Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on PD‐like phenotypes induced by 6‐OHDA in rats. (A) Schematic representation showing the experiment process of this study. (B, C) RT‐qPCR and western blotting analysis revealed that IGFBP2 expression was downregulated in the substantia nigra pars compacta (SNpc) of 6‐OHDA‐induced PD rats. (D) Behavioral tests showing the effect of IGFBP2 on 6‐OHDA‐induced behavioral impairment (Rotation, F (2, 15) = 745.7; Latency time, F (2, 15) = 59.48; T ‐turn time, F (2, 15) = 33.01; T ‐total time, F (2, 15) = 95.94). (E) Representative images showed IHC staining of TH in the SNpc of 6‐OHDA‐lesioned rats, followed by quantification of the number of TH‐positive cells ( F (2, 15) = 188.2). (F) Western blotting analysis depicting α‐synuclein expression in the SNpc tissues ( F (2, 15) = 102.3). Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemistry, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Differential expression of mRNAs in PD rats. (A) The rats were injected with 6‐OHDA to induce the PD animal model. Apomorphine‐induced rotational behavior was tested at 2 and 3 weeks after modeling. (B) IHC staining of TH in OHDA‐lesioned rats (3 weeks). Bar = 100 µm. (C) Bidimensional principal component analysis (PCA) showing distinct clustering of gene profiles in 6‐OHDA‐induced PD rats and control rats. (D, E) Volcano plot and heatmap showing differential expression of mRNAs (|log 2 fold change (FC)| > 1 and p < 0.05). (F) Venn graph depicting the common genes of downregulated differentially expressed genes (DEGs, log 2 FC < −1.5 and p < 0.001) and parkinson‐related genes from the GeneCards database. (G) The FPKM value of IGFBP2 was shown. Group sizes were: N = 3 animals per group. ** p < 0.01, *** p < 0.001. * = control vs. 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Expressing, Injection, Animal Model, Immunohistochemistry, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on 6‐OHDA‐induced oxidative stress and mitochondrial impairment in rats. (A) ROS production, MDA content, and SOD and GSH‐Px activities in the SNpc were tested using corresponding commercial assay kits (ROS, F (2, 15) = 37; MDA, F (2, 15) = 42.56; SOD, F (2, 15) = 25.42; GSH‐Px, F (2, 15) = 45.15). (B) JC‐1 staining indicating the changes in mitochondrial membrane potential (Ψm; Q2‐2, F (2, 15) = 175.4; Q2‐4, F (2, 15) = 175.5). (C) ATP level was assessed by ATP detection kit ( F (2, 15) = 26.63). (D) Western blotting analysis revealing cytochrome c expression in cytoplasm and mitochondria in the SNpc of rats (cytochrome c in cytoplasm, F (2, 15) = 38.49; cytochrome c in mitochondria, F (2, 15) = 193.7). (E) Representative images of TUNEL‐positive staining in the SNpc tissues. Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Staining, Membrane, Western Blot, Expressing, TUNEL Assay, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on IGF‐1R/AKT signaling pathway. (A, B) Relative expression levels of IGF‐1R, p‐IGF‐1R, AKT, and p‐AKT in the SNpc were measured by western blotting (p‐IGF‐1R, F (2, 15) = 345.4; IGF‐1R, F (2, 15) = 229.7; p‐IGF‐1R/IGF‐1R, F (2, 15) = 92.01; p‐AKT, F (2, 15) = 811.5; AKT, F (2, 15) = 0.2335; p‐AKT/AKT, F (2, 15) = 851.2). Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. *** p < 0.001, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on apoptosis induced by 6‐OHDA in differentiated PC12 cells. The differentiated PC12 cells were co‐treated with rIGFBP2 and rIGF‐1 for 24 h, and then subjected to 6‐OHDA for 24 h. (A) CCK‐8 assay was performed to determine cell viability ( F (4, 10) = 45.19). (B) LDH content was evaluated using a LDH detection kit ( F (4, 10) = 45.50). (C) Apoptosis induced by 6‐OHDA was determined with Hoechst 33258 staining ( F (4, 10) = 160.7). (D) Bax, Bcl‐2, cleaved caspase‐9, and cleaved caspase‐3 expression were determined by western blotting in PC12 cells (Bax, F (4, 10) = 82.57; Bcl‐2, F (4, 10) = 136.6; cleaved caspase‐9, F (4, 10) = 53.36; cleaved caspase‐3, F (4, 10) = 65.57). Data were expressed as mean ± SD. Group sizes were: N = 3 wells per group. * p < 0.05, *** p < 0.001, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001, + p < 0.05, +++ p < 0.001. * = control versus 6‐OHDA, ^ = 6‐OHDA versus rIGFBP2 + 6‐OHDA, # = 6‐OHDA versus rIGF‐1 + 6‐OHDA, + = rIGFBP2 + 6‐OHDA versus rIGF‐1 + rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: CCK-8 Assay, Staining, Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on oxidative stress and mitochondrial function in 6‐OHDA‐lesioned PC12 cells. (A) ROS and MDA levels, SOD and GSH‐Px activities were determined by the commercial assay kits (ROS, F (4, 10) = 37.55; MDA, F (4, 10) = 41.91; SOD, F (4, 10) = 55.79; GSH‐Px, F (4, 10) = 75.03). (B) Changes in Ψm were evaluated by JC‐1 staining. (C) Relative expression of Cytochrome c in cytoplasm and mitochondria was detected by western blotting analysis (cytochrome c in cytoplasm, F (4, 10) = 47.39; cytochrome c in mitochondria, F (4, 10) = 62). Data were expressed as mean ± SD. Group sizes were: N = 3 wells per group. *** p < 0.001, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, ## p < 0.01, ### p < 0.001, ++ p < 0.01, +++ p < 0.001. * = control versus 6‐OHDA, ^ = 6‐OHDA versus rIGFBP2 + 6‐OHDA, # = 6‐OHDA versus rIGF‐1 + 6‐OHDA, + = rIGFBP2 + 6‐OHDA versus rIGF‐1 + rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: RT-qPCR primers.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques:

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: The BLF ( n = 20) and blood samples ( n = 20) were collected from NSCLC patients. A The IGFBP2 level in BLF and serum was assessed by ELISA assay. B The mRNA level of IGFBP2 in lung tissues was detected by qRT-PCR. C The expression of IGFBP2 in NSCLC tissues based on TCGA database. D The immunoreactivity of IGFBP2 in lung tissues was detected by IHC analysis. E The secretion of IGFBP2 in culture medium was detected by ELISA assay. F , G The mRNA and protein levels of IGFBP2 were detected by qRT-PCR and western blot, respectively. In A , B , E , F, G , Student’s t -test. In C , Dunnett’s test of one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.005. BLF bronchoalveolar lavage fluid, TCGA The Cancer Genome Atlas.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: NCI-H1650 and NCI-H1975 cells were either treated with 1 μg/mL human recombinant IGFBP2 (hr IGFBP2) or transfected with OE-IGFBP2/si-IGFBP2, followed by the treatment of gefitinib (10 nM). A Cell viability was monitored by CCK-8 assay. B Colony formation was detected by colony formation assay. C The scheme of the in vivo experiment design. Representative photos and in vivo imaging of xenograft tumors/lung metastases with quantitative analysis. The immunoreactivity of IGFBP2 in lung tissues was detected by IHC analysis. D The protein levels of N-cadherin and vimentin were detected by western blot. Dunnett’s test of one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001. hr human recombinant, OE-NC overexpression vector alone, si-NC negative control siRNA.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: Recombinant, Transfection, CCK-8 Assay, Colony Assay, In Vivo, In Vivo Imaging, Western Blot, Over Expression, Plasmid Preparation, Negative Control

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: Heatmap ( A ), GO ( B ) and KEGG enrichment pathway ( C ) and Reactome analysis ( D ) for RNA-seq. E Changes of GPCR signaling molecules were detected by sequencing. NCI-H1650 and NCI-H1975 cells were transfected with si- IGFBP2 or IGFBP2 overexpression construct. F , G The mRNA and protein levels of CXCL1 were detected by qRT-PCR and western blot, respectively. Dunnett’s test of one-way ANOVA. ** P < 0.01; *** P < 0.001. OE-NC overexpression vector alone, si-NC negative control siRNA.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: RNA Sequencing Assay, Sequencing, Transfection, Over Expression, Construct, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Negative Control

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: A , C The mRNA and protein levels of CXCL1 were detected by qRT-PCR and western blot, respectively. B The secretion of CXCL1 was detected by ELISA assay. D The immunoreactivity of CXCL1 in lung tissues was detected by IHC analysis. NCI-H1650 and NCI-H1975 cells were transfected with CXCL1 overexpression construct, si-CXCL1 or/and IGFBP2 overexpression construct. E Cell proliferation was monitored by CCK-8 assay. F Colony forming ability was assessed by colony formation assay with quantitative analysis. G , H Cell invasion and migration were detected by Transwell and wound healing assays with quantitative analysis, respectively. In A – C , Student’s t -test. In E – H , Dunnett’s test of one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001. OE-NC, overexpression vector alone; si-NC, negative control siRNA.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Over Expression, Construct, CCK-8 Assay, Colony Assay, Migration, Plasmid Preparation, Negative Control

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: The interaction relation between IGFBP2 and ITGA5 through A BioGRID and B String databases. C , D The protein levels of ITGA5, STAT3, p-STAT3 and CXCL1 were detected by western blot.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: NCI-H1650 or NCI-H1975 cells were transfected with IGFBP2 overexpression construct or/and si-ITGA5. A Colony formation was detected by colony formation assay. B , C Cell invasion and migration were measured by Transwell and wound healing assays with quantitative analysis, respectively. D The protein levels of N-cadherin and vimentin were detected by western blot. Dunnett’s test of one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001. OE-NC overexpression vector alone, si-NC negative control siRNA.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: Transfection, Over Expression, Construct, Colony Assay, Migration, Western Blot, Plasmid Preparation, Negative Control

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: A The putative interaction between IGFBP2 and ITGA5 was validated by yeast two hybrid assay. B The interaction between IGFBP2 and ITGA5 in lung metastases and NSCLC cells was detected by co-IP. Whole cell lysates or normal IgG served as an input or negative control, respectively. C The in vitro binding of GST-tagged IGFBP2 and His-tagged ITGA5 was assessed by GST pull-down assay.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: Y2H Assay, Co-Immunoprecipitation Assay, Negative Control, In Vitro, Binding Assay, Pull Down Assay

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: A The scheme of the in vivo experiment design. Representative photos of lung metastases with quantitative analysis and in vivo imaging of lung tissues. The immunoreactivities of IGFBP2, STAT3 and CXCL1 in lung metastases were detected by IHC analysis. Dunnett’s test of one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques: In Vivo, In Vivo Imaging

Journal: Cell Death & Disease

Article Title: IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer

doi: 10.1038/s41419-024-06843-y

Figure Lengend Snippet: IGFBP2/ITGA5 promoted gefitinib resistance via activating STAT3/CXCL1 axis.

Article Snippet: For human recombinant IGFBP2 (hr IGFBP2, ab63223, Abcam) treatment, NSCLC cells were treated with 1 μg/mL hr IGFBP2 for 24 h. sh-IGFBP2, si-CXCL1, sh-STAT3, and si-ITGA5 were purchased from General BIOL.

Techniques:

Journal: Molecular Cancer

Article Title: Dynamic altruistic cooperation within breast tumors

doi: 10.1186/s12943-023-01896-7

Figure Lengend Snippet: miR-125b dichotomizes via NF-κB signaling into altruistic fitness benefits and disadvantage. A Schema showing cell cycle process and regulators. Those indicated with red boxes are involved in G1-S transition and which were selected for further investigation as inhibitory targets of miR-125b. B Cell cycle analysis of miR-125b m or Control m -transfected MCF7 or MDA-MB-468 cells, or control LNA or miR-125b LNA inhibitor-transfected MDA-MB-231 cells. C Box plots of fold change in enrichment of indicated gene transcripts pulled-down using biotinylated miR-125b. D Relative luciferase activities of HEK293T cells following transfection of wild-type or mutant reporter construct for indicated genes and mimics. E Homogeneous and heterogeneous mixtures of MCF7 (Left) or MDA-MB-468 (Right) were transfected with siRNA against indicated genes, exposed to sublethal 0.8 nM docetaxel, and the RF α measured. Blue or red dotted line/box plot indicate respective levels of RF α for homogenous mixture or heterogenous mixture with control KD. IKBKB gene codes for IKKβ. F Immunoblotting to detect for indicated proteins extracted from indicated cell lines transfected with combination of indicated mimics and siRNAs for IKBKB . G, H Conditioned media was harvested from cell culture of miR-125b m or Control m -transfected MCF-7 cells and analyzed using cytokine array kit (G) and iTRAQ (H). I Immunoblotting to detect CCL28 and IGFBP2 in conditioned media from mimic or LNA inhibitor-transfected, or EGFP reporter-sorted cell fractions of indicated cell lines. Ponceau S was used to visualize protein load. Quantification of band intensities (relative to Control m / Control LNA / Mi/EGFP Low ) is shown. See Fig. S F for results of plasmid-transfected MCF7 cells. J Viability of indicated cell lines with indicated exposure to docetaxel and/or neutralizing antibodies to IGFBP2 and/or CCL28. K Percentage change in size of MDA-MB-231 xenografted tumors in NSG mice with indicated treatment of docetaxel and/or neutralizing antibodies to IGFBP2 and/or CCL28. L Immunoblotting to detect for CCL28 and IGFBP2 in conditioned media from indicated cell lines transfected with combination of indicated mimics and siRNA. Ponceau S for protein load normalization. Quantification of band intensities (relative to first band of each set) is shown. M RS α of homogeneous and heterogeneous mixture of MCF7 (Left) or MDA-MB-468 (Right) cells transfected with indicated siRNA and exposed to indicated docetaxel concentration. N Schema showing proposed mechanism of how miR-125b dichotomizes into fitness benefit and disadvantage of cancer cell altruism. O Schematic showing the different components of the NF-κB signaling pathway and how the oncogenic (fitness benefits) and tumor suppressive (fitness disadvantage) events may be mediated by different parts of the same signaling pathway. Experiment repeated two times, representative result shown (B,F,L). Data are mean ± s.d. from three independent biological sets of triplicates (C,D,E,M). Experiment performed once (G), and results validated in (I). Representative blots from three independent replicates (I). Data are mean ± s.d. from three independent biological duplicates (H) or quadruplicates (J). n = 8-9 independent animals (K). Statistical analysis was performed using two-tailed one sample t -test against 1 (C,M) or 100 (D) or one-way ANOVA with post-hoc Tukey HSD (J,K). NT: no treatment; DTX: docetaxel treatment. Schematic created using Biorender (A,O). Exact P values are shown.

Article Snippet: 4’,6-Diamidino-2-Phenylindole (DAPI) from Thermo Fisher Scientific; IDEAL miRNA assays from MiRXES; Superscript VILO enzyme from Thermo Fisher Scientific; RPMI and DMEM from Thermo Fisher Scientific; McCoy’s 5A modified medium, Leibovitz L-15 medium and EMEM from Lonza; Trichostatin A (T8552), 5-azacytidine (A2385), curcumin (C1386), anacardic acid (05506), MB-3 (M2449) from Sigma-Aldrich; nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) tablets from Roche; Lipofectamine 3000 from Thermo Fisher Scientific; mirVana miR-125b oligonucleotide mimic (4464066) and negative control oligonucleotide mimic (4464058) tagged with fluorescein from Thermo Fisher Scientific; siRNA against PCAF (sc-36198), HDAC3 (sc-35538), PCAF (sc-36198), p53 (sc-29435), Ah receptor (sc-29654), Sp1 (sc-29487), TIP60 (sc-37966), FOXO3 (sc-37887), KLF2 (sc-35818), IKKβ (sc-35644), β-catenin (CTNNB1; sc-29209), SMO (sc-40161), GSK-3β (sc-35527), NFκB p65 (sc-29410), NFκB p50 (sc-29408) and control (sc-37007) from Santa Cruz; azidohomoalanine (AS-63669) from AnaSpe; docetaxel from Sanofi-Aventis; VivoGlo Luciferin (P1043) from Promega; Recombinant IGFBP2 (350-06B-20) and Recombinant CCL28 (300-57-20) from Peprotech; PI3K inhibitor LY294002 (HY-10108) from MedChemExpress,; Akt Inhibitor IV (sc-203809) from Santa Cruz Biotechnology, ; control (339121) and miR-125b miRNA LNA inhibitor (339126) from Qiagen.

Techniques: Cell Cycle Assay, Control, Transfection, Luciferase, Mutagenesis, Construct, Western Blot, Cell Culture, Multiplex sample analysis, Plasmid Preparation, Concentration Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: Dynamic altruistic cooperation within breast tumors

doi: 10.1186/s12943-023-01896-7

Figure Lengend Snippet: Altruistic cancer cells regenerate via epigenetic mechanism. A Mathematical model explains the persistence of altruistic subclone in breast cancer cell population (Upper), and a spatial model simulates changes in the percentage of altruistic cells over time for hypothesized genetic- and epigenetics-mediated altruism (Lower). Dotted lines link events in upper panel to corresponding points in lower panel. See Supplementary Note & . B Indicated cancer cell lines were sorted according to Mi/EGFP levels and the fluorescence monitored over indicated time. C Mi/EGFP fluorescence of non-sorted MDA-MB-231 miR-125bprom-EGFP cells, treated as indicated, as determined by FACS. D Mi/EGFP Low cells were transfected with indicated siRNAs and the Mi/EGFP fluorescence determined after four days. E Schema showing putative consensus sites for KLF2 binding along the hsa-miR-125b-1 promoter and gRNA targeting sequence of CRISPRi. F Mi/EGFP Low cells were transduced with lentivirus for expression of CRISPRi targeting KBS-1 or non-specific sequence, and Mi/EGFP fluorescence determined after four days. G,H Changes in Mi/EGFP fluorescence as treated in (F) were monitored in indicated cell lines (G). Percentage effect of KBS-1 CRISPRi expression on cell viability relative to control CRISPRi was also measured. Cancer cells were exposed to indicated concentration of docetaxel (H). I MDA-MB-231 miR-125bprom-EGFP cells, as treated in (F), were grown as xenografts in NSG mice. Upper: excised tumors at end point of monitoring period. Lower: Percentage change in tumor size with and without docetaxel treatment. J Immunoblotting to detect for IGFBP2 and CCL28 in conditioned media from MDA-MB-231 miR-125bprom-EGFP cells as treated in (F) and used to establish xenograft model for (I). Ponceau S was used to visualize protein load. Quantification of band intensities (relative to Control CRISPRi) is shown. Experiments repeated two times (B, C, D, F, G, H, J). Representative data are shown for (J). Mean percentage ± s.d. cells for technical triplicates of representative set are shown in same colours as corresponding histograms (C, D, F, G) or in black only (B). Data are mean ± s.d. from two independent biological sets of triplicates (H). n = 4 independent animals per group (I). Statistical analysis was performed using two-tailed one sample t -test against 0 (H) and two-tailed unpaired t -test (I lower). NT: no treatment; DTX: docetaxel treatment. Exact P values are shown

Article Snippet: 4’,6-Diamidino-2-Phenylindole (DAPI) from Thermo Fisher Scientific; IDEAL miRNA assays from MiRXES; Superscript VILO enzyme from Thermo Fisher Scientific; RPMI and DMEM from Thermo Fisher Scientific; McCoy’s 5A modified medium, Leibovitz L-15 medium and EMEM from Lonza; Trichostatin A (T8552), 5-azacytidine (A2385), curcumin (C1386), anacardic acid (05506), MB-3 (M2449) from Sigma-Aldrich; nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) tablets from Roche; Lipofectamine 3000 from Thermo Fisher Scientific; mirVana miR-125b oligonucleotide mimic (4464066) and negative control oligonucleotide mimic (4464058) tagged with fluorescein from Thermo Fisher Scientific; siRNA against PCAF (sc-36198), HDAC3 (sc-35538), PCAF (sc-36198), p53 (sc-29435), Ah receptor (sc-29654), Sp1 (sc-29487), TIP60 (sc-37966), FOXO3 (sc-37887), KLF2 (sc-35818), IKKβ (sc-35644), β-catenin (CTNNB1; sc-29209), SMO (sc-40161), GSK-3β (sc-35527), NFκB p65 (sc-29410), NFκB p50 (sc-29408) and control (sc-37007) from Santa Cruz; azidohomoalanine (AS-63669) from AnaSpe; docetaxel from Sanofi-Aventis; VivoGlo Luciferin (P1043) from Promega; Recombinant IGFBP2 (350-06B-20) and Recombinant CCL28 (300-57-20) from Peprotech; PI3K inhibitor LY294002 (HY-10108) from MedChemExpress,; Akt Inhibitor IV (sc-203809) from Santa Cruz Biotechnology, ; control (339121) and miR-125b miRNA LNA inhibitor (339126) from Qiagen.

Techniques: Fluorescence, Transfection, Binding Assay, Sequencing, Transduction, Expressing, Control, Concentration Assay, Western Blot, Two Tailed Test

Journal: Molecular Cancer

Article Title: Dynamic altruistic cooperation within breast tumors

doi: 10.1186/s12943-023-01896-7

Figure Lengend Snippet: Lateral inhibition maintains a sparse spatial organization of altruists. A Immunoblotting to detect indicated proteins extracted from Mi/EGFP High and Mi/EGFP Low fractions from indicated cancer cell lines. B,C Mi/EGFP Low and Mi/EGFP High cells from indicated cancer cell lines were exposed to conditioned media harvested from separate batches of Mi/EGFP Low and Mi/EGFP High cells. After four days, the formers’ fluorescence levels were analyzed by FACS (B) and extracted protein of exposed Mi/EGFP Low cell studied by immunoblotting (C). D, E Mi/EGFP Low cells from indicated cell lines were treated with indicated recombinant proteins in combination with neutralizing antibodies and the fluorescence level analyzed by FACS after four days. F-H Mi/EGFP Low cell fractions from indicated cancer cell lines, transfected with control or GAB1 siRNA, were exposed to recombinant IGFBP2 or CCL28. After four days, their fluorescence levels were analyzed by FACS (F, IGFBP2; G, CCL28) and their extracted protein by immunoblotting (H). CM-R: Mi/EGFP Low recipient cells exposed to recombinant protein. I Box plots of fold change in enrichment of GAB1 mRNA pulled-down using biotinylated miR-125b mimics. J Relative luciferase activities of HEK293T cells following transfection of wild-type or mutant reporter construct for GAB1 and indicated mimics. WT: wild type. K Immunoblotting to detect expression of indicated proteins extracted from miR-125b m or Control m -transfected MCF7 or MDA-MB-468 cells with or without docetaxel treatment (Upper), or miR-125b LNA-inhibitor- or control-LNA-transfected MDA-MB-231 or MDA-MB-415 cells (Lower). L,M Schema of lateral inhibition model mediated by diffusible IGFBP2 and CCL28 secreted by miR-125b High altruists. Upon exposure to IGFBP2 and CCL28, heightened PI3K-AKT signaling is induced in the recipient cells, resulting in reduction in miR-125b expression and adoption of the non-altruistic social fate (L). Hypothesized level of the diffusible proteins, extent of PI3K activation and probability of altruist arising as the distance from altruist increases is depicted, in association with the altruist’s ability to influence social fates beyond the immediate neighboring cells (M). N Simulation of lateral inhibition dynamics showing pattern generation when diffusion coefficient d = 1 (as in the case of Notch-Delta signaling) and d>1 (mediated by diffusible proteins such as IGFBP2 and CCL28) (Left column). Spatial patterns of Mi/EGFP High and Mi/EGFP Low cells in indicated cell lines, with or without IGFBP2 & CCL28 antibodies treatment, are shown (Center and Right columns). Experiments repeated two times, representative data are shown for (A-H, K, N). Mean percentage ± s.d. cells for technical triplicates of representative set are shown in same colour as corresponding histograms (B,D-G). Data are mean ± s.d. from three independent biological sets of triplicates (I, J). Statistical analysis was performed using two-tailed one sample t -test against 1 (I) or 100 (J). NT: no treatment; DTX: docetaxel treatment. Exact P values are shown

Article Snippet: 4’,6-Diamidino-2-Phenylindole (DAPI) from Thermo Fisher Scientific; IDEAL miRNA assays from MiRXES; Superscript VILO enzyme from Thermo Fisher Scientific; RPMI and DMEM from Thermo Fisher Scientific; McCoy’s 5A modified medium, Leibovitz L-15 medium and EMEM from Lonza; Trichostatin A (T8552), 5-azacytidine (A2385), curcumin (C1386), anacardic acid (05506), MB-3 (M2449) from Sigma-Aldrich; nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) tablets from Roche; Lipofectamine 3000 from Thermo Fisher Scientific; mirVana miR-125b oligonucleotide mimic (4464066) and negative control oligonucleotide mimic (4464058) tagged with fluorescein from Thermo Fisher Scientific; siRNA against PCAF (sc-36198), HDAC3 (sc-35538), PCAF (sc-36198), p53 (sc-29435), Ah receptor (sc-29654), Sp1 (sc-29487), TIP60 (sc-37966), FOXO3 (sc-37887), KLF2 (sc-35818), IKKβ (sc-35644), β-catenin (CTNNB1; sc-29209), SMO (sc-40161), GSK-3β (sc-35527), NFκB p65 (sc-29410), NFκB p50 (sc-29408) and control (sc-37007) from Santa Cruz; azidohomoalanine (AS-63669) from AnaSpe; docetaxel from Sanofi-Aventis; VivoGlo Luciferin (P1043) from Promega; Recombinant IGFBP2 (350-06B-20) and Recombinant CCL28 (300-57-20) from Peprotech; PI3K inhibitor LY294002 (HY-10108) from MedChemExpress,; Akt Inhibitor IV (sc-203809) from Santa Cruz Biotechnology, ; control (339121) and miR-125b miRNA LNA inhibitor (339126) from Qiagen.

Techniques: Inhibition, Western Blot, Fluorescence, Recombinant, Transfection, Control, Luciferase, Mutagenesis, Construct, Expressing, Activation Assay, Diffusion-based Assay, Two Tailed Test

Journal: Molecular Cancer

Article Title: Dynamic altruistic cooperation within breast tumors

doi: 10.1186/s12943-023-01896-7

Figure Lengend Snippet: Breast tumor as a dynamic social system manifesting altruistic cooperation. A In altruistic cooperation, a small subpopulation of altruistic cells (blue) confers communal protection against taxane exposure by secreting trophic factors (IGFBP2 and CCL28) that activate PI3K/AKT signaling and thus leading to heightened chemotolerance in neighboring cells (yellow). During post-treatment expansion phase, the altruistic subpopulation, saddled with a fitness disadvantage due to miR-125b-mediated cell cycle impediment, risks becoming extinct due to competition from the faster growing neighboring non-altruists. B Conferment of survival benefits to others (an oncogenic event) and incurring of fitness cost to self (a tumor suppressive event), both of which are defining attributes of altruism, are found to be commonly mediated by heightened miR-125b expression, via differential NF-κB signaling, in the altruistic cancer cells. C The altruistic subpopulation persists, due to phenotypic conversion from the neighboring non-altruists via a KLF2/PCAF-mediated epigenetic mechanism acting on the promoter of the hsa-miR-125b-1 gene. D The tumor cell population actively self-organizes via a lateral inhibition mechanism mediated by IGFBP2/CCL28-induced GAB1-PI3K-AKT-miR-125b signaling circuit. This limits the altruistic subpopulation to a minority presence and a sparse spatial arrangement. A closer look at the lateral inhibition model (below) shows inhibition of GAB1 by high miR-125b expression in the altruist, which prevents self-activation of PI3K/AKT by the altruists-secreted IGFBP2 and CCL28, thus averting self-benefiting and instability of the altruistic phenotype. E The lateral inhibition mechanism, coupled with epigenetic regenerability of the altruists, permits stable co-existence of functionally distinct subpopulations: an altruistic miR-125b High minority confers costly communal protection during chemotherapeutic crisis while the miR-125b Low majority undergoes aggressive proliferation post-crisis to re-colonize the tumor. Cooperation between these different phenotypes suggests the existence of division of labor, a hallmark of complex biological societies, within the breast tumor. F One possible explanation of the origin of altruistic tumor society is evolution from a homogeneous population of generalist cancer cells. The composition of the resulting altruistic society of cancer cells can theoretically be perturbed with varying ecological consequences. Without epigenetic regeneration of altruists, non-altruists or “cheats” would dominate and deplete existing resources, leading to a situation called the “tragedy of the commons” . Conversely, without lateral inhibition, altruists would dominate the population, hence inflicting a fitness burden on the tumor. Breast tumor may thus constitute a potential model to study how tumor-specific ecological factors can affect evolution and manifestation of altruistic cooperation. G Examples of altruistic social systems and how the social dynamics is regulated. Above: In honeybee ( Apis Mellifera ), the queen bee secretes primer pheromone such as CHCs and other glandular compounds that suppress worker ovarian development, thus maintaining the workers as reproductive altruists . In Dictyostelium amoeba, the pre-spores secrete differentiation-inducing factor-1 (DIF-1) to prevent the altruistic pre-stalk cells from developing into spores, thus maintaining a 80:20 spore-to-stalk cell ratio in the fruiting body that is formed eventually . Such secretion-mediated regulation of cell fate is similarly observed in the altruistic breast cancer cells. Below: Epigenetic regulation is known to underlie behavioral plasticity in Apis Mellifera , and we likewise observed how epigenetic mechanism regulates social fate plasticity in breast cancer cells

Article Snippet: 4’,6-Diamidino-2-Phenylindole (DAPI) from Thermo Fisher Scientific; IDEAL miRNA assays from MiRXES; Superscript VILO enzyme from Thermo Fisher Scientific; RPMI and DMEM from Thermo Fisher Scientific; McCoy’s 5A modified medium, Leibovitz L-15 medium and EMEM from Lonza; Trichostatin A (T8552), 5-azacytidine (A2385), curcumin (C1386), anacardic acid (05506), MB-3 (M2449) from Sigma-Aldrich; nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) tablets from Roche; Lipofectamine 3000 from Thermo Fisher Scientific; mirVana miR-125b oligonucleotide mimic (4464066) and negative control oligonucleotide mimic (4464058) tagged with fluorescein from Thermo Fisher Scientific; siRNA against PCAF (sc-36198), HDAC3 (sc-35538), PCAF (sc-36198), p53 (sc-29435), Ah receptor (sc-29654), Sp1 (sc-29487), TIP60 (sc-37966), FOXO3 (sc-37887), KLF2 (sc-35818), IKKβ (sc-35644), β-catenin (CTNNB1; sc-29209), SMO (sc-40161), GSK-3β (sc-35527), NFκB p65 (sc-29410), NFκB p50 (sc-29408) and control (sc-37007) from Santa Cruz; azidohomoalanine (AS-63669) from AnaSpe; docetaxel from Sanofi-Aventis; VivoGlo Luciferin (P1043) from Promega; Recombinant IGFBP2 (350-06B-20) and Recombinant CCL28 (300-57-20) from Peprotech; PI3K inhibitor LY294002 (HY-10108) from MedChemExpress,; Akt Inhibitor IV (sc-203809) from Santa Cruz Biotechnology, ; control (339121) and miR-125b miRNA LNA inhibitor (339126) from Qiagen.

Techniques: Expressing, Inhibition, Activation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reprogramming Macrophage Polarization, Depleting ROS by Astaxanthin and Thioketal-Containing Polymers Delivering Rapamycin for Osteoarthritis Treatment.

doi: 10.1002/advs.202305363

Figure Lengend Snippet: Fig. 3 Altruistic cancer cells regenerate via epigenetic mechanism. A Mathematical model explains the persistence of altruistic subclone in breast cancer cell population (Upper), and a spatial model simulates changes in the percentage of altruistic cells over time for hypothesized genetic- and epigenetics-mediated altruism (Lower). Dotted lines link events in upper panel to corresponding points in lower panel. See Supplementary Note 2 & 3. B Indicated cancer cell lines were sorted according to Mi/EGFP levels and the fluorescence monitored over indicated time. C Mi/EGFP fluorescence of non-sorted MDA-MB-231miR-125bprom-EGFP cells, treated as indicated, as determined by FACS. D Mi/EGFPLow cells were transfected with indicated siRNAs and the Mi/EGFP fluorescence determined after four days. E Schema showing putative consensus sites for KLF2 binding along the hsa-miR-125b-1 promoter and gRNA targeting sequence of CRISPRi. F Mi/EGFPLow cells were transduced with lentivirus for expression of CRISPRi targeting KBS-1 or non-specific sequence, and Mi/EGFP fluorescence determined after four days. G,H Changes in Mi/ EGFP fluorescence as treated in (F) were monitored in indicated cell lines (G). Percentage effect of KBS-1 CRISPRi expression on cell viability relative to control CRISPRi was also measured. Cancer cells were exposed to indicated concentration of docetaxel (H). I MDA-MB-231miR-125bprom-EGFP cells, as treated in (F), were grown as xenografts in NSG mice. Upper: excised tumors at end point of monitoring period. Lower: Percentage change in tumor size with and without docetaxel treatment. J Immunoblotting to detect for IGFBP2 and CCL28 in conditioned media from MDA-MB-231miR-125bprom-EGFP cells as treated in (F) and used to establish xenograft model for (I). Ponceau S was used to visualize protein load. Quantification of band intensities (relative to Control CRISPRi) is shown. Experiments repeated two times (B, C, D, F, G, H, J). Representative data are shown for (J). Mean percentage ± s.d. cells for technical triplicates of representative set are shown in same colours as corresponding histograms (C, D, F, G) or in black only (B). Data are mean ± s.d. from two independent biological sets of triplicates (H). n = 4 independent animals per group (I). Statistical analysis was performed using two-tailed one sample t-test against 0 (H) and two-tailed unpaired t-test (I lower). NT: no treatment; DTX: docetaxel treatment. Exact P values are shown

Article Snippet: 4’,6-Diamidino-2-Phenylindole (DAPI) from Thermo Fisher Scientific; IDEAL miRNA assays from MiRXES; Superscript VILO enzyme from Thermo Fisher Scientific; RPMI and DMEM from Thermo Fisher Scientific; McCoy’s 5A modified medium, Leibovitz L-15 medium and EMEM from Lonza; Trichostatin A (T8552), 5-azacytidine (A2385), curcumin (C1386), anacardic acid (05506), MB-3 (M2449) from Sigma-Aldrich; nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/ BCIP) tablets from Roche; Lipofectamine 3000 from Thermo Fisher Scientific; mirVana miR-125b oligonucleotide mimic (4464066) and negative control oligonucleotide mimic (4464058) tagged with fluorescein from Thermo Fisher Scientific; siRNA against PCAF (sc-36198), HDAC3 (sc-35538), PCAF (sc-36198), p53 (sc-29435), Ah receptor (sc-29654), Sp1 (sc-29487), TIP60 (sc-37966), FOXO3 (sc-37887), KLF2 (sc-35818), IKKβ (sc-35644), β-catenin (CTNNB1; sc-29209), SMO (sc-40161), GSK-3β (sc35527), NFκB p65 (sc-29410), NFκB p50 (sc-29408) and control (sc-37007) from Santa Cruz; azidohomoalanine (AS-63669) from AnaSpe; docetaxel from Sanofi-Aventis; VivoGlo Luciferin (P1043) from Promega; Recombinant IGFBP2 (350-06B-20) and Recombinant CCL28 (300-57- 20) from Peprotech; PI3K inhibitor LY294002 (HY-10108) from MedChemExpress,; Akt Inhibitor IV (sc-203809) from Santa Cruz Biotechnology, ; control (339121) and miR-125b miRNA LNA inhibitor (339126) from Qiagen.

Techniques: Fluorescence, Transfection, Binding Assay, Sequencing, Transduction, Expressing, Control, Concentration Assay, Western Blot, Two Tailed Test