shrna nontarget control  (Genechem)

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Preproghrelin, and thus obestatin, rescues autophagy flux in DMD cells. (A) Left panel : Immunofluorescence detection of MHC in human DMD cell models, DMD6 and DMD14, under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM) 72 h post stimulation. Right panel : Immunoblot analysis of p62, LC3, and Cathepsin L, in human DMD6 and DMD14 myotubes at the 72‐h point under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM). (B) DMD myotubes were transfected with siRNA targeting preproghrelin or siRNA‐scramble prior to induction of myogenesis for 96 h. The expression of expansion (p21, Ki‐67, fast‐MHC, slow‐MHC, myogenin), autophagy (p62, LC3, Beclin1, Bnip3, VPS34, AMBRA1) and mitochondrial homeostasis (DRP1, Parkin, TFAM, Mfn2) markers were evaluated by immunoblot analysis ( n = 3). Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM obtained from intensity scans of independent experiments. * p < 0.05. (C) Immunoblot analysis of p62, LC3, Cathepsin L, pAMPK(T172), AMPK, pmTOR(S2448), mTOR, and MHC in human DMD myotubes at the 24‐h point under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM). Cells were treated with the autophagy inhibitor chloroquine (20 µM) 6 h before collection. Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM obtained from intensity scans of three independent experiments *,# p < 0.05. (D) Left panel : Immunofluorescence detection of microtubule‐associated protein 1 light chain 3 isoform B (LC3B) in human DMD cell model under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM) at the 24 h point after stimulation. Cells were treated with the autophagy inhibitor chloroquine. Right panel : the changes in LC3 puncta/area from DMD myotube cells are shown. Data were expressed as mean ± SEM ( n = 3 per group). * p < 0.05. (E) Immunoblot analysis of DRP1, TFAM, Mfn2, and Pink1 in human DMD myotubes at the 24 h point under obestatin treatment (10 nM). Cells were treated with chloroquine before collection. Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM ( n = 3 per group; *,# p < 0.05). (F) Quantification of ROS levels in DMD myotube cells using CellROX TM fluorescent dye. DMD myotubes were treated with obestatin (10 nM), Dexa (1 µM), or vehicle (control) for 24 h. Results are represented as variation of MFI between control and obestatin‐ or Dexa‐treated cells. Data were expressed as mean ± SEM ( n = 3 per group). * p < 0.05.

Article Snippet: An ON‐TARGETplus nontargeting siRNA (Dharmacon) or control nontargeting shRNA plasmid (sc‐108060; Santa Cruz Biotechnology) was used as a control for siRNA or shRNA experiments.

Techniques: Immunofluorescence, Control, Western Blot, Transfection, Expressing

Journal: Cancer Research Communications

Article Title: OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

doi: 10.1158/2767-9764.CRC-25-0696

Figure Lengend Snippet: Silencing of OPG in 67NR-primed CD19 + B cells abrogates their regulatory activity and restores 4T1 osteolytic and metastatic phenotypes in vivo . A, Experimental scheme: CD19 + B cells were isolated from the iliac BM of BALB/c mice bearing 67NR tumors on day 11 after implantation. Cells were subjected to gene silencing using lentiviral vectors carrying shRNA targeting Tnfrsf11b (OPG; B 67NR sh OPG) or a nontargeting scrambled control (B 67NR sh scr). Naive mice were used as experimental controls (Nv). B, OPG levels in culture supernatants from CD19 + B cells were assessed by ELISA. C, Experimental setup for in vivo analysis: OPG-silenced (sh OPG) or control-transduced (sh scr) CD19 + B cells were adoptively transferred together with CD3 + T cells from 4T1 tumor-bearing mice into immunocompetent BALB/c mice implanted with 1 × 10 5 4T1 tumor cells. D, Primary tumor growth was monitored over time. E, Representative images of tumor-bearing mice after adoptive B cell transfer. Photographs were taken at the experimental endpoint to illustrate macroscopic differences in tumor burden among groups. F, Quantification of RANKL levels by ELISA in supernatants of in vitro restimulated LNs and BM cells. G, Metastatic burden in draining LNs and BM was assessed by clonogenic assays. Data are presented as mean ± SD from two independent experiments ( n = 5 mice/group). Statistical comparisons between more than two groups were performed using one-way ANOVA followed by Tukey post hoc test for pairwise comparisons when data were normally distributed. Statistical significance was considered when P < 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To silence OPG expression in BM–derived CD19 + B cells from 67NR tumor–bearing BALB/c mice (day 11), cells were transfected with a specific murine short hairpin (sh) RNA (shRNA) plasmid targeting OPG [sc-40153-SH, Santa Cruz Biotechnology] or with a nontargeting control shRNA plasmid (sc-108060).

Techniques: Activity Assay, In Vivo, Isolation, shRNA, Control, Enzyme-linked Immunosorbent Assay, In Vitro