Journal: Scientific Reports

Article Title: Regulation of degenerative spheroids after injury

doi: 10.1038/s41598-020-71906-x

Figure Lengend Snippet: NAD + acts on upstream of Rho activation to suppresses spheroid formation after injury. ( a ) Fluo4-AM calcium imaging of wild-type sympathetic axons 1 h after injury with or without drug treatment. Scale bar = 10 µm. ( b ) Quantification of axonal spheroid number per 100 µm of wild-type sympathetic axons at the indicated times after injury in the absence and presence of NAD + or CN03. Total number of n = 23 (Control), n = 13 (NAD + ), n = 21 (NAD + , CN03) axons from cultured neurons harvested from 3 independent litters were quantified. ( c ) Fluo4-AM calcium imaging of Sarm1 −/− sympathetic axons 1 h after injury with or without drug treatment. Scale bar = 10 µm. ( d ) Quantification of axonal spheroid number per 100 µm of Sarm1 −/− sympathetic axons at the indicated times after injury in the absence and presence of NAD + or CT04. Total number of n = 30 (Control), n = 16 (NAD + ), n = 25 (CT04) Sarm1 −/− axons from cultured neurons harvested from 3 independent litters were quantified. ( e ) Fluo4-AM calcium imaging of Wld s sympathetic axons 1 h after injury in the presence and absence of CN03. Scale bar = 10 µm. ( f ) Quantification of axonal spheroid number per 100 µm of Wld s sympathetic axons at the indicated times after injury in the absence and presence of CN03. Total number of n = 25 (Control), n = 24 (CN03) Wld s axons from cultured neurons harvested from 3 independent litters were quantified. ( g ) Representative images and ( h ) quantification of degeneration of wild-type and Wld s distal sympathetic axons immuno-stained for β3-tubulin with different treatments. Scale bar = 50 µm. For the “NAD + ” group, axons were incubated in SCG media containing 1 mM NAD + supplement overnight prior to injury. For the “CN03” and CT04″ groups, axons were incubated in SCG media containing 1 µg/mL Rho activator CN03 and Rho inhibitor CT04 for 2 h prior to injury, respectively. Data are reported as mean ± SEM, * p < 0.05; ** p < 0.001; *** p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: CT04 and CN03 were the products of Cytoskeleton Inc. Cytochalasin D (C2618), cytosine arabinofuranoside (Ara-C), poly-D-lysine, and paraformaldehyde were purchased from Millipore Sigma.

Techniques: Activation Assay, Imaging, Control, Cell Culture, Staining, Incubation

Journal: Biochemical and biophysical research communications

Article Title: Rho/ROCK mechanosensor in adipocyte stiffness and traction force generation.

doi: 10.1016/j.bbrc.2022.03.078

Figure Lengend Snippet: Fig. 1. ROCK inhibition decreases while Rho activation increases Young's modulus of preadipocytes and differentiated adipocytes. (A) AFM probe approaching a preadipocyte. Arrow: nucleus; arrowhead: cytoplasm. Representative force-displacement curves of preadipocytes when probing on nucleus (B) and cytoplasm (C). ROCK inhibition and Rho activation were conducted using Y-27632 and CN01, respectively. (D) AFM probing on differentiated adipocytes. Arrow: nucleus; arrowhead: lipid droplet. Representative force- displacement curves of differentiated adipocytes when probing on nucleus (E) and lipid droplet (F). For both preadipocytes (G) and differentiated adipocytes (H), ROCK inhibi- tion by Y-27632 decreased while Rho activation by CN01 increased cellular Young's modulus. *: p < 0.05, **: p < 0.01, and ***: p < 0.005 based on repeated AFM probing (n numbers). Scale bar: 20 mm.

Article Snippet: ForRho activation, CN01 (Cytoskeleton Inc.)wasdissolved into 20ml dimethyl sulfoxideanddiluted inDMEM at a concentration of 1 unit/ml.

Techniques: Inhibition, Activation Assay

Journal: Biochemical and biophysical research communications

Article Title: Rho/ROCK mechanosensor in adipocyte stiffness and traction force generation.

doi: 10.1016/j.bbrc.2022.03.078

Figure Lengend Snippet: Fig. 2. ROCK inhibitor (Y-27632) disrupts and Rho activator (CN01) strengthens stressed actin filament formation in adipose cells. Actin was immunostained with rhodamine phalloidin and nuclei with DAPI. Scale bar: 20 mm.

Article Snippet: ForRho activation, CN01 (Cytoskeleton Inc.)wasdissolved into 20ml dimethyl sulfoxideanddiluted inDMEM at a concentration of 1 unit/ml.

Techniques:

Journal: Biochemical and biophysical research communications

Article Title: Rho/ROCK mechanosensor in adipocyte stiffness and traction force generation.

doi: 10.1016/j.bbrc.2022.03.078

Figure Lengend Snippet: Fig. 3. Traction forces in adipose cells are decreased by ROCK inhibition, and traction force dependency on cell spreading area is increased by Rho activation. (AeC) Representative TFM images of preadipocytes from control, Y-27632 treatment, and CN01 treatment, respectively. (D) Histogram of traction forces from TFM traction force vectors measured for preadipocytes. (E) Average traction force for each cell obtained from entire traction force vectors. ROCK inhibition by Y-27632 significantly decreased traction force in preadipocytes, while Rho activation by CN01 did not induce significant change. (FeH) Representative TFM images of differentiated adipocytes. (I) Traction force histogram for differentiated adipocytes. (J) Differentiated adipocytes displayed substantially lower traction forces than preadipocytes. Trends of Y-27632 and CN01 exposure on the traction force of differ- entiated adipocytes were similar to those in preadipocytes. (K,L) Traction force dependency on projected cell spreading area is increased by Rho activation, and such dependency is less distinct in differentiated adipocytes. **: p < 0.01, ***: p < 0.001, and n.s.: non-significant. Scale bar: 5 mm.

Article Snippet: ForRho activation, CN01 (Cytoskeleton Inc.)wasdissolved into 20ml dimethyl sulfoxideanddiluted inDMEM at a concentration of 1 unit/ml.

Techniques: Inhibition, Activation Assay, Control

Journal: Biological Procedures Online

Article Title: Circ_0003570 Suppresses the progression of hepatocellular carcinoma through miR-182-5p/STARD13 regulatory axis

doi: 10.1186/s12575-022-00176-w

Figure Lengend Snippet: The primers in the study

Article Snippet: For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C.

Techniques:

Journal: Biological Procedures Online

Article Title: Circ_0003570 Suppresses the progression of hepatocellular carcinoma through miR-182-5p/STARD13 regulatory axis

doi: 10.1186/s12575-022-00176-w

Figure Lengend Snippet: The sequence of luciferase reporter plasmids

Article Snippet: For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C.

Techniques: Sequencing, Luciferase

Journal: Biological Procedures Online

Article Title: Circ_0003570 Suppresses the progression of hepatocellular carcinoma through miR-182-5p/STARD13 regulatory axis

doi: 10.1186/s12575-022-00176-w

Figure Lengend Snippet: Circ_0003570 acts as a sponge of miR-182-5p to regulate STARD13. A Bioinformatics analysis showed the binding site between circ_0003570 and miR-182-5p. B A dual luciferase reporter assay was applied to verify the targeted binding effect between circ_0003570 and miR-182-5p. ** P < 0.01. C Bioinformatics analysis showed the binding site between miR-182-5p and STARD13 3′‐UTR. D A dual luciferase reporter assay was applied to verify the targeted binding effect between circ_0003570 and miR-182-5p. ** P < 0.01. E , F qRT-PCR analysis of miR-182-5p expression following transfected HCC cells with siRNA or overexpressed lentiviral vector of circ_0003570. * P < 0.05; ** P < 0.01. G The STARD13 mRNA levels after miR-182-5p overexpression or downregulation were deceted by qRT-PCR. * P < 0.05. H The STARD13 protein levels after miR-182-5p overexpression or downregulation were deceted by western blot. Student’s t-test was performed to compare the difference between two groups

Article Snippet: For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C.

Techniques: Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Over Expression, Western Blot

Journal: Biological Procedures Online

Article Title: Circ_0003570 Suppresses the progression of hepatocellular carcinoma through miR-182-5p/STARD13 regulatory axis

doi: 10.1186/s12575-022-00176-w

Figure Lengend Snippet: Circ_0003570 suppresses HCC proliferation, migration and invasion via the miR-182-5p/STARD13 axis. A The results of MTT assay showed that miR-182-5p mimics restored the inhibition of LV-circ_0003570 on cell proliferation over time. HCC cells grew faster in LV-circ_0003570 and miR-182-5p mimics co‐transfection group than LV-circ_0003570 group. * P < 0.05. B Colony formation assay showed that the proliferative capability of HCC cells was increased by miR-182-5p mimics compared with LV-circ_0003570 group. * P < 0.05. C , D The migration and invasion capacity restrained by upregulation of circ_0003570 could be reversed by miR‐182‐5p mimics. Cells migrated and invaded were increased in LV-circ_0003570 and miR-182-5p mimics co‐transfection group. * P < 0.05; ** P < 0.01. E , F In wound healing assay, miR‐182‐5p mimics reversed the inhibitory role of LV-circ_0003570 on migration in HepG2 and MHCC-97L cells. * P < 0.05. G In protein level, the upregulation of STARD13 regulated by LV-circ_0003570 could be rescued by miR‐182‐5p mimics. One-way ANOVA was performed to compare the difference between two groups

Article Snippet: For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C.

Techniques: Migration, MTT Assay, Inhibition, Cotransfection, Colony Assay, Wound Healing Assay

Journal: Biological Procedures Online

Article Title: Circ_0003570 Suppresses the progression of hepatocellular carcinoma through miR-182-5p/STARD13 regulatory axis

doi: 10.1186/s12575-022-00176-w

Figure Lengend Snippet: Overexpression of circ_0003570 represses tumor growth and lung metastasis in vivo. A The transfection efficiency of HepG2 cells stably transfected with LV-circ_0003570 was detected by qRT‐PCR, and the result showed that the expression of circ_0003570 was evidently overexpressed. ** P < 0.01. B Representative images of nude mice modles and formed tumors that were subcutaneously injected with LV- circ_0003570 and NC cells. C , D Effect of circ_0003570 overexpression on HCC growth in vivo according to the tumor growth curve and tumor weight. * P < 0.05; ** P < 0.01. E Representative images of HE and IHC staining patterns for STARD13 in tumor xenografts of nude mice (Left:10 × ; Right: 40 ×). F , G Representative images of pulmonary metastatic models and HE staining of metastatic nodules in the lungs (20 ×). Black arrows indicate metastatic nodules. ** P < 0.01.Student’s t-test was performed to compare the difference between two groups

Article Snippet: For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C.

Techniques: Over Expression, In Vivo, Transfection, Stable Transfection, Quantitative RT-PCR, Expressing, Injection, Immunohistochemistry, Staining

Journal: Biological Procedures Online

Article Title: Circ_0003570 Suppresses the progression of hepatocellular carcinoma through miR-182-5p/STARD13 regulatory axis

doi: 10.1186/s12575-022-00176-w

Figure Lengend Snippet: Schematic diagram depicting the biological function of circ_0003570 on hepatocellular carcinoma development in this research. Circ_0003570 suppressed HCC tumorigenesis by competitively binding miR-182-5p and modulating STARD13 expression

Article Snippet: For IHC, primary antibody of STARD13 (Proteintech) at dilution rate 1:200 were incubated overnight at 4 °C.

Techniques: Binding Assay, Expressing

Journal: Oncogene

Article Title: Smoking-associated upregulation of CBX3 suppresses ARHGAP24 expression to activate Rac1 signaling and promote tumor progression in lung adenocarcinoma

doi: 10.1038/s41388-021-02114-8

Figure Lengend Snippet: a Venn diagram showed number of genes negatively correlated with CBX3 in TCGA-LUAD, GSE68465 and CPTAC-LUAD datasets. b , c Differential expression analyses of ARHGAP24 between tumor and normal tissues in TCGA-LUAD ( b ) and CPTAC-LUAD ( c ) datasets. d , e Kaplan–Meier analysis with two-sided log-rank test was conducted to evaluate the differences in RFS ( d ) and OS ( e ) between the patients with high and low expression of ARHGAP24 in TCGA-LUAD dataset. The dashed lines in panel ( d ) and ( e ) represented the 95% confidence intervals of the KM curves. f Volcano diagram showed differential expressed genes caused by the knockdown of CBX3 in A549 cell line. g – i ARHGAP24 was negatively correlated with CBX3 in TCGA-LUAD ( g ), GSE68465 ( h ) and CPTAC-LUAD ( i ) datasets. j , k The tissue microarray of lung adenocarcinoma was stained with CBX3 and ARHGAP24, respectively ( n = 59). The typical IHC images stained with CBX3 and ARHGAP24 were shown in panel ( j ). The size of the scale bar on microscopy images as indicated in the figure. The correlation of these two proteins was shown in panel ( k ). Spearman correlation was used to determine statistical significance, P = 0.0427.

Article Snippet: Antibodies against the following proteins were used: CBX3 (cat. no. 11650-2-AP, Proteintech; 1:1000 dilution); ARHGAP24 (cat. no. 18834-1-AP, Proteintech; 1:2500 dilution); Rac1 (cat. no. 66122-1-Ig, Proteintech; 1:2500 dilution); GAPDH (cat. no. 60004-1-Ig, Proteintech; 1:5000 dilution); TRIM24 (cat. no. 14208-1-AP.

Techniques: Quantitative Proteomics, Expressing, Knockdown, Microarray, Staining, Microscopy

Journal: Oncogene

Article Title: Smoking-associated upregulation of CBX3 suppresses ARHGAP24 expression to activate Rac1 signaling and promote tumor progression in lung adenocarcinoma

doi: 10.1038/s41388-021-02114-8

Figure Lengend Snippet: a , b A549 and H1299 cells were infected with shControl, shCBX3 #1, or shCBX3 #2 for 72 h. Cells were collected for Western blotting analysis ( a ) and RT-qPCR analysis ( b ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. ** P < 0.01. c , d A549 and H1299 cells were transfected with indicated plasmids for 24 h. Cells were collected for Western blotting analysis ( c ) and RT-qPCR analysis ( d ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. ** P < 0.01; *** P < 0.001. e , f The ChIP-seq of CBX3 on the promoter region of ARHGAP24. g The ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 and H1299 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. NS not significant; ** P < 0.01; *** P < 0.001. h The ChIP-qPCR of H3K9me3 on the promoter region of ARHGAP24 in A549 and H1299 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. NS not significant; * P < 0.05; *** P < 0.001. i A549 cells were infected with shControl or shARHGAP24 for 48 h. Then, cells were transfected with pcDNA3.1 or Flag-CBX3 as indicated. After 24 h, cells were harvested for Western blotting analysis. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For quantification of active Rac1, data presented as Mean ± SD with two replicates. NS not significant; * P < 0.05; ** P < 0.01. j , k A549 cells were infected with indicated shRNAs for 72 h. Cells were collected for Western blotting analysis ( j ) and MTS assay ( k ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For quantification of active Rac1, data presented as Mean ± SD with two replicates. For MTS assay, data presented as Mean ± SD with three replicates. NS not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. l – n A549 cells were infected with indicated shRNAs. After 72 h puromycin selection, cells were harvested and subcutaneously injected into nude mice for xenografts assay. The image of tumor was shown in panel ( l ). The tumor growth curve was indicated in panel ( m ). The tumor mass was demonstrated in panel ( n ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with five replicates. NS not significant; *** P < 0.001.

Article Snippet: Antibodies against the following proteins were used: CBX3 (cat. no. 11650-2-AP, Proteintech; 1:1000 dilution); ARHGAP24 (cat. no. 18834-1-AP, Proteintech; 1:2500 dilution); Rac1 (cat. no. 66122-1-Ig, Proteintech; 1:2500 dilution); GAPDH (cat. no. 60004-1-Ig, Proteintech; 1:5000 dilution); TRIM24 (cat. no. 14208-1-AP.

Techniques: Infection, Western Blot, Quantitative RT-PCR, Transfection, ChIP-sequencing, ChIP-qPCR, MTS Assay, Selection, Injection

Journal: Oncogene

Article Title: Smoking-associated upregulation of CBX3 suppresses ARHGAP24 expression to activate Rac1 signaling and promote tumor progression in lung adenocarcinoma

doi: 10.1038/s41388-021-02114-8

Figure Lengend Snippet: a PPI network between CBX3 and proteins correlated with CBX3. b The whole cell lysates (WCL) of A549 were collected to undergo immunoprecipitation by using the IgG and CBX3 antibodies. Western blotting analysis was using used to detect the RBBP4, TRIM24, TRIM28 and CBX3. c – e The whole cell lysates (WCL) of A549 were collected to undergo immunoprecipitation by using the IgG and TRIM28, TRIM2, or RBBP4 antibodies, respectively. Western blotting analysis was using used to detect the RBBP4, TRIM24, TRIM28 and CBX3. f , g A549 cells were infected with shControl, shTRIM28 #1, or shTRIM28 #2 for 72 h. Cells were collected for Western blotting analysis ( f ) and RT-qPCR analysis ( g ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. * P < 0.05; ** P < 0.01; *** P < 0.001. h , i A549 cells were infected with shControl, shTRIM24 #1, or shTRIM24 #2 for 72 h. Cells were collected for Western blotting analysis ( h ) and RT-qPCR analysis ( i ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. * P < 0.05; ** P < 0.01; *** P < 0.001. j , k A549 cells were infected with shControl, shRBBP4 #1, or shRBBP4 # 2 for 72 h. Cells were collected for Western blotting analysis ( j ) and RT-qPCR analysis ( k ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. * P < 0.05; ** P < 0.01; *** P < 0.001. l The ChIP-qPCR of TRIM28 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates ( n = 3). NS not significant; *** P < 0.001. Primer I indicated the pair of primer located in the common binding peak of RBBP4, TRIM24, TRIM28, CBX3 and H3K9me3; Primer O indicated the the pair of primer located outside the common binding peak of RBBP4, TRIM24, TRIM28, CBX3 and H3K9me3. m The ChIP-qPCR of TRIM24 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was deter m ined by two-side Student t test. Data presented as Mean ± SD with three replicates ( n = 3). NS not significant; *** P < 0.001. n The ChIP-qPCR of RBBP4 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates ( n = 3). NS not significant; ** P < 0.01. o A549 cells were infected with shControl and shTRIM28 for 72 h. Cells were collected for the ChIP-qPCR of CBX3 on the promoter regi o n of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates ( n = 3). * P < 0.05; ** P < 0.01. p A549 cells were infected with shControl and shTRIM24 for 72 h. Cells were collected for the ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. *** P < 0.001. q A549 cells were infected with shControl and shRBBP4 for 72 h. Cells were collected for the ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. * P < 0.05; ** P < 0.01; *** P < 0.001. r – t A549 cells were infected with indicated shRNAs for 72 h. Cells were harvested for Western blotting analysis.

Article Snippet: Antibodies against the following proteins were used: CBX3 (cat. no. 11650-2-AP, Proteintech; 1:1000 dilution); ARHGAP24 (cat. no. 18834-1-AP, Proteintech; 1:2500 dilution); Rac1 (cat. no. 66122-1-Ig, Proteintech; 1:2500 dilution); GAPDH (cat. no. 60004-1-Ig, Proteintech; 1:5000 dilution); TRIM24 (cat. no. 14208-1-AP.

Techniques: Immunoprecipitation, Western Blot, Infection, Quantitative RT-PCR, ChIP-qPCR, Binding Assay

Journal: Oncogene

Article Title: Smoking-associated upregulation of CBX3 suppresses ARHGAP24 expression to activate Rac1 signaling and promote tumor progression in lung adenocarcinoma

doi: 10.1038/s41388-021-02114-8

Figure Lengend Snippet: The overexpressed CBX3 coupled with TRIM28, TRIM24 or RBBP4 to repress ARHGAP24 and indirectly increase the amount of active Rac1, which finally promotes the progression of lung adenocarcinoma.

Article Snippet: Antibodies against the following proteins were used: CBX3 (cat. no. 11650-2-AP, Proteintech; 1:1000 dilution); ARHGAP24 (cat. no. 18834-1-AP, Proteintech; 1:2500 dilution); Rac1 (cat. no. 66122-1-Ig, Proteintech; 1:2500 dilution); GAPDH (cat. no. 60004-1-Ig, Proteintech; 1:5000 dilution); TRIM24 (cat. no. 14208-1-AP.

Techniques: