sirna sequences  (Thermo Fisher)

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A) Timeline of shRNA infection and analysis of mRNA and protein expression. B) Western blot shows expression of eIF3B protein in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. C) mRNA expression of Eif3b in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05. D) Flow cytometry results show percent of cells labeled with CD61-PE antibody compared to isotype control at a range of differentiation timepoints following stable infection with the indicated shRNAs. Error bars show standard deviation for all replicates at each time point. E) mRNA expression of Itgb3 in Ocy454 cells at differentiation day 10 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05. F) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Itgb3-targeting shRNA. G) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Itgb3-targeting shRNA.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: shRNA, Infection, Expressing, Western Blot, Flow Cytometry, Labeling, Control, Standard Deviation, Fluorescence, Stable Transfection

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A-D) mRNA expression of targeted genes at differentiation day 7 in Ocy454 cells stably expressing the indicated shRNAs. Line shows median of three biologic replicates. *p<0.05 compared to FLuc. E-H) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and the indicated targeting shRNA for one representative sample. I) Summary of flow cytometry results showing median fluorescence intensity (MFI) of CD61-PE at differentiation day 7 in cells stably expressing the indicated shRNAs. Error bars show standard deviation of all replicates. *p<0.05 compared to FLuc.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Stable Transfection, Fluorescence, Control, shRNA, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A) mRNA expression of Astn1 in Ocy454 cells at differentiation day 7 following stable infection with the indicated shRNAs. Line shows median of three biologic replicates. B) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and Astn1-targeting shRNA for one representative sample. C) Summary of flow cytometry results showing median fluorescence intensity (MFI) of CD61-PE at differentiation day 7 in cells stably expressing the indicated shRNAs. Error bars show standard deviation of all replicates. *p<0.05 compared to FLuc. D-L) Histograms show intensity of CD61-PE fluorescence compared to isotype control at differentiation day 7 for cells stably expressing FLuc-targeting shRNA and the indicated targeting shRNA for one representative sample.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Infection, Fluorescence, Control, Stable Transfection, shRNA, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

doi: 10.1101/2025.10.23.683957

Figure Lengend Snippet: A-G) mRNA expression of osteocyte maturity genes at differentiation day 10 in Ocy454 cells stably expressing the indicated shRNAs. *p<0.05 compared to FLuc. H) Alpha-tubulin immunofluorescence or isotype control immunofluorescence (red) and DAPI (blue) in Ocy454 cells without shRNA infection. Scale bars = 20 μm. I) Ocy454 cells labeled with phalloidin (green) and DAPI (blue) following stable expression of the indicated shRNAs. Scale bars = 20 μm. J-K) Cell Profiler measurements of form factor and perimeter. Each plotted point represents metrics of one cell. Lines designate the median and interquartile range of n=47-158 cells. *p<0.05.

Article Snippet: CRISPRi sgRNA sequences and shRNA sequences were designed using Broad Institute tools CRISPick ( https://portals.broadinstitute.org/gppx/crispick/public ) and TRC shRNA Design ( https://portals.broadinstitute.org/gpp/public/seq/search ).

Techniques: Expressing, Stable Transfection, Immunofluorescence, Control, shRNA, Infection, Labeling

Journal: Biomedical Reports

Article Title: Silencing of AEBP1 inhibits proliferation and promotes apoptosis via the AKT signaling pathway in osteosarcoma

doi: 10.3892/br.2025.2006

Figure Lengend Snippet: AEBP1 is a novel target for osteosarcoma and is markedly expressed in sarcoma and bone cancer cell lines. (A) Upregulated genes in 3 datasets ( GSE16088 , GSE197158 and GSE63631 ). (B) Downregulated genes in 3 datasets ( GSE16088 , GSE197158 and GSE63631 ). (C) The fold change of 25 upregulated genes of 3 datasets ( GSE16088 , GSE197158 and GSE63631 ). (D) Expression of AEBP1 across TCGA tumors. The black arrow indicates sarcoma. (E) Expression of AEBP1 in 33 types of cancer cell lines. The black arrow indicates bone cancer cell lines. (F) Expression of AEBP1 in bone cancer cell lines. Adipocyte enhancer-binding protein 1; TCGA, The Cancer Genome Atlas; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain lower grade glioma; OV, ovarian serous cystadenocarcinoma; MESO, mesothelioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PAAD, pancreatic adenocarcinoma; PRAD, prostate adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; LAML, acute myeloid leukemia; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; STAD, stomach adenocarcinoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Article Snippet: siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Expressing, Binding Assay

Journal: Biomedical Reports

Article Title: Silencing of AEBP1 inhibits proliferation and promotes apoptosis via the AKT signaling pathway in osteosarcoma

doi: 10.3892/br.2025.2006

Figure Lengend Snippet: mRNA and protein level determination of AEBP1 in OS cell lines after AEBP1 knockdown. (A and B) The mRNA level of AEBP1 was examined by reverse transcription-quantitative PCR in U2OS and HOS cells, and β-actin was used as an internal control. (C and D) The protein level of AEBP1 was examined by western blot analysis, and β-actin was used as an internal control. * P<0.05 and ** P<0.01. AEBP1, adipocyte enhancer-binding protein 1; OS, osteosarcoma; siNC, negative control group, siAEBP1, AEBP1-knockdown group.

Article Snippet: siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Western Blot, Binding Assay, Negative Control

Journal: Biomedical Reports

Article Title: Silencing of AEBP1 inhibits proliferation and promotes apoptosis via the AKT signaling pathway in osteosarcoma

doi: 10.3892/br.2025.2006

Figure Lengend Snippet: Effect of silencing of AEBP1 on OS cell proliferation. An MTT assay was used to determine the proliferation of siAEBP1 and the negative control in (A) U2OS and (B) HOS cells for 5 days. Cell clones of siAEBP1-treated cells and negative control cells in (C) U2OS and (D) HOS cells were observed by clone formation assay. An EdU assay was performed in (E) U2OS and (G) HOS cells. Analysis of the results of the EdU assay in (F) U2OS and (H) in HOS cells. Scale bar, 100 µm. * P<0.05 and ** P<0.01. AEBP1, adipocyte enhancer-binding protein 1; OS, osteosarcoma; siAEBP1, AEBP1-knockdown group; siNC, negative control group; EdU, 5-ethynyl-2'-deoxyuridine.

Article Snippet: siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: MTT Assay, Negative Control, Clone Assay, Tube Formation Assay, EdU Assay, Binding Assay, Knockdown

Journal: Biomedical Reports

Article Title: Silencing of AEBP1 inhibits proliferation and promotes apoptosis via the AKT signaling pathway in osteosarcoma

doi: 10.3892/br.2025.2006

Figure Lengend Snippet: Effect of AEBP1-knockdown on the OS cell cycle. The percentage of siAEBP1-treated cells and negative control cells in the different phases of the cell cycle was determined and analyzed by flow cytometry in (A and B) U2OS and (C and D) HOS cells. ** P<0.01. AEBP1, adipocyte enhancer-binding protein 1; OS, osteosarcoma; siAEBP1, AEBP1-knockdown group; siNC, negative control group.

Article Snippet: siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Knockdown, Negative Control, Flow Cytometry, Binding Assay

Journal: Biomedical Reports

Article Title: Silencing of AEBP1 inhibits proliferation and promotes apoptosis via the AKT signaling pathway in osteosarcoma

doi: 10.3892/br.2025.2006

Figure Lengend Snippet: AEBP1 knockdown promotes the apoptosis of OS cells. (A-D) A TUNEL assay revealed that apoptosis was increased after AEBP1 knockdown in (A and B) U2OS cells and (C and D) HOS cells. (E-H) Flow cytometry demonstrated that apoptosis was increased after AEBP1 knockdown in (E and F) U2OS cells and (G and H) HOS cells. The white arrows indicate the TUNEL-positive cells. (I) The protein expression level of Bcl-2 was downregulated and that of Bax was upregulated after AEBP1 knockdown in U2OS cells. (J) Statistical analysis of I. Scale bar, 100 µm. ** P<0.01. AEBP1, adipocyte enhancer-binding protein 1; OS, osteosarcoma; siAEBP1, AEBP1-knockdown group; siNC, negative control group; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Article Snippet: siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Knockdown, TUNEL Assay, Flow Cytometry, Expressing, Binding Assay, Negative Control, End Labeling

Journal: Biomedical Reports

Article Title: Silencing of AEBP1 inhibits proliferation and promotes apoptosis via the AKT signaling pathway in osteosarcoma

doi: 10.3892/br.2025.2006

Figure Lengend Snippet: Regulatory mechanism of AEBP1 in the proliferation of OS. Western blot analysis was carried out to detect the expression of cell cycle checkpoint proteins in U2OS cells. (A) The protein expression level of p-AKT (the phosphate site was Thr308) was downregulated while the expression level of total AKT was not altered after AEBP1 knockdown. In addition, the protein expression level of cyclin D1 was decreased after AEBP1 knockdown. (B) The protein expression level of p-AKT was upregulated after the addition of SC79, a stimulator of the AKT signaling pathway. SC79 could rescue the inhibitory effect induced by AEBP1 knockdown. (C) Statistical analysis of A. ** P<0.01 vs. the corresponding group indicated. (D) The statistical analysis of B. * P<0.05 and ** P<0.01 vs. the corresponding group indicated. AEBP1, adipocyte enhancer-binding protein 1; OS, osteosarcoma; siAEBP1, AEBP1-knockdown group; siNC, negative control group; p-, phosphorylated.

Article Snippet: siRNAs targeting the AEBP1 sequence (sense, 5'-GGUGGUGGCUCGUUUCAUC-3' and antisense, 5'-GAUGAAACGAGCCACCACC-3') and non-silencing sequences (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-GUGACACGUUCGGAGAATT-3') were procured from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was carried out using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.) following the manufacturer's instructions.

Techniques: Western Blot, Expressing, Knockdown, Binding Assay, Negative Control