Journal: Experimental & Molecular Medicine

Article Title: Lnc Tmem235 promotes repair of early steroid-induced osteonecrosis of the femoral head by inhibiting hypoxia-induced apoptosis of BMSCs

doi: 10.1038/s12276-022-00875-0

Figure Lengend Snippet: a , b JC-1 was used to detect the mitochondrial membrane potential ( n = 5); bone-marrow mesenchymal stem cells (BMSCs), 5,5′,6,6′ - tetrachloro − 1,1′,3,3′ - tetraethyl - imidacarbocyanine iodide (JC-1), and 4′,6 - diamidino - 2 - phenylindole (DAPI); c ATP content ( n = 6); adenosine triphosphate (ATP); d , e The content of ROS was detected by DCFH-DA (n = 5); reactive oxygen species (ROS) and 2′,7′ - dichlorofluorescin diacetate (DCFH-DA); f , g Apoptosis was detected by Annexin V/PI ( n = 5); fluorescein isothiocyanate (FITC) and propidium iodide (PI); h – k The expression levels of Bcl-2, Bax, and CASP3 were analyzed by western blotting ( n = 3); B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3 (CASP-3); l Cluster analysis of lncRNAs ( n = 3); m Cluster analysis of mRNAs ( n = 3); n Volcano map of lncRNA expression profile ( n = 3); fold change (FC), the base of logFC is 2; o . Volcano map of mRNA expression profile ( n = 3); the base of logFC is 2; p GO analysis ( n = 3); Gene Ontology (GO); q KEGG analysis ( n = 3); Kyoto Encyclopedia of Genes and Genomes (KEGG); r Coexpression analysis and gene-position relationships were used to screen candidate lncRNAs; s The expression levels of Lnc Tmem235 and Lnc LOC102553514 were verified by qPCR ( n = 6); real-time quantitative polymerase chain reaction (qPCR); t Apoptotic changes occurred as a function of hypoxia ( n = 5); u Lnc Tmem235 changed with the degree of hypoxia ( n = 6); v Lnc LOC102553514 changed with the degree of hypoxia. In ( b , c , e , g , i – k , s – v ), the data are normally distributed, and the variance is homogeneous. Data are presented as the means ± standard deviations (SDs). In ( b , c , e , g , i – k , s ), statistical significance was calculated by Student’s t tests; in ( t – v ), statistical significance was calculated by one-way ANOVA with Tukey’s post- hoc tests; * P < 0.05.

Article Snippet: The following antibodies were used for primary antibody reactions: rabbit anti-BIRC5 (1:3000; ab469; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:500; bs-0032R; Bioss, Beijing, China), rabbit anti-Caspase-3 (1:500; ab4051; Abcam), rabbit anti-Bax (1:1500; bs-28034R; Bioss), mouse anti-β-actin (1:2500; ab8226; Abcam), rabbit anti-CASP-3/Cleaved CASP-3 (1:500; ab13847; Abcam), rabbit anti-CASP-9/Cleaved CASP-9 (1:1000; ab25758; Abcam), rabbit anti-PARP1/Cleaved PARP1 (1:10000; ab191217; Abcam), rabbit anti-Argonaute 2 (1:800; ab5072; Abcam), mouse anti-OCN (10 µg/mL; ab13420; Abcam), rabbit anti-OPG (1:100; bs-20624R; Bioss), rabbit anti-Runx2 (1:200; AV36678; Sigma-Aldrich), and rabbit anti-OPN (1:200; ab8448; Abcam).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Experimental & Molecular Medicine

Article Title: Lnc Tmem235 promotes repair of early steroid-induced osteonecrosis of the femoral head by inhibiting hypoxia-induced apoptosis of BMSCs

doi: 10.1038/s12276-022-00875-0

Figure Lengend Snippet: a Localization of Lnc Tmem235 in the genome; b Western blot detection of myc fusion protein to evaluate the coding ability of Lnc Tmem235 ( n = 3), and KLF4 was used as the coding protein control; Kruppel-like factor-4 (KLF4); c RNA-FISH detection of subcellular localization of Lnc Tmem235 ( n = 5), and 18 S and U6 were used as positive controls for the cytoplasm and nucleus, respectively; RNA fluorescence in situ hybridization (RNA-FISH); d The expression of Lnc Tmem235 was detected by qPCR ( n = 6); empty vector (GV367); e – h The expression levels of Bcl-2, Bax, and CASP3 were analyzed by western blotting ( n = 3); i , k Apoptosis was detected by TUNEL/DAPI ( n = 5); TdT-mediated dUTP nick-end labeling (TUNEL); j , l Apoptosis was detected by Annexin V/PI ( n = 5). In ( d , f – h , k – l ), the data are normally distributed, and the variance is homogeneous. Data are presented as the means ± SDs; statistical significance was calculated by one-way ANOVA with Tukey’s post- hoc tests; * P < 0.05.

Article Snippet: The following antibodies were used for primary antibody reactions: rabbit anti-BIRC5 (1:3000; ab469; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:500; bs-0032R; Bioss, Beijing, China), rabbit anti-Caspase-3 (1:500; ab4051; Abcam), rabbit anti-Bax (1:1500; bs-28034R; Bioss), mouse anti-β-actin (1:2500; ab8226; Abcam), rabbit anti-CASP-3/Cleaved CASP-3 (1:500; ab13847; Abcam), rabbit anti-CASP-9/Cleaved CASP-9 (1:1000; ab25758; Abcam), rabbit anti-PARP1/Cleaved PARP1 (1:10000; ab191217; Abcam), rabbit anti-Argonaute 2 (1:800; ab5072; Abcam), mouse anti-OCN (10 µg/mL; ab13420; Abcam), rabbit anti-OPG (1:100; bs-20624R; Bioss), rabbit anti-Runx2 (1:200; AV36678; Sigma-Aldrich), and rabbit anti-OPN (1:200; ab8448; Abcam).

Techniques: Western Blot, Fluorescence, In Situ Hybridization, Expressing, Plasmid Preparation, TUNEL Assay, End Labeling

Journal: Experimental & Molecular Medicine

Article Title: Lnc Tmem235 promotes repair of early steroid-induced osteonecrosis of the femoral head by inhibiting hypoxia-induced apoptosis of BMSCs

doi: 10.1038/s12276-022-00875-0

Figure Lengend Snippet: a MRI, micro-CT, and HE staining were used to evaluate the SONFH model; magnetic resonance imaging (MRI), microcomputed tomography (micro-CT), and steroid-induced osteonecrosis of the femoral head (SONFH); b – c The expression of HIF-1α in the femoral head necrotic area was detected by western blotting ( n = 6); hypoxia inducible factor 1 alpha (HIF-1α); d Direct detection of oxygen concentration in the femoral head necrotic area ( n = 6); e At 0 and 2 days after surgery, the DiR fluorescence intensity in the transplanted area was detected by live imaging of small animals ( n = 8); 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR); f At 0 and 2 days after surgery, apoptosis of transplanted cells was detected by TUNEL staining ( n = 6); g Quantitative analysis of the DiR fluorescence intensity in the transplanted area as shown in e ( n = 8); h Quantitative analysis of the proportion of TUNEL positive cells in the transplanted area as shown in f ( n = 6); i At 0 and 2 days after surgery, the expression levels of BIRC5, Bcl-2, Bax, and CASP3 in the femoral head necrotic area were detected by western blotting ( n = 6); j The expression levels of Lnc Tmem235 in the femoral head necrotic area were detected by qPCR ( n = 6); k The expression levels of BIRC5 mRNA in the femoral head necrotic area were detected by qPCR ( n = 6); l Quantitative analysis of BIRC5 expression as shown in i ( n = 6); m Quantitative analysis of Bcl-2 expression as shown in i ( n = 6); n Quantitative analysis of Bax expression as shown in i ( n = 6); o Quantitative analysis of CASP-3 expression as shown in i ( n = 6); p Scanning electron microscope observation of tissue-engineered bone XACB/BMSCs ( n = 8); xenogeneic antigen-extracted cancellous (XACB) and tissue-engineered bone (XACB/BMSCs). q At 12 weeks after surgery, micro-CT analysis of the repair of the necrotic area of the femoral head ( n = 6); red circle indicates the transplantation area; r At 12 weeks postsurgery, H&E and Masson staining were used to evaluate the repair of the necrotic area ( n = 6); hematoxylin-eosin (H&E); s Quantitative analysis of the number of trabeculae as shown in q ( n = 6); t Quantitative analysis of the trabecular thickness as shown in q ( n = 6); u Quantitative analysis of the volume of new bone tissue as shown in q ( n = 6); v Quantitative analysis of the volume fraction of new bone tissue as shown in q ( n = 6); w At 12 weeks after surgery, the expression levels of OPG, OCN, and Runx2 in the femoral head necrotic area were detected by western blotting ( n = 6); osteoprotegerin (OPG), osteocalcin (OCN), and runt-related transcription factor 2 (Runx2); x Quantitative analysis of OPG expression as shown in w ( n = 6); y Quantitative analysis of OCN expression as shown in w ( n = 6); z Quantitative analysis of Runx2 expression as shown in w ( n = 6). In ( c , d , g , h , j – o , s – v , x – z ), the data are normally distributed, and the variance is homogeneous. Data are presented as the means ± SDs; statistical significance was calculated by Student’s t test; * P < 0.05.

Article Snippet: The following antibodies were used for primary antibody reactions: rabbit anti-BIRC5 (1:3000; ab469; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:500; bs-0032R; Bioss, Beijing, China), rabbit anti-Caspase-3 (1:500; ab4051; Abcam), rabbit anti-Bax (1:1500; bs-28034R; Bioss), mouse anti-β-actin (1:2500; ab8226; Abcam), rabbit anti-CASP-3/Cleaved CASP-3 (1:500; ab13847; Abcam), rabbit anti-CASP-9/Cleaved CASP-9 (1:1000; ab25758; Abcam), rabbit anti-PARP1/Cleaved PARP1 (1:10000; ab191217; Abcam), rabbit anti-Argonaute 2 (1:800; ab5072; Abcam), mouse anti-OCN (10 µg/mL; ab13420; Abcam), rabbit anti-OPG (1:100; bs-20624R; Bioss), rabbit anti-Runx2 (1:200; AV36678; Sigma-Aldrich), and rabbit anti-OPN (1:200; ab8448; Abcam).

Techniques: Micro-CT, Staining, Magnetic Resonance Imaging, Tomography, Expressing, Western Blot, Concentration Assay, Fluorescence, Imaging, TUNEL Assay, Microscopy, Transplantation Assay