mcl1 protein band quantity  (Bio-Rad)

Journal: iScience

Article Title: Developing therapeutic strategies to target MCL1 and BCLXL in lethal prostate cancer

doi: 10.1016/j.isci.2025.113985

Figure Lengend Snippet: MCL1 is highly expressed in castration-resistant prostate cancer transcriptomes and associates with worse clinical outcome (A) Prostate Cancer Foundation-Stand Up To Cancer (PCF-SU2C) castration-resistant prostate cancer (CRPC) transcriptome analysis for MCL1 RNA expression compared with the 15,000 highest expressed genes divided into very high (upper 25% expressed genes), medium high (50%–75% expressed genes), medium low (25%–50% expressed genes), and very low (lower 25% expressed genes) ( n = 159). (B) Quantification of MCL1 RNA expression in each CRPC patient transcriptome in the PCF-SU2C CRPC cohort ( n = 141). Biopsies (red dots) with MCL1 RNA expression >80 th percentile (dotted line) are shown. (C) Kaplan-Meier curves for overall survival (OS) from CRPC biopsy split by > 80th percentile (red, n = 28) or ≤80th percentile (gray, n = 113) MCL1 RNA expression in the PCF-SU2C transcriptome cohort. Median OS is shown. Hazard ratio (HR) with 95% confidence intervals and p values for univariate cox survival model are shown. (D) Gene set enrichment analysis shows MCL1 RNA level association with hallmark pathways in the PCF-SU2C transcriptome cohort ( n = 159). Normalized enrichment scores and false discovery rates are shown. (E and F) Association between MCL1 RNA and either RELA RNA (E) or STAT3 RNA (F) in the PCF-SU2C transcriptome cohort ( n = 159). Spearman r- and p values are shown. FPKM - Fragments per kilobase of transcript per million mapped reads.

Article Snippet: Densitometric analysis was used to evaluate the MCL1 protein band quantity (Image Lab Software, Bio-Rad Laboratories).

Techniques: RNA Expression

Journal: iScience

Article Title: Developing therapeutic strategies to target MCL1 and BCLXL in lethal prostate cancer

doi: 10.1016/j.isci.2025.113985

Figure Lengend Snippet: Prostate cancer cell line models demonstrate varying dependency on MCL1 for survival (A) MCL1 RNA expression was downloaded from publicly available RNA-sequencing data. Protein expression was determined across prostate cancer (PCa) cell line models. Basal MCL1, AR, AR-V7, and GAPDH protein expression was determined by western blot. Densitometry of MCL1 protein expression normalized to GAPDH protein expression is shown above each band (A, top). MCL1 RNA expression was determined across multiple publicly available RNA-sequencing experiments and presented as individual data points (A, bottom). FPKM - Fragments per kilobase of transcript per million mapped reads. (B) The impact of MCL1 (siMCL1) and non-targeting control (siControl) siRNA (50 nM) on cell viability was determined using CellTiter-Glo in PCa cell line models. Cell viability of siMCL1 compared to siControl was determined after 3, 5 and 7 days. Mean cell viability and standard deviation is shown. The experiment was performed in three biological replicates, each with three technical replicates. The unpaired Student’s t test was used to compare siMCL1 with siControl on each specific day for each cell line. ∗ p value ≤ 0.05. (C) The impact of AZD5991 (1 μM) or Vehicle (DMSO 0.01%) on cell viability (CellTiter-Glo, bottom) and caspase 3/7 activity (Caspase-Glo 3/7, top) was determined in PCa cell line models. Cell viability and caspase 3/7 activity of AZD5991 compared to Vehicle is shown at 7 days and 6 h, respectively. Mean cell viability and standard deviation is shown for three biological replicates, each performed with three technical replicates. Mean caspase 3/7 activity and standard deviation is shown for one single experiment performed in sextuplets. The unpaired Student’s t test was used to compare AZD5991 with Vehicle. ∗ p value ≤ 0.05.

Article Snippet: Densitometric analysis was used to evaluate the MCL1 protein band quantity (Image Lab Software, Bio-Rad Laboratories).

Techniques: RNA Expression, RNA Sequencing, Expressing, Western Blot, Control, Standard Deviation, Activity Assay

Journal: iScience

Article Title: Developing therapeutic strategies to target MCL1 and BCLXL in lethal prostate cancer

doi: 10.1016/j.isci.2025.113985

Figure Lengend Snippet: siRNA targeting of UCHL3 downregulates MCL1 protein expression, but the phenotype is driven through an off-target effect (A) LNCaP95 and 22Rv1 prostate cancer (PCa) cells were transfected with siRNA for (one of) 104 deubiquitinating enzymes (siDUBs) or non-targeting control (50 nM) for 72 h. The impact on MCL1 protein expression and GAPDH was determined by western blot. MCL1 protein expression was quantified using densitometry, normalized to GAPDH, and compared with non-targeting control (siControl). Fold change in MCL1 protein expression (Log2 siDUB vs. siControl) is shown. Mean fold change and range (effect in LNCaP95 and 22Rv1 PCa cells) is shown for a single experiment performed in both cell lines. UCHL3 is highlighted (red bar). (B) Various PCa cell line models were transfected with UCHL3 (siUCHL3), MCL1 (siMCL1) or non-targeting control (siControl) siRNA (50 nM) for 72 h and MCL1, UCHL3, and GAPDH protein expression was determined by western blot from one experiment. (C) The impact of UCHL3 (siUCHL3) and non-targeting control (siControl) siRNA (50 nM) on cell viability was determined using CellTiter-Glo in PCa cell line models. Cell viability of siUCHL3 compared to siControl was determined after 3, 5, and 7 days. Mean cell viability and standard deviation is shown for three biological replicates, each performed with three technical replicates. The unpaired Student’s t test was used to compare siUCHL3 with siControl on each specific day for each cell line. ∗ p value ≤ 0.05. (D) LNCaP95 PCa cells were transfected with non-targeting control (siControl), UCHL3 pool (siUCHL3) and single siRNAs (siUCHL3 5, 6, 7, and 8) making the UCHL3 pool (50 nM) for 72 h. The effect of each condition on UCHL3, MCL1, and GAPDH protein expression was determined by western blot. Western blot from one experiment performed in biological triplicate. (E) C4-2 PCa cell shControl and shUCHL3 clones were transfected with non-targeting control (siControl), UCHL3 pool (siUCHL3), and single siRNAs (siUCHL3 5, 6, 7, and 8) making the UCHL3 pool (50 nM) for 72 h. The effect of each condition on UCHL3, MCL1, and GAPDH protein expression was determined by western blot. Western blot from one experiment performed in biological duplicate. (F) LNCaP95 PCa cells were transfected with non-targeting control (siControl), MCL1 (siMCL1), single siRNA UCHL3 5 (siUCHL3 (5)) and siUCHL3 5 mouse seed siblings (BACE2, SRA1, PTPN9, IGFBPL1, and SLC7A15) at 50 nM. The effect of each condition on UCHL3, MCL1, and GAPDH protein expression was determined by western blot at 72 h from one experiment.

Article Snippet: Densitometric analysis was used to evaluate the MCL1 protein band quantity (Image Lab Software, Bio-Rad Laboratories).

Techniques: Expressing, Transfection, Control, Western Blot, Standard Deviation, Clone Assay

Journal: iScience

Article Title: Developing therapeutic strategies to target MCL1 and BCLXL in lethal prostate cancer

doi: 10.1016/j.isci.2025.113985

Figure Lengend Snippet: BCL2L1 is highly expressed in castration-resistant prostate cancer and MCL1 knockdown (with siUCHL3) sensitizes prostate cancer cells to BCLXL targeting (A) Prostate Cancer Foundation-Stand Up To Cancer (PCF-SU2C) castration-resistant prostate cancer (CRPC) transcriptome analysis for BCL2L1 (the gene encoding BCLXL) RNA expression compared with the 15,000 highest expressed genes divided into very high (upper 25% expressed genes), medium high (50%–75% expressed genes), medium low (25%–50% expressed genes), and very low (lower 25% expressed genes) ( n = 159). (B) BCLXL RNA was downloaded from publicly available RNA-sequencing data. Protein expression was determined across prostate cancer cell line models. Basal BCLXL and GAPDH protein expression was determined by western blot. Densitometry of BCLXL normalized to GAPDH is shown above each band (B, top). BCLXL RNA expression was determined across multiple publicly available RNA-sequencing experiments and presented as individual data points (B, bottom). FPKM - Fragments per kilobase of transcript per million mapped reads. (C) The relative importance of specific gene expression on MCL1 dependency in cell lines (top panel—RNAi and bottom panel—CRISPR) from the DepMap database. The top 10 most important genes are shown. (D) C4-2 prostate cancer (PCa) cells were transfected with 50 nM siRNA for UCHL3 (siUCHL3) or non-targeting control (siControl). 72 h after transfection, C4-2 PCa cells were treated with various concentrations of navitoclax (BCLXL/BCL2 inhibitor) or Vehicle (DMSO 0.01%) and cell viability was determined using CellTiter-Glo after 24 h. Mean cell viability and standard deviation for siControl (gray line) and siUCHL3 (red line) compared with Vehicle is shown for three biological replicates, each performed with three technical replicates. (E) C4-2 PCa cells were transfected with 50 nM siRNA for UCHL3 (siUCHL3) or non-targeting control (siControl). 72 h after transfection, the cells were treated with two concentrations of navitoclax (100 and 500 nM) or Vehicle (DMSO 0.01%) for 6 h. The effect of each condition on caspase 3/7 activation (E, left) was determined using Caspase-Glo 3/7 and the effect of each condition on UCHL3, MCL1, PARP/cleaved PARP, cleaved caspase 3 (cC3), and GAPDH protein expression (E, right) was determined by western blot. Mean caspase 3/7 activity and standard deviation compared to siControl and Vehicle is shown for three biological replicates, each performed with three technical replicates. The unpaired Student’s t test was used to compare siUCHL3 with siControl for each treatment. ∗ p value ≤ 0.05. Western blot from one experiment performed in biological triplicate. (F) C4-2 PCa cells were transfected with siRNA for UCHL3, BAX plus BAK, or UCHL3 plus BAX plus BAK siRNA, or non-targeting control (25 nM of each siRNA; total 75 nM). 72 h after transfection, the cells were treated with navitoclax (500 nM) or Vehicle (DMSO 0.01%). The impact of each condition on caspase 3/7 activation (F, left) was determined using Caspase-Glo 3/7 and the effect of each condition on cell viability (F, right) was determined by CellTiter-Glo at 6 and 24 h, respectively. Mean caspase 3/7 activity and cell viability with standard deviation compared to siControl and Vehicle is shown for three biological replicates, each performed with three technical replicates. A one-way ANOVA with post-hoc Tukey test was used to compare siUCHL3 with siControl and siUCHL3 with siUCHL3/BAX/BAK for each treatment ∗ p value ≤ 0.05.

Article Snippet: Densitometric analysis was used to evaluate the MCL1 protein band quantity (Image Lab Software, Bio-Rad Laboratories).

Techniques: Knockdown, RNA Expression, RNA Sequencing, Expressing, Western Blot, Gene Expression, CRISPR, Transfection, Control, Standard Deviation, Activation Assay, Activity Assay

Journal: iScience

Article Title: Developing therapeutic strategies to target MCL1 and BCLXL in lethal prostate cancer

doi: 10.1016/j.isci.2025.113985

Figure Lengend Snippet: Combined MCL1 and BCLXL/BCL2 blockade drives apoptotic cell death in prostate cancer patient-derived xenograft-organoids and prostate cancer pre-clinical mouse modeling platform-organoids (A and B) Prostate cancer patient-derived xenograft-organoids (PDX-O) were treated with Vehicle or combined MCL1 (1 μM AZD5991) and BCLXL/BCL2 (1 μM navitoclax) inhibitors. The impact of each condition on caspase 3/7 activation (A, left) was determined using Caspase-Glo 3/7 and the effect of each condition on cell viability (A, right) was determined by CellTiter-Glo at 6 and 24 h respectively. Mean caspase 3/7 activity and cell viability with standard deviation compared to Vehicle is shown for three separate experiments performed in quintuplet. The unpaired Student’s t test was used to compare Vehicle with treatment for each model. ∗ p value ≤ 0.05. Representative images of PDX-Os in all conditions are shown (B). Scale bar, 100 μm. (C and D) Prostate cancer pre-clinical mouse modeling platform-organoids (ProMPt-O) were treated with Vehicle or combined MCL1 (5 μM AZD5991) and BCLXL/BCL2 (1 μM navitoclax) inhibitors. The impact of each condition on caspase 3/7 activation (C, left) was determined using Caspase-Glo 3/7 and the effect of each condition on cell viability (C, right) was determined by CellTiter-Glo at 6 and 24 h, respectively. Mean caspase 3/7 activity and cell viability with standard deviation compared to Vehicle is shown for three biological replicates, each performed with three technical replicates. The unpaired Student’s t test was used to compare Vehicle with treatment for each model. ∗ p value ≤ 0.05. Representative images of ProMPt-Os in all conditions are shown (D). Scale bar, 100 μm.

Article Snippet: Densitometric analysis was used to evaluate the MCL1 protein band quantity (Image Lab Software, Bio-Rad Laboratories).

Techniques: Derivative Assay, Activation Assay, Activity Assay, Standard Deviation