Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: a, Schematic for the surface immunoprecipitation (IP) of BCL2 family proteins. Anti-apoptotic proteins (BCL2, BCLxL, MCL1) in cell extracts were lysed with mild detergent and immobilized onto the surface of the reaction chamber. b, Schematic of sandwich-type immunoassay selectively detecting PPI complex (left) and the total level of surface bait (right). The fluorescence signals from detection antibodies were imaged by a single-molecule co-IP platform. c, Comparing the formation of BCL2-BIM BH3 PPI complex in an intracellular environment by BCL2-mCherry/BIM BH3 -eGFP co-transfection (red) and in vitro mixing of two individual extracts expressing BCL2-mCherry and BIM BH3 -eGFP (black). d, Relative changes of active BAX level from the STS treated HL60 cells after lysed with different detergent types. The monoclonal antibody 6A7 was used to detect the activated BAX . Spurious BAX activation by Triton X-100 was indicated (blue). e, Surface-immobilization of the matching IP antibodies immunoprecipitate BCL2-eGFP from the crude extract with high specificity. f, Comparison of signal to noise ratio (SNR) from single molecule counting analysis (red) and total fluorescence signal integrating analysis (black). The raw data to calculate the SNRs are described in Extended Data Fig. 2. g, Counts of endogenous BCL2-related PPI complexes (BCL2-BIM, BCL2-BAX) from the 30,000 HL60 cells by using immunoassay. Error bars represent mean ± s.d. from independent inter-chip measurement ( n ≥ 2). (Two-sided two-sample t -test, P =1.46x10 - , 1.11x10 - respectively). h , Counts of endogenous BCL2-BIM PPI complexes and inter-chip CVs from the fixed numbers of HL60 cells ( n ≥ 2). i , Inter-chip CVs obtained from independent inter-chip measurement for all the immunoassays and cell numbers ( n ≥ 2).

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Immunoprecipitation, Fluorescence, Co-Immunoprecipitation Assay, Cotransfection, In Vitro, Expressing, Activation Assay, Single Molecule Counting

Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: Changes of endogenous PPI profiles of HL60 cells through the apoptosis progression by 2 μM of STS. (a) Active BAX/BAK level, (b) Total levels of anti-apoptotic proteins (BCL2, BCLxL, MCL1), (c) BIM complexes with anti-apoptotic proteins, (d) BAX complexes with anti-apoptotic proteins. e , Schematic for the PPI probe binding assay (PBA) for measuring unoccupied populations of surface-immobilized anti-apoptotic proteins. f, Changes of BIM BH3 PBAs for anti- apoptotic proteins from HL60 cells through the apoptosis progression by STS. g, Schematic for the comparison of the BCL2 PPI complex compositions in different AML cell lines. The density of surface-immobilized BCL2 was constantly maintained by optimizing the total protein concentration of crude cell extracts for all AML cell lines (HL60, THP-1, NB4, U937). h, Compositions of the BCL2 complexes (BIM, BAX, BAD, BAK) in four different AML cell lines. The labeling efficiencies of the immunoassays were applied. i, BCL2-BIM BH3 PBA counts of four AML cell lines. j, The PPI complex compositions of BCL2 in HL60, NB4, and U937 cell extracts. k, Schematic of binding competition between two PPI probes, BIM BH3 -eGFP and BAD- mCherry, on surface immobilized BCL2 from HL60 cells. l, Changes of BCL2-BIM BH3 PBA and BCL2-BAD PBA with increasing amounts of BAD probe. BIM BH3 PPI probe was presented in 10 nM.

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Binding Assay, Protein Concentration, Labeling

Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: Changes of BCL2-related PPI profile in HL60 cells after ABT-199 (100 nM, 24 hours) treatment. (a) Total levels of anti-apoptotic proteins, (b) Active BAX/BAK level, (c) Total levels of BAX/BAK, (d) BCL2 complexes (BIM, BAX), (e) BCL2-BIM BH3 PBA. f-h, Changes of PPI profile of HL60 cells after higher concentration of ABT-199 (300 nM) treatment. (f) BCL2 complexes (BIM, BAX), (g) Active BAX/BAK level, (h) Total levels of BAX/BAK. i, Schematic model of mode of action of ABT-199. j-l , Changes of MCL1-related PPI profile of U937 cells after AZD-5991 (100 nM, 24 hours) treatment. (j) Active BAX/BAK level, (k) MCL1 complexes (BIM, BAK), (l) MCL1-BIM BH3 PBA. The decrease of MCL1-BAK complex after AZD-5991 treatment was indicated (blue). m, Changes of BCLxL complexes (BIM, BAX, BAD, BAK) from U937 cells after AZD-5991 (100 nM, 24 hours) treatment. The increase of BCLxL-BAK complex after AZD-5991 treatment was indicated (green). n, Comparison of relative changes of BAK complexes from U937 cells after AZD-5991 treatment. The relative changes were obtained from the indicated data in (k) and (m). o, Schematic model of mode of action of AZD-5991. (d, k, m) The labeling efficiencies of the immunoassays were applied.

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Concentration Assay, Labeling

Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: a, Pearson’s correlations between ex vivo AUC of AZD-5991 and PPI profiles for primary AML samples ( n =27). b-c , Correlations between ex vivo AUC and single PPI metrics. (b) MCL1 total level, (c) MCL1-BIM BH3 PBA. d , Negative correlation between ex vivo AUC for AZD-5991 and BCLxL-BIM BH3 PBA. e, Comparison of BCLxL-BIM BH3 PBA counts for the AZD-5991 responsive ( ex vivo AUC > 0.6, n =12) and non-responsive ( ex vivo AUC < 0.6, n =15) AML samples (Two-sided two-sample t -test, mean difference: 3.90, P =9.09x10 - ). f, Correlation between ex vivo AUC and combination of multiple PPI metrics (MCL1 total level, BCLxL-BIM BH3 PBA, BCLxL-BAK CPX). The statistical indicators (coefficient. p-value) of each metric were displayed.

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Ex Vivo

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Mcl-1 protein expression in the lineage-related ARCaP E and ARCaP M cells. (B) Ectopic expression of Mcl-1 in ARCaP M cells and the effects on docetaxel cytotoxicity in ARCaP M cells. pCMV: vector control. (C) Left panel: The dose- and time-dependent effects of PDGF-BB on Mcl-1 mRNA expression in ARCaP M cells; middle panel: qRT-PCR analysis of Mcl-1 mRNA expression in response to PDGF-BB treatment in ARCaP M cells (24 h); right panel: The effects of PDGF-BB treatment (20ng/ml, 72 h) on Mcl-1 protein expression in ARCaP M cells. ImageJ was used to quantitate the relative expression of Mcl-1 protein. (D) The effects of exogenous PDGF-BB on the cytotoxicity of docetaxel in ARCaP M cells, as determined by the MTS assay.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, MTS Assay

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Expression profile of PDGFR signaling components in PCa cells, as analyzed by RT-PCR and Western blotting. (B) The effects of PDGF-BB (20 ng/ml) on the phosphorylation of PDGFR-α and -β in ARCaP M cells. (C) The effects of depleting PDGFR-α or/and -β on Mcl-1 protein expression in ARCaP M cells. The cells were transfected with either isotype-specific siRNAs targeting PDGFR-α (left panel, 30 nM) or PDGFR- β (central panel, 100 nM), or a mixture of PDGFR-α and -β siRNAs (right panel) for 48 h, serum-starved overnight, and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 72 h. (D) Upper panel: The time-dependent effects of AG-17 (100 nM) on Mcl-1 mRNA expression in ARCaP M cells; bottom panel: The effects of AG-17 treatment on the expression of Mcl-1 and cleaved PARP in the presence (20 ng/ml) or absence of PDGF-BB (20 ng/ml) in ARCaP M cells. (E) The effects of AG-17 treatment (100 nM, 72 h) on the viability of ARCaP E and ARCaP M cells.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Incubation

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Expression profile of β-catenin-TCF signaling components in PCa cells. (B) TCF reporter activity in the LNCaP-C4-2 and ARCaP E -ARCaP M cells. (C) Upper panel: The effects of PDGF-BB (20 ng/ml) on the nuclear translocation of β-catenin in ARCaP M cells; Bottom panel: The effects of PDGF-BB (20 ng/ml) on TCF reporter activity in the presence (100 nM) or absence of AG-17. (D) The effects of ectopic expression of β-catenin (72 h) on Mcl-1 expression at both mRNA and protein levels. (E) The effects of β-catenin depletion on PDGF-BB regulation of Mcl-1 expression in ARCaP M cells. The cells were transfected with β-catenin siRNA or control siRNA (30 nM) for 48 h, serum-starved overnight, and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 72 h. (F) The effects of β-catenin depletion on Mcl-1 reporter activity in ARCaP M cells. The cells were transfected with β-catenin or control siRNA (30 nM) for 48 h, and further transfected with a human Mcl-1 reporter for 24 h. Following serum starvation overnight, the cells were incubated in the presence or absence of PDGF-BB (20 ng/ml) for 48 h.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Activity Assay, Translocation Assay, Transfection, Incubation

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) Expression of c-Abl and p68 in PCa cells. (B) Left panel: The effects of PDGF-BB (20 ng/ml) on the phosphorylation of c-Abl; middle panel: The effects of PDGF-BB (20 ng/ml) on the phosphorylation of p68 and the expression of β-catenin in the p68 immunoprecipitates in ARCaP M cells. Phosphorylation of p68 on the tyrosine sites was detected using a pan-phosphorylated-Tyr antibody; right panel: The effects of PDGF-BB (20 ng/ml) on the expression of p68 in the β-catenin immunoprecipitates in ARCaP M cells. (C) The effects of PDGF-BB (20 ng/ml) on the nuclear translocation of p68 in ARCaP M cells. (D) Confocal microscopy analysis of the effects of PDGF-BB on the co-localization of β-catenin and p68 in the nucleus in a time course experiment in ARCaP M cells. (E) The effects of p68 depletion on PDGF regulation of Mcl-1 in ARCaP M cells. The cells were transfected with p68 or control siRNA (30 nM) for 48 h, serum-starved overnight, and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 24 h (upper panel) or 72 h (bottom panel). Upper panel: RT-PCR analysis of mRNA expression of p68 and Mcl-1; bottom panel: Western blot analysis of protein expression of p68, β-catenin and Mcl-1. (F) The effects of p68 depletion on Mcl-1 reporter activity in ARCaP M cells. The cells were transfected with p68 or control siRNA (30 nM) for 48 h, and further transfected with human Mcl-1 reporter for 24 h. Following serum starvation overnight, the cells were incubated in the presence or absence of PDGF-BB (20 ng/ml) for 48 h.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Translocation Assay, Confocal Microscopy, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) The effects of HIF-1α depletion on Mcl-1 expression in ARCaP M cells. The cells were transfected with HIF-1α or control siRNA (30 nM) for 72 h, and analyzed for Mcl-1 expression by immunoblotting. (B) Western blot analysis of the effects of PDGF-BB (20 ng/ml) on the nuclear translocation of β-catenin and HIF-1α in ARCaP M cells. (C) Co-immunoprecipitation assays of the effects of PDGF-BB (20 ng/ml) on the interaction between β-catenin and HIF-1α in the nucleus in ARCaP M cells. (D) Confocal microscopy of the effects of PDGF-BB (20 ng/ml) on the co-localization of β-catenin and HIF-1α in the nucleus in ARCaP M cells.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Expressing, Transfection, Western Blot, Translocation Assay, Immunoprecipitation, Confocal Microscopy

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: (A) The effects of PDGF-BB on the HIF-1 reporter activity in ARCaP M cells. The cells were transiently transfected with HIF-1 reporter or pGL3 for 24 h, serum-starved and incubated in the presence or absence of PDGF-BB (20 ng/ml) for 48 h. (B) Schematic diagram of human Mcl-1 promoter and its deletion mutation at the putative HRE site. (C) The effects of deleting the putative HRE site on PDGF regulation of Mcl-1 promoter activity in ARCaP M cells. IL-6 (200 ng/ml) was included as the positive control. (D) ChIP-Re-ChIP assay of the effects of PDGF-BB treatment (20 ng/ml) on the binding of β-catenin and HIF-1α to human Mcl-1 promoter region in ARCaP M cells.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Activity Assay, Transfection, Incubation, Mutagenesis, Positive Control, Binding Assay

Journal: PLoS ONE

Article Title: PDGF Upregulates Mcl-1 Through Activation of β-Catenin and HIF-1α-Dependent Signaling in Human Prostate Cancer Cells

doi: 10.1371/journal.pone.0030764

Figure Lengend Snippet: The engagement of PDGF-BB to PDGFR dimers activates the c-Abl-p68 cascade, which subsequently stablizes β-catenin and promotes its nuclear translocation. In the nucleus, interaction between β-catenin and HIF-1α increases the binding of HIF-1α to the HRE site within Mcl-1 promoter, thereby activating the transcription of Mcl-1 gene. Upregulation of Mcl-1 antagonizes apoptotic signals and confers survival advantages to metastatic PCa cells. Furthermore, tumor-derived and locally expressed PDGF may mediate the interactions between PCa and bone microenvironment. Co-targeting the PDGF signaling in PCa cells (autocrine) and microenvironment (paracrine) could provide a new strategy to disrupt the “vicious cycle” and efficaciously treat metastatic PCa.

Article Snippet: Human Mcl-1 expression vector (pCMV-Mcl-1) was obtained from Origene, Inc. Human β-catenin expression plasmid was provided by Dr. Zhi-Ren Liu.

Techniques: Translocation Assay, Binding Assay, Derivative Assay

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: Expression of miR-193b and Mcl-1 in melanoma tissue samples. A: miR-193b expression was measured by Agilent miRNA microarray. The black line indicates the mean value for each group. B: Mcl-1 staining was scored in metastatic melanomas, primary melanomas, and benign nevi. Data shown are mean ± SEM. C: Pearson correlation analysis was performed to determine the relationship between miR-193b expression and Mcl-1 expression. The results show an inverse correlation (r = −0.7 and P < 0.001). *P < 0.0001 was calculated using the independent samples t-test.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Microarray, Staining

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: miR-193b down-regulates Mcl-1 and sensitizes melanoma cells to ABT-737. A: Western blot analysis of Mcl-1 and cleaved PARP expression in Malme-3M, MeWo, SK-MEL-2, and SK-MEL-28 cells transfected with miR-193b or negative control. Cells were transfected with 5 nmol/L miRNA precursor (miR-193b or negative control) and were harvested 72 hours after transfection. B: Western blot analysis of cleaved PARP and Mcl-1 in Malme-3M cells transfected with miR-193b or negative control, as indicated, incubated for 56 hours, and treated with 10 mmol/L ABT-737, as indicated. Cells were harvested 72 hours after transfection. C: Western blot analysis of MeWo and SK-MEL-28 cells, as indicated. The procedure was performed as described in B. D: One microgram of Mcl-1 plasmid (pMcl-1) was cotransfected with 5 nmol/L miRNA precursors, as indicated, and treated with 10 nmol/L ABT-737 as described in B. Gamma tubulin was used as the loading control. Representative data from one of three independent experiments are shown.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Expressing, Transfection, Negative Control, Incubation, Plasmid Preparation

Journal: The American Journal of Pathology

Article Title: miR-193b Regulates Mcl-1 in Melanoma

doi: 10.1016/j.ajpath.2011.07.010

Figure Lengend Snippet: miR-193b directly targets the 3′ UTR of Mcl-1. A: miR-193b and its predicted seed binding site in the 3′ UTR of Mcl-1 (top). The underlined 8-nucleotide sequence indicates the miR-193b seed region. Relative luciferase activity is shown for reporter constructs containing 3′ UTR or 3′ UTR M (with deleted seed pairing region) (bottom) in cells transfected with miR-193b or negative control. B: As in A, except reporter constructs carried the indicated fragment of the Mcl-1 3′ UTR (top). Four MREs predicted by RNA22 in the 3′ UTR of Mcl-1 (middle). C: As in A, except reporter constructs carried UTR1 M1 or UTR1 M2 (top). UTR1 M1 contains two point mutations, as indicated in bold italic. UTR1 M2 contains no seed binding region. Renilla luciferase activity was measured 24 hours after transfection using the Dual-Glo luciferase assay system (Promega). Data were normalized to firefly luciferase. Data shown are the mean ± SEM of three replicates and are representative of three independent experiments. *P < 0 0.05 and **P < 0.001 were calculated using the paired samples t-test.

Article Snippet: A sequence-validated Mcl-1 expression plasmid (SC315538) was purchased from OriGene Technologies (Rockville, MD).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Construct, Transfection, Negative Control