2316s parp cl cleaved parp asp214 d64e10 cell signaling technology 5625s casp3 cl cleaved caspase 3  (Cell Signaling Technology Inc)

Journal: Biomedicines

Article Title: Cyclophilin Inhibitor Rencofilstat Combined with Proteasome Inhibitor Ixazomib Increases Proteotoxic Cell Death in Advanced Prostate Cancer Cells with Minimal Effects on Non-Cancer Cells

doi: 10.3390/biomedicines13102442

Figure Lengend Snippet: RCF + Ixz increases apoptotic cell death in PCa/CRPC cells. ( A ) Increased cell death in RCF (10 μM) + Ixz (2238, active form) (25 nM; 50 nM in LNCaP)-treated LNCaP, 22Rv1, and PC3 compared to RCF-, Ixz-, and control-treated cells (trypan blue assay; 72 h). QVD (10 μM; apoptosis inhibitor) decreased RCF + Ixz cell death in LNCaP, 22Rv1, and PC3. p -Values are near the bars. ( B – D ) Increased cl-PARP, poly-Ub, XBP1s, and LC3B in RCF + Ixz-treated ( B ) LNCaP, ( C ) 22Rv1, and ( D ) PC3 (24, 48 h) compared to RCF-, Ixz-, and control-treated cells (Western blot). CypB decreased with RCF and RCF + Ixz (24, 48 h), whereas there were variable effects on p62. No changes were observed in CypA. QVD decreased cl-PARP in RCF + Ixz. Molecular weight markers in kDa are shown to the left. Protein refers to loading control (Coomassie blue) after completion of analysis.

Article Snippet: The following antibodies were used: cl-PARP (9541), XBP1s (D2C1F), LC3B (2775), eIF2α (9722), phospho (P)-eIF2α (Ser51), PERK (C33E1), IRE1α (14C10), ERK1/2 (9102), and P-ERK1/2 (9101) from Cell Signaling Technology (Danvers, MA, USA); Ub (P4D1); p62 (D3), GRP78 (A10), AR (441), CD147 (8D6), CypD (G9, referred as CypF or PPIF), actin (C-11), mouse anti-rabbit IgG-HRP (2357), and m-IgG-Fc BP-HRP (525409) from Santa Cruz Biotechnology; CypB (16045) and P-PERK (T982) from Abcam (Cambridge, MA, USA); CypA (SA296) from Enzo Life Sciences (Farmingdale, NY, USA); and and P-IRE1α (Ser724) from Novus Biologicals (Centennial, CO, USA).

Techniques: Control, Western Blot, Molecular Weight

Journal: Biomedicines

Article Title: Cyclophilin Inhibitor Rencofilstat Combined with Proteasome Inhibitor Ixazomib Increases Proteotoxic Cell Death in Advanced Prostate Cancer Cells with Minimal Effects on Non-Cancer Cells

doi: 10.3390/biomedicines13102442

Figure Lengend Snippet: Maintaining protein synthesis is important for RCF + Ixz cell death in CRPC cells. ( A ) Chx (10 μM) + RCF (10 μM) + Ixz (2238, active form) (25 nM) significantly decreased cell death in 22Rv1 and PC3 compared to RCF + Ixz (trypan blue assay; 72 h). p -Values are above the bars. ( B ) In 22Rv1 and PC3, Chx + RCF + Ixz decreased cl-PARP, poly-Ub, and LC3B compared to RCF + Ixz (Western blot). Chx decreased RCF + Ixz’s effect on XBP1s in 22Rv1 (24 h) but not in PC3, and the effects on p62 were reduced (decreased in 22Rv1 and increased in PC3). There were no changes in CypA or B. ( C ) In 22Rv1 (24 h) and PC3 (48 h), RCF + Ixz decreased T-PERK and P-eIF2α compared to RCF, Ixz, and control (Western blot). RCF + Ixz increased GRP78, a marker of ER stress. Molecular weight markers in kDa are shown to the left. Protein refers to loading control (Coomassie blue) after completion of analysis.

Article Snippet: The following antibodies were used: cl-PARP (9541), XBP1s (D2C1F), LC3B (2775), eIF2α (9722), phospho (P)-eIF2α (Ser51), PERK (C33E1), IRE1α (14C10), ERK1/2 (9102), and P-ERK1/2 (9101) from Cell Signaling Technology (Danvers, MA, USA); Ub (P4D1); p62 (D3), GRP78 (A10), AR (441), CD147 (8D6), CypD (G9, referred as CypF or PPIF), actin (C-11), mouse anti-rabbit IgG-HRP (2357), and m-IgG-Fc BP-HRP (525409) from Santa Cruz Biotechnology; CypB (16045) and P-PERK (T982) from Abcam (Cambridge, MA, USA); CypA (SA296) from Enzo Life Sciences (Farmingdale, NY, USA); and and P-IRE1α (Ser724) from Novus Biologicals (Centennial, CO, USA).

Techniques: Western Blot, Control, Marker, Molecular Weight

Journal: Biomedicines

Article Title: Cyclophilin Inhibitor Rencofilstat Combined with Proteasome Inhibitor Ixazomib Increases Proteotoxic Cell Death in Advanced Prostate Cancer Cells with Minimal Effects on Non-Cancer Cells

doi: 10.3390/biomedicines13102442

Figure Lengend Snippet: RCF + Ixz does not affect cell death in non-cancer RWPE-1 prostate epithelial cells. ( A ) RCF (5 μM) + Ixz (2238, active form) (25 nM) did not increase cell death in RWPE-1 cells (3.2%) compared to RCF-, Ixz-, and control-treated cells, but it greatly increased cell death in H660 NEPC cells (96%) (trypan blue assay; 72 h). ( B ) RCF + Ixz had minimal effects on cl-PARP and XBP1s, unlike the greater increases in PC3 (Western blot). There was a similar increase in poly-Ub in both RWPE-1 and PC3. P62 was greatly increased in RWPE-1, suggesting inhibition of autophagy. There were no changes in CypA/B and LC3B. Vertical line refers to the removal of empty lanes between RWPE-1 and PC3 samples from the same blot. ( C ) Unlike in PCa cells, RCF + Ixz had no effects on PERK or P-eIF2α in RWPE-1 cells (Western blot). No clear difference in GRP78 was noted. Molecular weight markers in kDa are shown to the left. Protein refers to loading control (Coomassie blue) after completion of analysis.

Article Snippet: The following antibodies were used: cl-PARP (9541), XBP1s (D2C1F), LC3B (2775), eIF2α (9722), phospho (P)-eIF2α (Ser51), PERK (C33E1), IRE1α (14C10), ERK1/2 (9102), and P-ERK1/2 (9101) from Cell Signaling Technology (Danvers, MA, USA); Ub (P4D1); p62 (D3), GRP78 (A10), AR (441), CD147 (8D6), CypD (G9, referred as CypF or PPIF), actin (C-11), mouse anti-rabbit IgG-HRP (2357), and m-IgG-Fc BP-HRP (525409) from Santa Cruz Biotechnology; CypB (16045) and P-PERK (T982) from Abcam (Cambridge, MA, USA); CypA (SA296) from Enzo Life Sciences (Farmingdale, NY, USA); and and P-IRE1α (Ser724) from Novus Biologicals (Centennial, CO, USA).

Techniques: Control, Western Blot, Inhibition, Molecular Weight

Journal: Signal Transduction and Targeted Therapy

Article Title: Precise targeting of transcriptional co-activators YAP/TAZ annihilates chemoresistant brCSCs by alteration of their mitochondrial homeostasis

doi: 10.1038/s41392-025-02133-x

Figure Lengend Snippet: Silencing YAP/TAZ disrupts mitochondrial homeostasis and induces cell death in mammospheres. a Western blot analyses were performed to assess the expression of mitochondrial fission markers (DRP1, pDRP1, FIS1), fusion markers (OPA1, MFN1), ETC complex proteins (NDUFA9, UQCRC2, OSCP) and apoptosis markers (CL-caspase 3, CL-PARP) in YAP/TAZ depleted mammospheres. Mitochondrial fission, fusion and ETC complex markers are denoted in green, blue, and red respectively ( n = 3). b The cytoplasmic and mitochondrial fractions were analysed for the expression of DRP1 and BAX in mammospheres ( n = 3). c Molecular docking model depicting the DRP1/YAP and DRP1/TAZ complexes. d Co-immunoprecipitation experiments utilizing control IgG or anti-YAP/anti-TAZ antibody, for assessing the interaction between YAP/DRP1 and TAZ/DRP1 in patient samples ( n = 5) and ( e ) in mammospheres ( n = 3). f Co-immunoprecipitation experiments using either control IgG or anti-YAP antibodies, followed by western blot analyses in the adherent and mammosphere culture of MDA-MB-231 ( n = 3). g YAP deletion mutant (ΔTBD) was transfected into MDA-MB-231 adherent cells and mammospheres. Co-immunoprecipitation experiments were performed with anti-FLAG antibody and immunoblots were probed with anti-DRP1 or anti-FLAG antibodies ( n = 3). h Representative confocal microscopy images depicting mitochondrial morphology of MDA-MB-231 mammospheres after treatment with control siRNA and dual knockdown with YAP and TAZ -specific siRNA. Indirect immunofluorescence for TOM20 was utilized to visualize mitochondrial morphology (mitochondria, red; nuclei stained with DAPI, cyan) ( n = 3). Scale bar: 20 µm. i Quantification of length and j connectivity of mitochondria in mammospheres ( n = 3; Each point represents individual cells). k Mitochondrial respiration as reflected by the oxygen consumption rate (OCR) in MDA-MB-231 mammospheres after treatment with control and YAP/TAZ -specific siRNA under basal conditions and after addition of oligomycin (2 μM), FCCP (1 μM) or rotenone/antimycin (0.5 μM) ( n = 3). l Bar plots representing basal respiration rate and m ATP-linked OCR in control mammospheres in comparison to YAP/TAZ- depleted mammospheres ( n = 3). n Relative cell viability was measured using MTT assay in mammospheres after treatment with YAP/TAZ -specific siRNA ( n = 3). o Representative plots and p quantitative analysis of the percentage of apoptotic cells in MDA-MB-231-derived mammospheres following YAP/TAZ depletion as analyzed by flow-cytometry using Annexin-V/PI analysis ( n = 3). Cytosolic, nuclear and mitochondrial protein expressions were normalized against β-tubulin, H2B and COX IV respectively. The data are presented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance was assessed using unpaired Student’s t -test, and the associated two-tailed p -value is indicated in the bar plots. Compared to their respective untreated control group: * p < 0.05 and *** p < 0.001. 231 MDA-MB-231, 468 MDA-MB-468, siCTL control siRNA, siY+siT siYAP+siTAZ, CL-CASP3 cleaved-caspase 3, CL-PARP cleaved-PARP, β-TUB β-tubulin, ACE Atomic contact energy, 3D Mammospheres, OCR Oxygen consumption rate, O oligomycin, F FCCP, R/A rotenone/antimycin, C Cytoplasm, Nuc Nucleus, IP Immunoprecipitate, IB Immunoblot, M Mitochondria

Article Snippet: The antibodies used for western blot analysis were as follows: SOX2, YAP, TAZ, β-TUBULIN, BAX, NRF-2, KEAP1, FIS1, UQCRC2, OSCP, cl-caspase3, cl-PARP (Santa Cruz Biotechnology, USA); ALDH1A1, NANOG, COXIV, MFN1 (Abcam); Hippo Signaling Antibody Sampler Kit, pTAZs89, pDRP1, H2B (Cell Signaling Technologies); DRP1, OPA1 (BD Biosciences); NDUFA9 (Invitrogen).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Mutagenesis, Transfection, Confocal Microscopy, Knockdown, Immunofluorescence, Staining, Comparison, MTT Assay, Derivative Assay, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Precise targeting of transcriptional co-activators YAP/TAZ annihilates chemoresistant brCSCs by alteration of their mitochondrial homeostasis

doi: 10.1038/s41392-025-02133-x

Figure Lengend Snippet: YAP/TAZ depletion disrupts cellular homeostasis in TNBC patient-derived spheroids. a Co-immunoprecipitation (Co-IP) analyses using either control IgG or antibodies against YAP and TAZ. Western blot analyses were performed to analyse differential interaction pattern in the nucleus of CD44 + /CD24 − cell population (representing CSCs) in comparison with rest of the cell population (representing non-CSCs) isolated from patient breast tumors ( n = 5). b The mRNA expression levels of transcriptional co-activators YAP / TAZ , transcription factor SOX2 , and SOX2 -target genes NANOG and CCND1 following YAP/TAZ knockdown in patient-derived spheroids using semi-quantitative PCR ( n = 5). c Western blot analyses depicting the expression of stemness markers SOX2, ALDH1A1, and NANOG following YAP and TAZ knockdown in patient-derived spheroids ( n = 5). The hippo pathway associated markers are indicated in blue and stemness markers are indicated in pink. d Graphical representation of sphere forming efficiency post siRNA treatment in patient-derived spheroids ( n = 5). e Representative plots and f quantitative analysis of the percentage of CD44 + /CD24 − populations in patient-derived spheroids following YAP/TAZ depletion as analyzed by flow-cytometry ( n = 5). g , h Assessment of alterations in reactive oxygen species (ROS) levels following genetic depletion of YAP and TAZ in patient-derived spheroids was measured using H2DCFDA assay ( n = 5). i Western blot analyses of NRF2 and KEAP1 expression in patient-derived spheroids post YAP/TAZ knockdown ( n = 5). j Evaluation of changes in the levels of SOD (superoxide dismutase) and k GSH (glutathione) in patient-derived spheroids after treatment with control siRNA and YAP/TAZ -specific siRNA, either individually or in combination ( n = 5). l Western blot analyses were performed to assess the expression of mitochondrial fission markers (DRP1, pDRP1, FIS1) and fusion markers (OPA1, MFN1) in CSCs compared to NCSCs ( n = 5). Mitochondrial fission and fusion markers are denoted in green and blue respectively. m Relative cell viability was measured using MTT assay in patient-derived spheroids after treatment with OXPHOS inhibitors: rotenone, antimycin, CCCP and oligomycin ( n = 5). n Western blot analyses were performed to assess the expression of mitochondrial fission markers (DRP1, pDRP1) and apoptosis markers (CL-caspase 3, CL-PARP) in YAP/TAZ depleted spheroids. Mitochondrial fission and apoptosis markers are denoted in blue and pink respectively ( n = 5). o The cytoplasmic and mitochondrial fractions were analysed for the expression of DRP1 and BAX in spheroids ( n = 5). p Co-immunoprecipitation experiments utilizing control IgG or anti-YAP/anti-TAZ antibody, for assessing the interaction between YAP/DRP1 and TAZ/DRP1 in the cytoplasm of CD44 + /CD24 − cell population (representing CSCs) in comparison with rest of the cell population (representing non-CSCs) isolated from patient breast tumors ( n = 5). q Representative confocal microscopy images depicting mitochondrial morphology of patient-derived spheroids after treatment with control siRNA and dual knockdown with YAP and TAZ -specific siRNA. Indirect immunofluorescence for TOM20 was utilized to visualize mitochondrial morphology (mitochondria, red; nuclei stained with DAPI, cyan) ( n = 5). Scale bar: 20 µm. r Quantification of length and s connectivity of mitochondria in patient-derived spheroids ( n = 5; Each point represents individual cells). t Quantification of ATP levels in spheroids post siRNA treatment ( n = 5). u Relative cell viability was measured using MTT assay in patient-derived spheroids after treatment with YAP/TAZ -specific siRNA ( n = 5). mRNA expressions were normalized against 18S rRNA. Protein expressions in nuclear, cytoplasmic and mitochondrial fraction were normalized against H2B, β-tubulin and COXIV respectively. The data are presented as mean ± standard deviation (SD), with “ n ” representing the number of biological replicates per experimental group. Significance was assessed using an unpaired Student’s t -test, and the associated two-tailed p -value is indicated in the bar plots. Compared to the untreated control group: * p < 0.05, ** p < 0.01 and *** p < 0.001. IP immunoprecipitate, IB immunoblot, NCSC non-cancer stem cells, CSC cancer stem cells, siCTL control siRNA, siY+siT siYAP+siTAZ, β-TUB β-tubulin, Cont control, Rote Rotenone, Anti Antimycin, Olig Oligomycin, M mitochondria, C Cytoplasm

Article Snippet: The antibodies used for western blot analysis were as follows: SOX2, YAP, TAZ, β-TUBULIN, BAX, NRF-2, KEAP1, FIS1, UQCRC2, OSCP, cl-caspase3, cl-PARP (Santa Cruz Biotechnology, USA); ALDH1A1, NANOG, COXIV, MFN1 (Abcam); Hippo Signaling Antibody Sampler Kit, pTAZs89, pDRP1, H2B (Cell Signaling Technologies); DRP1, OPA1 (BD Biosciences); NDUFA9 (Invitrogen).

Techniques: Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Comparison, Isolation, Expressing, Knockdown, Real-time Polymerase Chain Reaction, Flow Cytometry, MTT Assay, Confocal Microscopy, Immunofluorescence, Staining, Standard Deviation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Precise targeting of transcriptional co-activators YAP/TAZ annihilates chemoresistant brCSCs by alteration of their mitochondrial homeostasis

doi: 10.1038/s41392-025-02133-x

Figure Lengend Snippet: Verteporfin in combination with paclitaxel induces apoptosis-mediated cell death in CSCs from cell lines and patient-derived organoids. a Western blot analyses depict the expression levels of YAP/TAZ, stemness markers (SOX2 and ALDH1A1) and apoptosis markers (cleaved-caspase 3 and cleaved-PARP) in VP-treated MDA-MB-231 mammospheres ( n = 3). Markers associated with Hippo signaling pathway are depicted in blue, while markers linked to stemness are represented in pink. b Assessment of the percentage viable cells and c relative cell viability using trypan blue exclusion assay and MTT assay, respectively, in mammospheres after treatment with VP ( n = 3). d Representative plots of the percentage apoptotic cells in mammospheres following VP treatment as analyzed by flow-cytometry using Annexin V/PI analysis ( n = 3). e Representative plots of apoptotic cell percentage in mammospheres following treatment with VP alone and in combination with paclitaxel, assessed by flow cytometry using Annexin V/PI analysis ( n = 3). f Representative plots of the percentage of CD44 + /CD24 − populations in patient-derived spheroids following VP treatment as analyzed by flow-cytometry ( n = 5). g Change in ROS levels following VP treatment of patient-derived spheroids were quantified using H2DCFDA assay ( n = 5). h Cell viability of untreated and 4 µM VP-treated TNBC patient-derived organoids following paclitaxel exposure. Organoids were pre-treated with VP for 48 h, then exposed to graded doses of paclitaxel. Viability was then quantified after 24 h ( n = 5). i Hypothetical model delineating possible mechanistic pathway of YAP/TAZ-mediated regulation of brCSCs on the left, where YAP/TAZ might promote persistence of brCSCs. On the right, an alternative mechanism illustrates the putative cascade that might promote the eradication of the brCSCs upon VP treatment, subsequent to the depletion of YAP/TAZ. This schematic was created using BioRender ( https://biorender.com ). All protein expressions were normalized against β-tubulin, which served as the internal loading control. The data are presented as the mean ± standard deviation (SD), with “ n ” representing the number of biological replicates per experimental group. Significance was assessed using an unpaired Student’s t -test, and the associated two-tailed p -value is indicated in the bar plots. Compared to the untreated control group: * p < 0.05, ** p < 0.01 and *** p < 0.001. 231 MDA-MB-231, 468 MDA-MB-468, VP verteporfin, PAX paclitaxel, CL-CASP3 cleaved-caspase 3, CL-PARP cleaved-PARP, β-TUB β-tubulin, brCSC breast cancer stem cells, ARE anti-oxidant response element, AOs anti-oxidants

Article Snippet: The antibodies used for western blot analysis were as follows: SOX2, YAP, TAZ, β-TUBULIN, BAX, NRF-2, KEAP1, FIS1, UQCRC2, OSCP, cl-caspase3, cl-PARP (Santa Cruz Biotechnology, USA); ALDH1A1, NANOG, COXIV, MFN1 (Abcam); Hippo Signaling Antibody Sampler Kit, pTAZs89, pDRP1, H2B (Cell Signaling Technologies); DRP1, OPA1 (BD Biosciences); NDUFA9 (Invitrogen).

Techniques: Derivative Assay, Western Blot, Expressing, Trypan Blue Exclusion Assay, MTT Assay, Flow Cytometry, Control, Standard Deviation, Two Tailed Test