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A Schematic representation of lentiviral vectors. Control (CTRL) and XIAP‐targeting shRNAs were expressed in the 3´untranslated region of dsRED in the background of a <t>miR30</t> cassette under control of the Pol II promoter. mTagBFP and eGFP were used to mark each cell population; the control shRNA expressing plasmid was transduced into mTagBFP + , the XIAP KD plasmid into eGFP + cells. B shRNA expression control. Equal expression levels of ds‐RED in the sh‐CTRL/mTagBFP and sh‐XIAP/eGFP subpopulation indicate identical expression levels of control and XIAP shRNA. One representative histogram out of three independent experiments is shown. C Expression of IAP. Western blot analysis of XIAP in NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of three independent experiments is shown. D Western blot analysis of NALM‐6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh‐CTRL. One representative immunoblot out of three independent experiments is shown. E Schematic representation of competitive in vivo assays. Cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into mice (± in vivo treatment); at indicated time points, mice were sacrificed, cells isolated from bone marrow and analyzed. F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. Four days after injection, mice were either treated with PBS ( n = 4), or with Vincristine (VCR, n = 8), 0.3 mg/kg i.v. once a week for 3 consecutive weeks. Seven days after last treatment, mice were sacrificed, and relative proportion of sh‐CTRL and sh‐XIAP subpopulations re‐isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** P ≤ 0.0001 by unpaired Student's t ‐test with Welch's correction. G Strong knockdown of XIAP impairs NALM‐6 growth in vivo . NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice ( n = 8). Thirty‐eight or 46 days after injection, mice were sacrificed, and relative proportion of sh‐CTRL and sh‐XIAP subpopulations re‐isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** P ≤ 0.0001 by unpaired Student's t ‐test. H PARP cleavage in NALM‐6 cells with strong XIAP inhibition. NALM‐6 cells were lentivirally transduced with sh‐CTRL or sh‐XIAP (SFFV). Four days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of three independent experiments is shown; ** P < 0.01 by unpaired Student's t ‐test. I XIAP reconstitution in NALM‐6 cells. NALM‐6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM‐6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of three independent experiments is shown; ** P < 0.01 by unpaired Student's t ‐test. Source data are available online for this figure.
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A Schematic representation of lentiviral vectors. Control (CTRL) and XIAP‐targeting shRNAs were expressed in the 3´untranslated region of dsRED in the background of a miR30 cassette under control of the Pol II promoter. mTagBFP and eGFP were used to mark each cell population; the control shRNA expressing plasmid was transduced into mTagBFP + , the XIAP KD plasmid into eGFP + cells. B shRNA expression control. Equal expression levels of ds‐RED in the sh‐CTRL/mTagBFP and sh‐XIAP/eGFP subpopulation indicate identical expression levels of control and XIAP shRNA. One representative histogram out of three independent experiments is shown. C Expression of IAP. Western blot analysis of XIAP in NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of three independent experiments is shown. D Western blot analysis of NALM‐6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh‐CTRL. One representative immunoblot out of three independent experiments is shown. E Schematic representation of competitive in vivo assays. Cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into mice (± in vivo treatment); at indicated time points, mice were sacrificed, cells isolated from bone marrow and analyzed. F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. Four days after injection, mice were either treated with PBS ( n = 4), or with Vincristine (VCR, n = 8), 0.3 mg/kg i.v. once a week for 3 consecutive weeks. Seven days after last treatment, mice were sacrificed, and relative proportion of sh‐CTRL and sh‐XIAP subpopulations re‐isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** P ≤ 0.0001 by unpaired Student's t ‐test with Welch's correction. G Strong knockdown of XIAP impairs NALM‐6 growth in vivo . NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice ( n = 8). Thirty‐eight or 46 days after injection, mice were sacrificed, and relative proportion of sh‐CTRL and sh‐XIAP subpopulations re‐isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** P ≤ 0.0001 by unpaired Student's t ‐test. H PARP cleavage in NALM‐6 cells with strong XIAP inhibition. NALM‐6 cells were lentivirally transduced with sh‐CTRL or sh‐XIAP (SFFV). Four days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of three independent experiments is shown; ** P < 0.01 by unpaired Student's t ‐test. I XIAP reconstitution in NALM‐6 cells. NALM‐6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM‐6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of three independent experiments is shown; ** P < 0.01 by unpaired Student's t ‐test. Source data are available online for this figure.

Journal: EMBO Molecular Medicine

Article Title: X‐linked inhibitor of apoptosis protein represents a promising therapeutic target for relapsed/refractory ALL

doi: 10.15252/emmm.202114557

Figure Lengend Snippet: A Schematic representation of lentiviral vectors. Control (CTRL) and XIAP‐targeting shRNAs were expressed in the 3´untranslated region of dsRED in the background of a miR30 cassette under control of the Pol II promoter. mTagBFP and eGFP were used to mark each cell population; the control shRNA expressing plasmid was transduced into mTagBFP + , the XIAP KD plasmid into eGFP + cells. B shRNA expression control. Equal expression levels of ds‐RED in the sh‐CTRL/mTagBFP and sh‐XIAP/eGFP subpopulation indicate identical expression levels of control and XIAP shRNA. One representative histogram out of three independent experiments is shown. C Expression of IAP. Western blot analysis of XIAP in NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP under the SFFV promoter; GAPDH was used as loading control. One representative immunoblot out of three independent experiments is shown. D Western blot analysis of NALM‐6 cells with moderate (69%, EF1α promoter) or strong (92%, SFFV promoter) KD of XIAP; GAPDH was used as loading control. The XIAP KD levels (% KD) were quantified relative to sh‐CTRL. One representative immunoblot out of three independent experiments is shown. E Schematic representation of competitive in vivo assays. Cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into mice (± in vivo treatment); at indicated time points, mice were sacrificed, cells isolated from bone marrow and analyzed. F Moderate knockdown of XIAP sensitizes towards chemotherapy. NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice. Four days after injection, mice were either treated with PBS ( n = 4), or with Vincristine (VCR, n = 8), 0.3 mg/kg i.v. once a week for 3 consecutive weeks. Seven days after last treatment, mice were sacrificed, and relative proportion of sh‐CTRL and sh‐XIAP subpopulations re‐isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** P ≤ 0.0001 by unpaired Student's t ‐test with Welch's correction. G Strong knockdown of XIAP impairs NALM‐6 growth in vivo . NALM‐6 cells expressing sh‐CTRL/mTagBFP or sh‐XIAP/eGFP were mixed at a 1:1 ratio and injected into groups of mice ( n = 8). Thirty‐eight or 46 days after injection, mice were sacrificed, and relative proportion of sh‐CTRL and sh‐XIAP subpopulations re‐isolated from the BM was quantified by flow cytometry. Data are depicted as mean ± SEM of all mice analyzed with **** P ≤ 0.0001 by unpaired Student's t ‐test. H PARP cleavage in NALM‐6 cells with strong XIAP inhibition. NALM‐6 cells were lentivirally transduced with sh‐CTRL or sh‐XIAP (SFFV). Four days later, cells were lysed and relative cleavage level of PARP (%) was analyzed by protein immunoassay (Simple Western). Mean ± SD of three independent experiments is shown; ** P < 0.01 by unpaired Student's t ‐test. I XIAP reconstitution in NALM‐6 cells. NALM‐6 cells were transduced with shCTRL, shXIAP.1 alone or shXIAP.1 together with the XIAP overexpression vector. At day 0, transduced cells were mixed in a 1:1 ratio with parental (untransduced) NALM‐6 cells. Distribution of the fluorochrome marker was used as a readout to determine growth variations of the respective subpopulation over time. Mean ± SD of three independent experiments is shown; ** P < 0.01 by unpaired Student's t ‐test. Source data are available online for this figure.

Article Snippet: XIAP over‐expression: For over‐expression of XIAP, the full‐length CDS of XIAP (gBlock, IDT) was cloned into the pCDH‐vector backbone (System Biosciences) using EcoRI and BamHI and linked by a T2A fragment to mTagBFP, used as marker. shRNA expression system: The construct encoding for dsRED and the miR30 KD cassette was generated by cloning the PCR‐amplified dsRED‐miR30 fragment (TRMPVIR vector; Addgene #27994) into the pCDH‐EF1α‐MCS‐T2A‐copGFP vector (SBI, CD521A‐1) using primers carrying MfeI and SalI as 5′ and 3′ restriction sites.

Techniques: shRNA, Expressing, Plasmid Preparation, Western Blot, In Vivo, Injection, Isolation, Flow Cytometry, Inhibition, Transduction, Over Expression, Marker