Journal: Cells

Article Title: Disclosing the Interactome of Leukemogenic NUP98-HOXA9 and SET-NUP214 Fusion Proteins Using a Proteomic Approach

doi: 10.3390/cells9071666

Figure Lengend Snippet: Proximity-dependent biotin identification (BioID) fusion protein expression and biotinylation of endogenous proteins. ( A ) Localization of NHA9-BioID, SN214-BioID, and BirA R118G was evaluated by immunostaining with anti-HA antibody. ( B ) Localization of GFP-tagged NUP98-HOXA9 and SET-NUP214 was evaluated by green fluorescent protein (GFP) fluorescence. NHA9-BioID and SN214-BioID exhibit the same distribution pattern in nuclear foci and at the nuclear envelope as their GFP-tagged counterparts. ( C ) Detection of protein biotinylation by Streptavidin-488. Cells were transfected with NHA9-BioID, SN214-BioID, and BirA R118G and probed with Streptavidin-Alexa Fluor™ 488 conjugate. Shown are representative confocal images. DNA was visualized with DAPI (blue). Scale bars, 10 µm. ( D ) For detection of protein biotinylation by NHA9-BioID, SN214-BioID, and BirA R118G , corresponding cell lysates were enriched on Streptavidin-coated magnetic beads and whole protein lysates and the bound fractions were analyzed by immunoblotting. Note, virtually no specific bands were detected in the bound fraction of BirA R118G , in contrast to the bound fractions of NHA9-BioID and SN214-BioID, which exhibited patterns of differentially biotinylated proteins.

Article Snippet: To generate the SN214-BioID construct, the SET-NUP214 coding sequence was subcloned from the pGEM-T backbone into the pcDNA3.1 MCS-BirA(R118G)-HA using AgeI and EcoRI restriction enzymes (New England Biolabs—NEB, Ipswich, MA, USA).

Techniques: Expressing, Immunostaining, Fluorescence, Transfection, Magnetic Beads, Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 3. Combination of Aloin CPT-11 downregulates IGF1R and its downstream MEK/ERK and PI3K/AKT/mTOR pathways. (A) The effect of the Aloin-CPT- 11 combination on IGF1R expression in CRC cells (Lovo, Caco2 and SW620). (B-C) The effect of Aloin and CPT-11 combination on the two main IGF1R downstream pathways determined by western blot analysis. (D) IGF1R was knocked down to determine its role in CRC in the context of cell viability and apoptosis. (E-F) Knockdown of IGF1R reduced cell viability. Co-treatment of Aloin-CPT combination with siRNA-IGF1R reduced cell viability even further. (G-H) Aloin-CPT-11 enhanced apoptosis. Treatment of Aloin-CPT-11 combination with IGF1R knockdown intensified apoptosis even further. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Expressing, Western Blot, Knockdown, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 4. MicroRNA-133b is upregulated by Aloin-CPT-11 combination, targets IGF1R and reduces cell viability in LoVo cells. (A) MiRNAs that are predicted to target IGF1R were identified using miRTARGETLink2.0 2. Forty-seven miRNAs were initially identified. (B) The 47 miRNAs were further filtered and trimmed to 17 miRNAs. These miRNA were screened by RT-qPCR in LoVo cells after treatment with Aloin-CPT-11 combination. (C) The three miRNAs that showed the best sig nificant upregulation with Aloin-CPT-11 combination were identified. (D).The statistically significant upregulated miRNA-133b was adopted and Target Scan indicated that miR-133b binds the 3′ UTR region between 4007 and 4013 of the IGF1R. (E) Cells were transfected with miR-133b mimic and inhibitor and the transfection efficiency was determined by RT-qPCR. (F) Transfection of miR-133b mimic downregulated IGF1R while inhibitor upregulated it. (G-H) miRNA-133b targets IGF1R. The increase in cell viability induced by miRNA-133b inhibitor was reversed by Aloin-CPT-11 combination treatment. (I) IGF1R downregulation by miRNA-133b mimic was further enhanced with an addition of Aloin-CPT-11 combination treatment. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Quantitative RT-PCR, Transfection, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 5. Over expression of miRNA-133b reversed the effect of IGF1R over expression. (A) Co-transfection of cells with pcDNA-IGF1R and miR-133b mimic resulted in the downregulation of pcDNA-induced IGF1R overexpression. (B) pcDNA- induced IGF1R overexpression was reversed by treatment with Aloin-CPT-11 combination. (C) IGF1R-overexpressed cells showed an increase in cell viability but this phenomenon was reversed when treated with Aloin-CPT-11 combination. (D) Flow cytometry: combination of Aloin and CPT-11 enhanced LoVo cell apoptosis in presence of IGF1R upregulation. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Over Expression, Cotransfection, Flow Cytometry, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 6. Aloin and CPT-11 attenuated and reversed CPT-11 resistance in LoVo CPT-11 resistant cells. (A). CPT-11-resistant cells were established. Their response to CPT-11 were compared with that of parental LoVo cells. MTT cell viability had both cells respond to CPT-11 dose dependently. The response by the resistant cell was poor with IC50 three times more than parental cell (35.6 μМ against 12.0 μM). (B) Cell resistant characteristics were confirmed by IGF1R expression and stem cell markers. (C) The response of resistant cells to CPT-11 was again confirmed by western blot with apoptotic markers. (D-E) Expression of miR-133b and IGF1R mRNA in resistant cells was determined by RT-qPCR. (F-G) The effect of Aloin-CPT-11 combination in resistant cells was assessed through western blot focusing on IGF1R expression and apoptosis. Results are presented as means ± standard deviation from three independent experiments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Aloin and CPT-11 combination activates miRNA-133b and downregulates IGF1R- PI3K/AKT/mTOR and MEK/ERK pathways to inhibit colorectal cancer progression.

doi: 10.1016/j.biopha.2023.115911

Figure Lengend Snippet: Fig. 7. Aloin and CPT-11 combination exhibited improved anti-tumor efficacy against murine colorectal cancer in BALB/c nude mice. (A) Schematic illustration of the animal model timeline and experimental plan. (B) Tumor volumes were recorded twice a week until the end of the study and were statistically compared between groups. (C) The physical sizes of the tumors were compared against each group after mice were sacrificed. (D-E) Tumor sizes and weights were measured and statistically compared. (F) Survival times and percentages were predicted and calculated using Kaplan-Meier estimator. (G-J). The in vivo effect of the Aloin-CPT-11 combination was determined by estimating the expression levels of IGF1R and its downstream pathways, as well as apoptotic markers in tumor xe nografts for each treatment group. (K) Eosin & Hematoxylin stain was used to asses mophological and poliferative changes in tumos. Immunohistochemestly was used to determine the expression levels of IGF1R and Ki67 in tumor xenograft. Results are shown as means ± standard deviation from three independent experi ments. ns: not significant, p < 0.05 (*p < 0.01, ** p < 0.001,*** p < 0.0001).

Article Snippet: MicroRNA inhibitors, mimics & controls were obtained from RiboBio (Guangzhou, China), IGF1R siRNA from Sigma-Aldrich (Germany), and p-cDNA -IGF1R (Plasmid# 109232) from Addgene.

Techniques: Animal Model, In Vivo, Expressing, Staining, Standard Deviation

Journal: bioRxiv

Article Title: ZDHHC5 directs Protocadherin 7 to the mitotic cell surface and cleavage furrow by a palmitoylation-dependent mechanism for a successful cell division

doi: 10.1101/2020.05.24.111831

Figure Lengend Snippet: A . The cell cycle-specific localization of PCDH7:BirA*-HA in interphase (top panel) and mitosis (bottom panel) cells, fixed and stained with PCDH7 antibody (red) and fluorescence conjugated streptavidin (green), DAPI in blue. B . Western blotting analysis shows the biotinylation efficiency of BioID constructs. Controls; non-transfected, backbone vector-transfected (BirA*-HA and mycBirA*). Samples; PCDH7:BirA*-HA, PCDH7Δcyt:BirA*-HA, myrPalm:BirA*-HA (an unrelated construct as negative control). Upper blot; Cells supplied with biotin. Lower blot; Cells not supplied with biotin. In the absence of biotin, only a basal level of biotinylation was observed for all cell lines (bottom panel). In the presence of biotin, while non-transfected cells showed a basal level of biotinylation, nonspecific biotinylation was observed for the empty BioID vector-transfected cells (top left panel). The inputs (I) obtained from PCDH7 BioID, truncated PCDH7 BioID and MyrPalm BioID transfected cells demonstrated different biotinylation patterns as expected. The biotinylated proteins were successfully enriched at the elute fractions (E) of each sample after streptavidin affinity pulldown (top right panel). C . Western blotting analysis shows the biotinylation specificity of BioID constructs. Upper blot; Cells supplied with biotin. Lower blot; Cells not supplied with biotin. Both PCDH7 and truncated PCDH7 (PCDH7Δcyt) are enriched in the elute fractions (E) after streptavidin pulldown only in the biotin-supplied cells (top panel). No PCDH7 enrichment is observed in the negative control. The full-length PCDH7 was only present in the elute (E) fraction of PCDH7 BioID transfected cells, but not in the truncated PCDH7 BioID transfected cells. PCDH7 was not detected at all in the elute of MyrPalm BioID sample as expected. (top panel). The absence of actin in the elute fractions confirmed the lack of cytosolic contamination (bottom panel). Scale bars: 10 μm

Article Snippet: PCDH7 sequence was amplified from PCDH7:eGFPN1 and cloned into BioID vector (pcDNA3.1 MCS-BirA* (R118G) Addgene; 36047).

Techniques: Staining, Fluorescence, Western Blot, Construct, Transfection, Plasmid Preparation, Negative Control