combined pUL97-Flag plus CDK7 kinase activity. Mean values ± SD derived from one representative experiment are given, as derived from measurements in tripli cates. Statistical analysis was performed using an ordinary two-way ANOVA: n. s., not significant.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: In Vitro, Activity Assay, Transfection, Negative Control, SDS Page, Derivative Assay, Immunoprecipitation, Control

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 7. Phosphorylation patterns of CDK7 depend on the state of HCMV infection and pUL97 kinase activity. (A) HFFs were seeded in T75 cell culture flasks and infected 1 d later with HCMV AD169, or pUL97 kinase-deficient mutant ORF-UL97 K355Δ at MOI of 0.5, or remained-mock infected. 24 h after infection HCMV AD169 infected cells were treated with 0.35 µM (i.e. EC50) pUL97-specific inhibitor maribavir (MBV), or with equal amounts of DMSO. Cells were harvested 3 d p.i. and samples were subjected to standard SDS-PAGE and Wb analysis. Proteins of interest and site-specific phosphorylation were stained using the indicated antibodies. (B) Mean values ± SD of phosphorylation intensity of CDK7 at sites Ser164 and Thr170, as well as CDK9 Thr186, was determined by quadruplicate densitometric quantitation of those Wbs depicted in (A) together with a second biological replicate (shown in Fig. S5). Statistical analysis was performed using an ordinary one-way ANOVA and post-hoc Tukey correction: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, Activity Assay, Cell Culture, Mutagenesis, SDS Page, Staining, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

doi: 10.1128/mcb.00807-09

Figure Lengend Snippet: FIG. 6. GnRH treatment stimulates SF-1 ubiquitination. (A) LT2 cell lysates after transfection with FLAG–SF-1 and exposure of some cells to 100 nM GnRH for 6 h were precipitated with anti-FLAG M2 beads. The IP samples were analyzed using anti-FLAG, Rb anti-FLAG, and anti-Ub Ab. (B) Western blotting detected phosphorylated ERK (pERK) in LT2 cells after exposure to 100 nM GnRH for 0 to 4 h and/or 1 M U0126; total ERK is shown as a loading control. (C and D) LT2 cell lysates after transfection with WT FLAG–SF-1 or SF-1 S203A, some of which were exposed to 100 nM GnRH and 1 M U0126 (C) or 15 M roscovitine (ROS) (D) for 0 or 4 h, were precipitated with anti-FLAG M2 beads. The input and IP samples were analyzed using anti-FLAG, anti-Ub, and anti-GAPDH Ab. (E) LT2 cell lysates after transfection with FLAG–SF-1 together with pcDNA3, pcDNA3 CDK7 HA, or pcDNA3 CDK7 D155A HA were precipitated with anti-FLAG M2 beads. The input and IP samples were analyzed by Western blotting. Monoubiquitinated SF-1 is marked with arrows on the left.

Article Snippet: Pitx1, SF-1, Egr-1, ubiquitin, and SUMO1 sequences were inserted into pxj40-HA, pxj40-FLAG, and pxj40-Myc (gifts from B. C. Low, National University of Singapore). pxj40-GFP was used to construct pxj40 GFP SF-1 and pxj40 GFP SF-1 K119R. pcDNA3 CDK7 HA (P#633) and pcDNA3 CDK7 D155A HA (P#812) were purchased from Addgene.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Pin1 Facilitates the Phosphorylation-Dependent Ubiquitination of SF-1 To Regulate Gonadotropin β-Subunit Gene Transcription

doi: 10.1128/mcb.00807-09

Figure Lengend Snippet: FIG. 10. Model of regulation of gonadotropin -subunit gene transcription by Pin1. GnRH activates PKC and PKA, either of which might phosphorylate Pin1 at Ser 16, causing its nuclear export. However, GnRH also elevates intracellular calcium levels and activates calcineurin, which dephosphorylates Pin1, allowing it to translocate back into the nucleus. Inside the nucleus Pin1, interacts with its target transcription factors, including SF-1, Pitx1, and Egr-1, enhancing their transcriptional activity toward the gonadotropin -subunit genes. DeSUMOylation of SF-1 at K194, which occurs through an unknown mechanism, results in enhanced phosphorylation of SF-1 at Ser203 by CDK7 (67). DeSUMOylation at K119 and GnRH-activated ERK1/2 also increase SF-1 phosphorylation. Phosphorylation of SF-1 and its subsequent isomerization by Pin1 promote ubiquitination of SF-1 at K119, facilitating the interaction between SF-1 and Pitx1 (and possibly other cofactors), to upregulate gonadotropin -subunit gene transcription. The proteasomal degradation of ubiquitinated SF-1 in the cytosol is a putative event, for which we do not yet have direct proof. Dashed lines represent pathways that have or may have (for PKA and PKC phosphorylation of Pin1) intermediate elements that are not shown.

Article Snippet: Pitx1, SF-1, Egr-1, ubiquitin, and SUMO1 sequences were inserted into pxj40-HA, pxj40-FLAG, and pxj40-Myc (gifts from B. C. Low, National University of Singapore). pxj40-GFP was used to construct pxj40 GFP SF-1 and pxj40 GFP SF-1 K119R. pcDNA3 CDK7 HA (P#633) and pcDNA3 CDK7 D155A HA (P#812) were purchased from Addgene.

Techniques: Activity Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: Cell & Bioscience

Article Title: CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

doi: 10.1186/s13578-024-01260-2

Figure Lengend Snippet: CDK7/CDK9 mediates the transcriptional regulation for paraptotic development. ( A – C ) MDA-MB-435 cells pretreated with the CDK7 inhibitor THZ1 (THZ, 0.2 µM) or the CDK9 inhibitor LY2857785 (LY, 1 µM) for 30 min were treated with CPYPP (10 µM), cyclosporin A (CsA, 20 µM), or curcumin (CUR, 30 µM) for 24 h. Cells were then collected for vacuolization ( A ), cell viability ( B ), and protein expression ( C ) analyses. ( D – F ) MDA-MB-435 cells were treated with control shRNA (Void), shCDK7, or shCDK9 for 4 days and then with CPYPP (10 µM) for 24 h. Cells were harvested for Western blot analysis ( D ) and vacuolization alterations ( E , F ). ( G , H) MDA-MB-435 cells were treated with CPYPP (10 µM) for the indicated time and then collected for Western blot and CDK7/CDK9 enzyme activity analysis. ( I , J ) MDA-MB-435 cancer cells were treated with CPYPP for 6–24 h, then harvested to isolate cytosolic and nuclear fractions for Rpb1 phosphorylation ( I ) and CDK7–Rpb1 complex activation by co-immunoprecipitation method ( J ). ( K ) The co-localization of Rpb1 and CDK9 was detected through co-immunofluorescence staining using anti-Rpb1 antibody (shown in green) and anti-CDK9 antibody (shown in red), followed by secondary antibody staining, along with DAPI (shown in blue) for nuclei counter staining. The images were captured using confocal microscopy at 630× magnification. The scale bar represents 25 μm. ( L ) The interaction and distribution between Rpb1 and CDK9 were assessed using the proximity ligation assay. ( M – O ) MDA-MB-435 cells pretreated with the CDK7 inhibitor THZ1 (THZ, 0.2 µM) or the CDK9 inhibitor LY2857785 (LY, 1 µM) for 30 min were treated with CPYPP (10 µM), cyclosporin A (CsA, 20 µM), or curcumin (CUR, 30 µM) for 24 h. Cells were collected for CDK7/CDK9 activity analysis ( M ) or to isolate cytosolic and nuclear fractions for Rpb1 phosphorylation ( N ) and CDK9–Rpb1 complex activation by co-immunoprecipitation method ( O ). ** P < 0.01, compared to the corresponding control group (CTL)

Article Snippet: Ten micrograms of proteins were used for CDK7 and CDK9 activity determination following the instructions enclosed in the kit (CDK7 Assay Kit, Cat# 79603; CDK9 Assay Kit, Cat# 79628, BPS Bioscience, San Diego, CA, USA).

Techniques: Expressing, Control, shRNA, Western Blot, Activity Assay, Activation Assay, Immunoprecipitation, Immunofluorescence, Staining, Confocal Microscopy, Proximity Ligation Assay

Journal: Cell & Bioscience

Article Title: CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

doi: 10.1186/s13578-024-01260-2

Figure Lengend Snippet: HSPs interact with the CDK7/CDK9–Rpb1 complex to prime stress-specific transcription in a forward loop and reciprocally regulated manner. ( A , B ) MDA-MB-435 cells were treated with CPYPP (10 µM) for 6–24 h, or with CPYPP, cyclosporin A (CsA, 20 μM), or curcumin (CUR, 30 μM) for 24 h, and then collected for cytosolic (Cyto) and nuclear (Nu) fraction isolation for Western blot analysis. ( C – G ) MDA-MB-435 and/or MDA-MB-231 cells were pretreated with MKT-077 (MKT) for 30 min followed by CPYPP (10 µM), cyclosporin A (CsA, 20 µM), or curcumin (CUR, 30 µM) for 24 h. Cells were then collected for cell viability ( C ), RNA polymerase II activation ( D ), ER stress, UPR, and protein ubiquitination ( E ), paraptotic vacuolization ( F ), and CDK7 and CDK9 activity ( G ) analysis. The results are expressed as the mean ± SD from three independent experiments. ** P < 0.01, compared to the corresponding control group. ( H , I ) Cancer cells treated with indicated concentrations of ML346 for 24 h promoted cell death ( H ) and paraptotic vacuolization (I, 10 µM). ( J , K , N) MDA-MB-435 cells were pretreated with THZ1 (THZ, 0.2 µM) or LY2857785 (LY, 1 µM) for 30 min followed by ML346 treatment for 24 h. Total cell lysates, cytosolic and nuclear fractions were used for CDK7 and CDK9 activity ( J ), protein expression ( K ), and the interaction of HSPs with CDKs by co-immunoprecipitation ( N ) analysis. ** P < 0.01, compared to the corresponding control group (CTL). ( L ) The co-localization of Rpb1, HSP40, and HSP70 was examined in CPYPP-treated MDA-MB-435 cells co-stained with anti-Rpb1 (shown in green), anti-HSP40 (shown in red), and anti-HSP70 (shown in magenta) antibodies, followed by individual secondary antibodies. DAPI staining (shown in blue) was used for nuclei counterstaining. Confocal microscopy was utilized to capture the images at 630× magnification. The scale bar represents 25 μm. ( M ) The interaction and distribution between Rpb1 and HSP40 were evaluated using the proximity ligation assay

Article Snippet: Ten micrograms of proteins were used for CDK7 and CDK9 activity determination following the instructions enclosed in the kit (CDK7 Assay Kit, Cat# 79603; CDK9 Assay Kit, Cat# 79628, BPS Bioscience, San Diego, CA, USA).

Techniques: Isolation, Western Blot, Activation Assay, Activity Assay, Control, Expressing, Immunoprecipitation, Staining, Confocal Microscopy, Proximity Ligation Assay

Journal: Cell & Bioscience

Article Title: CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

doi: 10.1186/s13578-024-01260-2

Figure Lengend Snippet: Overload of HSPs and the ubiquitin-proteasome system elicits unclear stress and promotes CDK7/CDK9–Rpb1 activation for transcriptional regulation. ( A ) MDA-MB-435 cells were treated with CPYPP (10 µM), cyclosporin A (CsA, 20 µM), or curcumin (CUR, 30 µM) for 24 h. Protein ubiquitination was visualized by immunofluorescence using an anti-ubiquitin antibody (shown in green), with DAPI (shown in blue) used for nuclei counterstaining. Results were imaged under a confocal microscopy at 630× magnification. Scale bar = 25 μm. ( B ) The subunits of the 19S and 20S proteasome complex. ( C , D , F , G , H , J) Cancer cells were treated with CPYPP, CsA, CUR, or ML346 (10 µM) for 6 h ( D , G ) or 24 h and then harvested for whole lysate collection or cytosolic and nuclear fractionation for proteasome subunit abundance determination by Western blot ( C , H ), 20 S proteasome activity ( D , F , G ), and protein ubiquitination analysis ( J ). ( E ) Cancer cells were treated with CPYPP for the indicated time period (h). The results are expressed as the mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, compared to the corresponding control group (CTL). ( I , K ) CPYPP-treated MDA-MB-435 cells were imaged by immunofluorescence staining for nuclear accumulation of proteasomes using an anti-20S core subunit antibody (green) and DAPI (blue) for nuclei counterstaining ( I ), or for colocalization of ubiquitins and aggresomes using an anti-ubiquitin antibody (green) with aggresome staining by ProteoStat® dye (red), and DAPI (blue) for nuclei counterstaining ( K )

Article Snippet: Ten micrograms of proteins were used for CDK7 and CDK9 activity determination following the instructions enclosed in the kit (CDK7 Assay Kit, Cat# 79603; CDK9 Assay Kit, Cat# 79628, BPS Bioscience, San Diego, CA, USA).

Techniques: Activation Assay, Immunofluorescence, Confocal Microscopy, Fractionation, Western Blot, Activity Assay, Control, Staining

Journal: Cell & Bioscience

Article Title: CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

doi: 10.1186/s13578-024-01260-2

Figure Lengend Snippet: ROS ignites the paraptotic process by CPYPP. ( A – F , H – L ) MDA-MB-435 and/or MDA-MB-231 cells were pretreated with NAC (3 mM) for 30 min followed by CPYPP (10 µM), cyclosporin A (CsA, 20 µM), or curcumin (CUR, 30 µM) treatment for 24 h. Cells were collected for analyses, including morphological vacuolization ( A ), ROS generation ( B ), abundance of ER stress and UPR proteins, HSPs ( C ), CDK7 and CDK9 activity ( D ), RNA polymerase II activation ( E ), and cell viability ( F ), proteasome subunit abundances ( H ), 20S proteasome activity ( I ), nuclear hydrogen peroxide generation ( J ), free cysteine-thiol levels ( K ), and CDK7 interaction with HSPs by co-immunoprecipitation ( L ). ( G ) Cancer cells pretreated with THZ1 (THZ, 0.2 µM) or LY2857785 (LY, 1 µM) for 30 min followed by CPYPP treatment for 24 h were assessed for cellular ROS generation. The results are expressed as the mean ± SD from three independent experiments. * P < 0.05 or ** P < 0.01, compared to the corresponding control group (CTL)

Article Snippet: Ten micrograms of proteins were used for CDK7 and CDK9 activity determination following the instructions enclosed in the kit (CDK7 Assay Kit, Cat# 79603; CDK9 Assay Kit, Cat# 79628, BPS Bioscience, San Diego, CA, USA).

Techniques: Activity Assay, Activation Assay, Immunoprecipitation, Control

Journal: Cell & Bioscience

Article Title: CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

doi: 10.1186/s13578-024-01260-2

Figure Lengend Snippet: Protein kinase R (PKR) interacts with the CDK7/CDK9–Rpb1 complex to enhance transcription activation and exacerbate paraptotic progression. ( A – F , I , J ) MDA-MB-435 cells were pretreated with PKR inhibitor (PKRi, 2 µM), trans-ISRIB (Trans, 3 µM), GCN2-IN-1 (GCN2i, 5 µM), or GSK2656157 (GSK, 5 µM) for 30 min followed by CPYPP (10 µM), CsA (20 µM), or curcumin (CUR, 30 µM) for 24 h. Cells were harvested and assessed for cell viability ( A ), morphological vacuolization ( B ), protein abundance of ER stress, UPR, and HSPs ( C ), CDK7 and CDK9 activity ( D ), RNA polymerase II activation ( E ), PKR interaction with CDK7/CDK9 by co-immunoprecipitation ( F ), cellular ROS ( I ), and nuclear hydrogen peroxide generation ( J ). The results are expressed as the mean ± SD from three independent experiments. * P < 0.05 or ** P < 0.01, compared to the corresponding control group (CTL). ( G , H ) MDA-MB-435 cells pretreated with ActD (1 µM), CHX (20 µM), THZ1 (0.2 µM), or LY2857785 (1 µM) for 30 min followed by CPYPP treatment for 24 h. Cytosolic and nuclear fractions were isolated for PKR abundance and activation analysis by Western blot. ( K , L ) MDA-MB-435 cells pretreated with NAC (3 mM) for 30 min followed by CPYPP treatment for 24 h were used for PKR abundance and activation ( K ) and interaction with CDK7 by co-immunoprecipitation ( L )

Article Snippet: Ten micrograms of proteins were used for CDK7 and CDK9 activity determination following the instructions enclosed in the kit (CDK7 Assay Kit, Cat# 79603; CDK9 Assay Kit, Cat# 79628, BPS Bioscience, San Diego, CA, USA).

Techniques: Activation Assay, Activity Assay, Immunoprecipitation, Control, Isolation, Western Blot

Journal: Cell & Bioscience

Article Title: CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

doi: 10.1186/s13578-024-01260-2

Figure Lengend Snippet: CPYPP and curcumin-induced paraptosis suppress tumor growth in xenograft mice. ( A – H) MDA-MB-231 breast cancer xenograft mouse models treated with vehicle (CTL) or CPYPP. n = 5. ( A ) Representative image of the tumor masses in mice treated with different regimens. ( B , C ) Tumor size and tumor weight. ( D ) Hematoxylin and eosin (H&E) staining, Ki67, CDK7, and CDK9 expression in tumor samples. ( E–G) CDK7, CDK9, and 20S proteasome activities in tumor samples. ( H ) Changes in mouse body weight throughout the 14-day experimental period. ( I , J ) Docetaxel-resistant head and neck cancer cell lines, OECM1-DTX and SAS-DTX, were treated with indicated concentrations of CPYPP or curcumin (CUR) for 24 h, followed by morphological vacuolization assessment ( I ) and cell viability analysis ( J ). Results are expressed as the mean ± SD from three independent experiments. ** P < 0.01, compared to the control group (CTL). ( K – Q ) OECM1-DTX tumor xenograft mouse models treated with vehicle (CTL), CPYPP, or curcumin (CUR). ( K ) Representative images of the tumor masses in mice receiving different treatments. ( L , M ) Tumor volume and tumor weight at experiment endpoint. n = 5. ( N – P ) CDK7, CDK9, and 20S proteasome activities in tumor samples. ( Q ) Changes in mouse body weight throughout the 12-day experimental period

Article Snippet: Ten micrograms of proteins were used for CDK7 and CDK9 activity determination following the instructions enclosed in the kit (CDK7 Assay Kit, Cat# 79603; CDK9 Assay Kit, Cat# 79628, BPS Bioscience, San Diego, CA, USA).

Techniques: Staining, Expressing, Control