Journal: Scientific Reports

Article Title: miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma

doi: 10.1038/s41598-022-07468-x

Figure Lengend Snippet: Expression levels of miR-96-5p and PTEN in patient samples. ( a ) The fold changes detected by microarray are denoted in a bar graph. Real-time RT-PCR validation of the miR-96-5p expression levels in the same patient samples used in the microarray. ( b ) Western blot (left) and quantification (right) of PTEN proteins in tumor tissues from sunitinib-resistant (R1, R2, and R3) and sunitinib-sensitive (S1, S2, and S3) CCRCC patients. Samples were collected from two different regions of tumors. GAPDH was used as a loading control. Data are presented as mean ± s.e.m. (n = 3). Significant results are presented as * P < 0.01. ( c ) The levels of miR-96-5p determined by qRT-PCR in fresh frozen tissue were compared with the corresponding PTEN proteins determined by Western blotting.

Article Snippet: To manipulate the expression of miR-96-5p and PTEN in cells, we purchased lentiviral miRNA-96 (pLV-hsa-mir-96 plasmid, mir-p069), miR-96-5p inhibitor (pLV-hsa-miR-96-5p locker plasmid, mir-hp0066), mir-control lentivirus (mir-LV000) from Biosettia (San Diego, CA), and PTEN lentivirus (#51304) from Addgene (Watertown, MA).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Biomarker Discovery, Western Blot, Control

Journal: Scientific Reports

Article Title: miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma

doi: 10.1038/s41598-022-07468-x

Figure Lengend Snippet: Associations of higher miR-96 and lower PTEN expression levels with a poor prognosis in CCRCC patients. ( a ) The correlation between miR-96 and PTEN expression in TCGA CCRCC patients (KIRC) was estimated using Pearson’s correlation test. ( b–d ) Kaplan–Meier curves of overall survival (OS) and progression-free survival (PFS) for miR-96 and PTEN expression in CCRCC patients. miR-96 and PTEN expression was dichotomized into high- and low-expression categories (b, c). CCRCC patients in KIRC were dichotomized based on the expression of miR-96 and PTEN and patients with higher expression levels of both miR-96 and PTEN or lower expression levels of both miR-96 and PTEN were identified for analysis ( d ).

Article Snippet: To manipulate the expression of miR-96-5p and PTEN in cells, we purchased lentiviral miRNA-96 (pLV-hsa-mir-96 plasmid, mir-p069), miR-96-5p inhibitor (pLV-hsa-miR-96-5p locker plasmid, mir-hp0066), mir-control lentivirus (mir-LV000) from Biosettia (San Diego, CA), and PTEN lentivirus (#51304) from Addgene (Watertown, MA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma

doi: 10.1038/s41598-022-07468-x

Figure Lengend Snippet: Modulation of sunitinib sensitivity by the miR-96-5p/PTEN axis in CCRCC patient-derived xenografts (PDXs). ( a ) H&E staining images of tumors from original sunitinib-resistant and sunitinib-sensitive patients and their corresponding PDXs (scale bar = 50 μm). ( b , c ) PDX models were divided into two treatment groups: vehicle and 40 mg/kg sunitinib. Body weight and tumor volumes were assessed for changes over time (Vehicle n = 4; sunitinib n = 4, mean ± s.e.m., * P < 0.05, *** P < 0.001, one-way ANOVA). ( d , e ) Comparison of the expression of PTEN between sunitinib-resistant (d) and sunitinib-sensitive PDX (e) at 0 and 28 days; GAPDH, internal control. ( f, g ) PTEN (f) and miR-96-5p (g) expression from tissue of sunitinib-resistant PDX, compared with the sunitinib-sensitive counterparts (mean ± s.e.m., * P < 0.05, *** P < 0.001, one-way ANOVA, n = 3).

Article Snippet: To manipulate the expression of miR-96-5p and PTEN in cells, we purchased lentiviral miRNA-96 (pLV-hsa-mir-96 plasmid, mir-p069), miR-96-5p inhibitor (pLV-hsa-miR-96-5p locker plasmid, mir-hp0066), mir-control lentivirus (mir-LV000) from Biosettia (San Diego, CA), and PTEN lentivirus (#51304) from Addgene (Watertown, MA).

Techniques: Derivative Assay, Staining, Comparison, Expressing, Control

Journal: Scientific Reports

Article Title: miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma

doi: 10.1038/s41598-022-07468-x

Figure Lengend Snippet: miR-96-5p directly represses PTEN expression. ( a ) Schematic representation of the predicted miR-96-5p target site sequence within the 3′ UTR of PTEN . Six nucleotides within the target site are complementary to the seed sequence of miR-96-5p. ( b ) Luciferase reporter assay. 293 T cells were co-transfected with luciferase reporters carrying either the wild-type PTEN 3′ UTR (PTEN WT) or the mutagenized PTEN 3′ UTR (PTEN Mut), as well as 50 nM negative control mimic (miR-Control) or miR-96-5p mimic. Data are presented as mean ± s.e.m. (n = 3). Significant results are presented as * P < 0.05.

Article Snippet: To manipulate the expression of miR-96-5p and PTEN in cells, we purchased lentiviral miRNA-96 (pLV-hsa-mir-96 plasmid, mir-p069), miR-96-5p inhibitor (pLV-hsa-miR-96-5p locker plasmid, mir-hp0066), mir-control lentivirus (mir-LV000) from Biosettia (San Diego, CA), and PTEN lentivirus (#51304) from Addgene (Watertown, MA).

Techniques: Expressing, Sequencing, Luciferase, Reporter Assay, Transfection, Negative Control, Control

Journal: Scientific Reports

Article Title: miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma

doi: 10.1038/s41598-022-07468-x

Figure Lengend Snippet: Downregulation of PTEN and upregulation of miR-96-5p are associated with sunitinib resistance in CCRCC cell lines. ( a ) Western blot (left) and quantification (right) of PTEN proteins in A498, TK10, and ACHN cells. GAPDH was used as a loading control. ( b , c ) The levels of PTEN mRNA ( b ) and miR-96-5p ( c ) were analyzed by qRT-PCR in A498, TK10, and ACHN cells. All data were normalized with GAPDH for mRNA or RNU6B for miRNA. ( d , e ) Dose-dependent effect of sunitinib on the viability ( d ) and death ( e ) of A498, TK10, and ACHN cells. Data are presented as mean ± s.e.m. (n = 3). Significant results are presented as * P < 0.05.

Article Snippet: To manipulate the expression of miR-96-5p and PTEN in cells, we purchased lentiviral miRNA-96 (pLV-hsa-mir-96 plasmid, mir-p069), miR-96-5p inhibitor (pLV-hsa-miR-96-5p locker plasmid, mir-hp0066), mir-control lentivirus (mir-LV000) from Biosettia (San Diego, CA), and PTEN lentivirus (#51304) from Addgene (Watertown, MA).

Techniques: Western Blot, Control, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: miR-96-5p targets PTEN to mediate sunitinib resistance in clear cell renal cell carcinoma

doi: 10.1038/s41598-022-07468-x

Figure Lengend Snippet: miR-96-5p-mediated downregulation of PTEN regulates sensitivity to sunitinib in CCRCC cell lines. ( a ) The expression level of PTEN was downregulated after overexpression of miR-96-5p mimic (miR-96-5p) in the A498 cell line. ( b , c ) PTEN downregulation affects the viability ( b ) and death ( c ) of A498 cell lines upon treatment with 20 μM sunitinib. ( d ) Representative images of Matrigel invasion assay of NC and miR-96-5p transfected A498 cells under 10 uM sunitinib treatment (scale bars, 100 μm). The number of invaded cells was quantified and statistically analyzed (*** P < 0.001). Error bars indicate the standard error of mean of four experiments. ( e ) The expression level of PTEN was upregulated after knockdown of miR-96-5p in the ACHN cell line. ( f , g ) The effects of PTEN upregulation on the viability ( f ) and death ( g ) upon 20 μM sunitinib treatment in ACHN. ( h ) The expression level of PTEN was verified after overexpression of PTEN in the miR-96-5p-expressing A498 cell line. ( i ) Upregulation of PTEN significantly sensitized cells to sunitinib treatment in miR-96-5p-expressing A498. Data are presented as mean ± s.e.m. (n = 3). Significant results are presented as * P < 0.05, ** P < 0.01, *** P < 0.001. NC: negative control.

Article Snippet: To manipulate the expression of miR-96-5p and PTEN in cells, we purchased lentiviral miRNA-96 (pLV-hsa-mir-96 plasmid, mir-p069), miR-96-5p inhibitor (pLV-hsa-miR-96-5p locker plasmid, mir-hp0066), mir-control lentivirus (mir-LV000) from Biosettia (San Diego, CA), and PTEN lentivirus (#51304) from Addgene (Watertown, MA).

Techniques: Expressing, Over Expression, Invasion Assay, Transfection, Knockdown, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

doi: 10.1073/pnas.2312499121

Figure Lengend Snippet: Fig. 1. Establishment of a protein switch for human hematopoietic stem and progenitor cell expansion using the MLL-ENL (ME) fusion gene and the Shield1 destabilization domain (DD). (A) Illustration of the procedure by which functional monocytes were derived from transduced and expanded human bone marrow CD34+ cells. (B) Gene ontology (GO) analysis of ME target genes (36) categorized by function (33–35). (C) A depiction of the retroviral vector containing ME fused to a DD destabilization domain (DD-ME). (D) Transduced CD34+ cells cultured in the presence of IL-3, IL-6, SCF, TPO, Flt3-L, and GM-CSF with or without Shield1 and analyzed for eGFP expression. (E) FSC/SSC profiles of DD-ME-expressing cells in the presence of Shield1 at day 44 posttransduction. (F) DD-ME signal intensity in the presence of Shield1 (+S) and after withdrawal of Shield1 (for 7 d) via intracellular staining. The data are presented as the mean plus the SD of three biological replicates. (G) Western blot analysis of HOXA9 expression using whole cell lysates of DD-ME-expressing cells before and after Shield1 removal. (H) Outgrowth of transduced, eGFP-expressing cells was determined via flow cytometry. #, terminally differentiated; FSC, forward scatter; SSC, side scatter.

Article Snippet: The sequences of HOXB8 (synthesized by Geneart) and HOXA9 (Addgene #97041) were cloned into the same retroviral backbone.

Techniques: Functional Assay, Derivative Assay, Retroviral, Plasmid Preparation, Cell Culture, Expressing, Staining, Western Blot, Flow Cytometry