Journal: OncoTargets and Therapy

Article Title: <em>TRIM44</em>, a crucial target of miR-410, functions as a potential oncogene in osteosarcoma

doi: 10.2147/ott.s163163

Figure Lengend Snippet: Figure 1 Upregulation of TRIM44 in human osteosarcoma tissues and cell lines. Notes: (A) Expression of TRIM44 was detected by immunohistochemistry in osteosarcoma tissues and their paired adjacent non-cancerous tissues. The samples were stained with an anti-TRIM44 antibody. (B) Statistical analysis of TRIM44 expression in osteosarcoma tissues and adjacent normal tissues. Data in bold indicates statistical difference (P,0.05). (C, D) Western blotting analysis of TRIM44 in eight paired osteosarcoma tissues and their corresponding non-cancerous tissues. GAPDH was used as a loading control, data shown were mean ± SEM values (n=3). (E, F) Expression of TRIM44 was upregulated in all four osteosarcoma cell lines compared with normal osteoblastic cell line NHOst, confirmed by Western blotting and RT-PCR. The average TRIM44 mRNA expression was normalized to the endogenous control GAPDH. Error bars represent the mean ± SEM values from three independent experiments, *P,0.05, **P,0.01 by two-tailed Student’s t-test.

Article Snippet: Written informed consent was obtained from every patient. immunohistochemistry Immunohistochemistry was performed as previously described.21 Briefly, slides went through antibody incubation overnight at 4°C with TRIM44 antibody (11511-1-AP; Proteintech, Chicago, IL, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: OncoTargets and Therapy

Article Title: <em>TRIM44</em>, a crucial target of miR-410, functions as a potential oncogene in osteosarcoma

doi: 10.2147/ott.s163163

Figure Lengend Snippet: Figure 2 TRIM44 knockdown inhibits cell proliferation, invasion, and EMT. Notes: (A) Western blotting was used to detect the protein expression of TRIM44 in MG-63 and U2OS cells upon siRNA transfection. (B) RT-PCR was performed to examine the mRNA expression of TRIM44 in MG-63/U2OS cells after transfection. (C) CCK8 assay revealed that knockdown of TRIM44 inhibited cell viability. (D, E) The wound healing assay showed that TRIM44 knockdown decreased the cell migration ability. (F, G) Representative images and quantification of invasive cells. (H) Western blotting analysis of EMT markers after transfection with TRIM44 siRNA. Error bars represent the mean ± SEM values of three independent experiments (*P,0.05, **P,0.01 by two-tailed Student’s t-test). Abbreviations: EMT, epithelial–mesenchymal transition; CCK8, Cell Counting Kit-8; OD, optical density; NC, negative control.

Article Snippet: Written informed consent was obtained from every patient. immunohistochemistry Immunohistochemistry was performed as previously described.21 Briefly, slides went through antibody incubation overnight at 4°C with TRIM44 antibody (11511-1-AP; Proteintech, Chicago, IL, USA).

Techniques: Knockdown, Western Blot, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, CCK-8 Assay, Wound Healing Assay, Migration, Two Tailed Test, Cell Counting, Negative Control

Journal: OncoTargets and Therapy

Article Title: <em>TRIM44</em>, a crucial target of miR-410, functions as a potential oncogene in osteosarcoma

doi: 10.2147/ott.s163163

Figure Lengend Snippet: Figure 3 miR-410 directly targets TRIM44, and miR-410 is downregulated in osteosarcoma tissues. Notes: (A) The putative miR-410-binding sequence in the 3′UTR of TRIM44. (B) 293T cells were co-transfected with the WT or MUT luciferase reporter plasmids and miR-410 mimics or negative control. The luciferase activity was evaluated 24 h post-transfection. (C, D) Western blot and qRT-PCR analysis of TRIM44 expression in osteosarcoma cells transfected with miR-410 mimics or negative control. (E) Detection of miR-410 expression in 20 paired osteosarcoma tissues and its corresponding non- cancerous tissues. All data are presented as the mean ± SEM of experiments performed in triplicate (**P,0.01). Abbreviations: WT, wild-type; MUT, mutant; NC, negative control; ns, non-significant.

Article Snippet: Written informed consent was obtained from every patient. immunohistochemistry Immunohistochemistry was performed as previously described.21 Briefly, slides went through antibody incubation overnight at 4°C with TRIM44 antibody (11511-1-AP; Proteintech, Chicago, IL, USA).

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Negative Control, Activity Assay, Western Blot, Quantitative RT-PCR, Expressing, Mutagenesis

Journal: OncoTargets and Therapy

Article Title: <em>TRIM44</em>, a crucial target of miR-410, functions as a potential oncogene in osteosarcoma

doi: 10.2147/ott.s163163

Figure Lengend Snippet: Figure 5 Ectopic expression of TRIM44 partially rescues the miR-410-induced effects on osteosarcoma cells. Notes: (A) Western blot analysis of TRIM44 expression after transfection with TRIM44 no-UTR and miR-410 mimics or negative control. CCK-8 and Transwell assays were performed to examine the cell viability (B) and invasive ability (C), respectively. (D) Western blot analysis of the EMT markers’ expression after transfection with TRIM44 no-UTR and miR-410 mimics or negative control. All data are presented as the mean ± SEM of experiments performed in triplicate (*P,0.05, **P,0.01). Abbreviations: EMT, epithelial–mesenchymal transition; CCK8, Cell Counting Kit-8; OD, optical density; NC, negative control.

Article Snippet: Written informed consent was obtained from every patient. immunohistochemistry Immunohistochemistry was performed as previously described.21 Briefly, slides went through antibody incubation overnight at 4°C with TRIM44 antibody (11511-1-AP; Proteintech, Chicago, IL, USA).

Techniques: Expressing, Western Blot, Transfection, Negative Control, CCK-8 Assay, Cell Counting

Journal: BMB Reports

Article Title: Regorafenib prevents the development of emphysema in a murine elastase model

doi: 10.5483/BMBRep.2023-0072

Figure Lengend Snippet: Effect of regorafenib on PPE-induced in inflammatory mediators in BAL fluid. (A) Representative membrane images of the antibody array. (B) Quantification of relative optical density for IL-1β, IL-6, CXCL1/KC, and TIMP-1 in the array membranes. (C-F) Measurement of IL-1β (C), IL-6 (D), CXCL1/KC (E), and TIMP-1 (F) levels in BAL fluid by ELISA. Error bars indicate the mean ± SEM. N = 8 per group, *P < 0.05, **P < 0.01, ****P < 0.0001, t -test.

Article Snippet: Anti-IL-6 antibody (ab208113) from abcam (Cambridge, UK), CXCL1 antibody (12335-1-AP) from Proteintech (Rosemont, IL, USA), and TIMP-1 antibody (#AF980) from R&D Systems were purchased respectively.

Techniques: Membrane, Ab Array, Enzyme-linked Immunosorbent Assay

Journal: BMB Reports

Article Title: Regorafenib prevents the development of emphysema in a murine elastase model

doi: 10.5483/BMBRep.2023-0072

Figure Lengend Snippet: Effect of regorafenib on PPE-induced expression of inflammatory mediators in lung parenchyme. (A) Experimental scheme. Mice were sensitized by treatment with regorafenib (5 mg/kg) for one day in the only PPE + Reg group. After PPE treatment, regorafenib was administered orally to mice for six days. On the seventh day, lung tissues were removed from mice, prepared in paraffin sections and stained with antibodies for inflammatory mediators. (B) Representative images of stained paraffin sections. Red arrows indicate the macrophages in inserted boxes. Scale bars indicate 50 μm. (C-E) Quantification of . IL-6 (C), CXCL1/KC (D), and TIMP-1 (E). Error bars indicate the mean ± SEM. N = 5 per group, *P < 0.05, **P < 0.01, t -test.

Article Snippet: Anti-IL-6 antibody (ab208113) from abcam (Cambridge, UK), CXCL1 antibody (12335-1-AP) from Proteintech (Rosemont, IL, USA), and TIMP-1 antibody (#AF980) from R&D Systems were purchased respectively.

Techniques: Expressing, Staining

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: The sequences of siRNAs used in this study.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Sequencing, Negative Control

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Molecular Weight, FLAG-tag

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A , B HeLa and SiHa cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 1 μM) and proteasome inhibitor MG132 (10 μM). Total cell proteins were collected for western blotting at different time points (0 h, 3 h, 6 h, and 12 h). The addition of MG132 slowed the degradation rate of the NCAPH protein, suggesting the involvement of the ubiquitin proteasome pathway. C Total protein in HeLa cells was immunoprecipitated by agarose beads containing IgG or NCAPH antibodies and silver stained after gel electrophoresis. The protein bands with the most significant differences were subjected to mass spectrometry analysis. D , E Coimmunoprecipitation was used to detect the interaction between TRIM21 and NCAPH. NCAPH and TRIM21 antibodies were used as separate baits; F Immunofluorescence assay showing the colocalization of the NCAPH and TRIM21 proteins in HeLa and SiHa cells. NCAPH, 488 nm, green fluorescence; TRIM21, 594 nm, red fluorescence; nucleus, DAPI, blue fluorescence. G Western blotting analysis showed that interference with NCAPH induced no changes in TRIM21 protein levels in cervical cancer cells. H – J Western blotting showed the efficiency of interference with three sets of siRNAs targeting TRIM21 and a significant increase in NCAPH protein levels after siTRIM21 treatment in HeLa and SiHa cells. *** P < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Staining, Nucleic Acid Electrophoresis, Mass Spectrometry, Immunofluorescence, Fluorescence

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A Construction of the TRIM21 truncation plasmid. Different colored boxes represent different structural domains, while discontinuous points represent deleted structural domains. B , C TRIM21 plasmids and corresponding domain mutants (labeled with HA) were cotransfected with NCAPH plasmids (labeled with Flag) into HEK 293T cells, and co-IP experiments were performed using Flag- or HA-labeled antibodies. Mutations in the PRY/SPRY and CC domains significantly influenced the binding between TRIM21 and NCAPH. D – G Cervical cancer cells were treated with cycloheximide (CHX), and total proteins were collected at different time points. Western blotting analysis revealed that, compared with the control group (siNC), knocking down TRIM21 significantly reduced the degradation rate of the NCAPH protein. H Western blotting analysis showed that MG132 treatment (10 μM) significantly reversed the decrease in NCAPH expression induced by TRIM21 overexpression. I HeLa and SiHa cells were transfected with siTRIM21 and HA-UB plasmids, respectively. Coimmunoprecipitation and western blotting assays showing a significant decrease in the ubiquitination level of NCAPH after interference with TRIM21. J , K HeLa and SiHa cells were transfected with siTRIM21 and cocultured with the HA-UB-K11, HA-UB-K48, or HA-UB-K63 plasmid. Coimmunoprecipitation and western blotting showing the effects of interfering with TRIM21: ubiquitination at the K11 position of NCAPH is significantly decreased. L Western blotting analysis showed that the mutation in the RING domain of TRIM21 resulted in the loss of regulation of NCAPH expression, suggesting that TRIM21 regulates NCAPH expression through its E3 ubiquitin ligase activity. M , N HeLa and SiHa cells were transfected with HA-TRIM21 or HA-TRIM21 Δ RING and cocultured with the His-UB plasmid. Coimmunoprecipitation and western blotting showed that TRIM21 overexpression significantly increased the ubiquitination level of NCAPH, while the elimination of the RING domain of TRIM21 significantly decreased it. The HA pcDNA3.1 vector was used as a control. * P < 0.05.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Plasmid Preparation, Labeling, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Control, Expressing, Over Expression, Transfection, Ubiquitin Proteomics, Mutagenesis, Activity Assay

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A – C Construction of a cervical cancer cell line with stable knockout of TRIM21. HeLa and SiHa cells were transfected with lentivirus for 72 h and then observed under a fluorescence microscope. The cells were screened with puromycin, and the knockdown efficiency was further confirmed by western blotting. Scale = 100 µM. D , E Cervical cancer cells were treated with 5 µM rapamycin for 2–4 h and then with 20 µM chloroquine for 12 h and then observed by transmission electron microscopy. The number of autophagosomes was calculated by quantitative analysis. Results showed that silencing TRIM21 significantly decreased the number of autophagosomes in HeLa and SiHa cells. Data are representative images and means ± SD of 3 fields (100 μm 2 per field). F – H Western blotting analysis showed that TRIM21 silencing decreased the expression of LC3B II and beclin-1 while increasing that of P62. I – L Cervical cancer cells were transfected with TRIM21 siRNA and then stained with AO and LysoTracker Red working solution. Hoechst 33342 (blue) was used to stain the nucleus. Statistical analysis revealed that knocking down TRIM21 had no effect on the fluorescence signals. M – P Western blotting analysis showed that 3-MA (2 mM) treatment almost completely reversed the changes in LC3B II caused by TRIM21 overexpression. In contrast, bafilomycin A1 (100 nM) treatment did not reverse the increase in LC3 II expression caused by TRIM21 overexpression. Q – S Western blotting analysis showed that knocking down TRIM21 expression significantly decreased the expression levels of autophagosome formation-related proteins (Beclin-1, ATG5, and ATG7). shTRIM21 and siTRIM21, TRIM21 interference group; shNC and siNC, negative control group. TRIM21, pcDNA3.1-TRIM21 plasmid; pcDNA3.1, mock vector. Scale = 25 μM. * P < 0.05, ** p < 0.01.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Knock-Out, Transfection, Fluorescence, Microscopy, Knockdown, Western Blot, Transmission Assay, Electron Microscopy, Expressing, Staining, Over Expression, Negative Control, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A – D Immunofluorescence staining was used to detect changes in the expression of LC3B and P62 in HeLa and SiHa cells. Interference with TRIM21 decreased the LC3B signal but increased the P62 signal. Knocking down NCAPH significantly reversed the changes caused by siTRIM21. Scale bar = 50 µm. E – H The mRFP-GFP-LC3 indicator system showed that interference with TRIM21 significantly decreased the spot ratio of mRFP to GFP, while silencing NCAPH dramatically reversed the changes in the spot ratio caused by siTRIM21 treatment. The autophagic flow process was observed and analyzed via confocal microscopy. ImageJ software was used to calculate the number of yellow and red puncta. Scale bar, 10 µm. I , J Western blotting analysis showed that interference with NCAPH eliminated the changes caused by knocking down TRIM21 ( I ). In contrast, overexpression of NCAPH eliminated the increase in LC3B II levels caused by overexpression of TRIM21 ( J ). The figure shows the representative results of three experiments. * P < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: Immunofluorescence, Staining, Expressing, Confocal Microscopy, Software, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A , B HeLa and SiHa cells were treated with RAPA (5 μM) for 24 h, and the EdU assay was used to detect cell proliferation capacity. C – F HeLa and SiHa cells were treated with RAPA (5 μM) or 3-MA (25 μM) for 24 h, and colony formation experiments were used to evaluate colony formation ability. G , I HeLa and SiHa cells were transfected with siNCAPH alone or in combination with 3-MA (25 μM). A colony formation assay was used to determine whether 3-MA could rescue the siNCAPH-induced reduction in proliferation. H , J HeLa and SiHa cells were transfected with the NCAPH overexpression plasmid alone or in combination with RAPA (5 μM). A colony formation assay was used to assess whether RAPA could rescue the increase in proliferation induced by ectopic NCAPH expression. K , M HeLa and SiHa cells were transfected with siTRIM21 alone or in combination with RAPA (5 μM) before the colony formation assay was performed. L , N HeLa and SiHa cells were transfected with the TRIM21 overexpression plasmid alone or treated in combination with 3-MA (25 μM) before performing the colony formation assay. The figure shows the representative results of three experiments. siTRIM21, TRIM21 interference group; NC, negative control group. TRIM21, pcDNA3.1-TRIM21 plasmid; pcDNA3.1, mock vector. * P < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: EdU Assay, Transfection, Colony Assay, Over Expression, Plasmid Preparation, Expressing, Negative Control

Journal: Cell Death & Disease

Article Title: NCAPH, ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy of cervical cancer through AKT/mTOR dependent signaling

doi: 10.1038/s41419-024-06932-y

Figure Lengend Snippet: A , B Silencing TRIM21 significantly increased the number of colonies of cervical cancer cells, while interference with NCAPH dramatically reversed the changes in colony numbers induced by siTRIM21. C , D The EdU assay was used to detect changes in cell proliferation capacity. Ectopic expression of NCAPH rescues the reduction in cell proliferation induced by the increase in TRIM21 expression. E Cells were treated with siTRIM21 alone or in combination with siNCAPH. Interference with TRIM21 expression increased p-AKT and p-mTOR protein levels but had no significant effect on total AKT or mTOR levels. NCAPH suppression reverses the effects of siTRIM21 on the protein levels of p-AKT and p-mTOR. F – H Western blotting analysis showing that treatment with an AKT/mTOR pathway inhibitor (MK2206) reversed the effects of TRIM21 silencing on the expression of p-AKT, p-mTOR and LC3B. I The proposed model in the study. NCAPH, which is ubiquitinated by TRIM21, promotes cell proliferation by inhibiting autophagy in cervical cancer through AKT/mTOR-dependent signaling. The figure shows the representative results of three experiments. * P < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: TRIM21 , 1:1000 (WB) 1:100 (IHC) , 55KD , Proteintech, China.

Techniques: EdU Assay, Expressing, Western Blot