oip5 shrna lentiviral plasmid  (Santa Cruz Biotechnology)

Journal: Molecular Oncology

Article Title: The Sp1/FOXC1/HOTTIP/LATS2/YAP/β‐catenin cascade promotes malignant and metastatic progression of osteosarcoma

doi: 10.1002/1878-0261.12760

Figure Lengend Snippet: FOXC1 played an essential role sustaining multiple malignant phenotypes of OS cells, both in vitro and in vivo . (A) Upon transfecting SW1353 (left panel) and 143B (right panel) cells with shFOXC1‐1, shFOXC1‐2, or control shRNA (shNC), the expression of FOXC1 protein was determined by western blot. The viability, long‐term proliferation, migration, and invasion of indicated cells at indicated time points were determined by MTT assay (B), colony‐forming assay (C), wound‐healing assay (D), and Transwell invasion assay (E), respectively, and compared between shNC and shFOXC1 cells. (F–H) shNC, shFOXC1‐1, or shFOXC1‐2 SW1353 and 143B cells were injected subcutaneously to establish xenograft tumors. The pictures of isolated xenografts after 28 days were shown in F, the growth curve in G, and the weights in H. (I–K) shNC, shFOXC1‐1, or shFOXC1‐2 SW1353 and 143B cells were injected intravenously to establish lung metastasis. The pictures of isolated lung tissues after 36 days were shown in I and microscopic metastasis detected upon HE staining shown in J and K. Error bars represent standard deviation. Student's t ‐test with four biological independent replicates was used to determine statistical significance; * P < 0.05, ** P < 0.01, *** P < 0.001, when compared to shNC cells. Scale bars: 1000 μm (D), 250 μm (E), and 100 μm (J).

Article Snippet: For HOTTIP shRNA (shHOTTIP), we designed four shRNA sequences shHOTTIP #1: 5′‐AAA AGC ATC TCA AAT TAA GCT TTG CCT CGA GGC AAA GCT TAA TTT GAG ATG C‐3′; shHOTTIP #2: 5′‐AAA AGG GAC TGA ATT CTT CGA GAT TTC TCG AGA AAT CTC AAG AAT TCA GTC CC‐3′; shHOTTIP #3: 5′‐AAA AGG TGC ACC TTA TTG ATC AAA TCT CGA GAT TTG AAT AAG GTG CAC C‐3′; shHOTTIP #4: 5′‐AAA AGC AGC CAA CAA ACT GAC TTG CCT CGA GGC AAG TCA GTT TGT TGG CTG C‐3′), cloned them into OIP5 shRNA lentiviral plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and generated lentiviral particles.

Techniques: In Vitro, In Vivo, Control, shRNA, Expressing, Western Blot, Migration, MTT Assay, Wound Healing Assay, Transwell Invasion Assay, Injection, Isolation, Staining, Standard Deviation

Journal: Molecular Oncology

Article Title: The Sp1/FOXC1/HOTTIP/LATS2/YAP/β‐catenin cascade promotes malignant and metastatic progression of osteosarcoma

doi: 10.1002/1878-0261.12760

Figure Lengend Snippet: HOTTIP recruited EZH1 and LSD1 to LATS2 promoter, enhanced promoter methylation, and suppressed the expression of LATS2. (A) The percentage distribution of HOTTIP in the nucleus and cytoplasm was examined by qRT‐PCR. GAPDH and U1 were detected as the marker for cytoplasm and nucleus, respectively. (B) EZH2 and LSD1 were identified as the potential interacting partner of HOTTIP using rpiseq software. (C) The interaction between HOTTIP and EZH2 or LSD1 was examined by RIP assay and presented as fold enrichment values relative to the amount of HOTTIP bound to IgG. (D, E) The expression of LATS2 in shHOTTIP‐1 and shHOTTIP‐2 vs. shNC cells was determined on the mRNA level by qRT‐PCR (D) and western blot (E), respectively. The levels of β‐catenin, YAP1, and p‐YAP1 were also detected by western blot (E). (F–H) SW1353 or 143B cells were transfected with siRNA targeting EZH2 (si‐EZH2‐1 and si‐EZH2‐2), LSD1 (si‐LSD1‐ and si‐LSD1‐2), or control (si‐NC). The expression levels of EZH2 (F), LSD1 (G), and LATS2 (H) were detected by qRT‐PCR. (I) The occupancy of EZH2, H3K27me3, LSD1, and H3K4me2 on LATS2 promoter in indicated cells was determined by ChIP analysis and presented as a fold value relative to the amount of IgG bound to the promoter. Error bars represent standard deviation. Student's t ‐test with three biological independent replicates was used to determine statistical significance; * P < 0.05, ** P < 0.01.

Article Snippet: For HOTTIP shRNA (shHOTTIP), we designed four shRNA sequences shHOTTIP #1: 5′‐AAA AGC ATC TCA AAT TAA GCT TTG CCT CGA GGC AAA GCT TAA TTT GAG ATG C‐3′; shHOTTIP #2: 5′‐AAA AGG GAC TGA ATT CTT CGA GAT TTC TCG AGA AAT CTC AAG AAT TCA GTC CC‐3′; shHOTTIP #3: 5′‐AAA AGG TGC ACC TTA TTG ATC AAA TCT CGA GAT TTG AAT AAG GTG CAC C‐3′; shHOTTIP #4: 5′‐AAA AGC AGC CAA CAA ACT GAC TTG CCT CGA GGC AAG TCA GTT TGT TGG CTG C‐3′), cloned them into OIP5 shRNA lentiviral plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and generated lentiviral particles.

Techniques: Methylation, Expressing, Quantitative RT-PCR, Marker, Software, Western Blot, Transfection, Control, Standard Deviation