Journal: Scientific Reports

Article Title: Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis

doi: 10.1038/s41598-019-40553-2

Figure Lengend Snippet: Treating differentiated murine C2C12 myotubes with 100 ng/mL TNFα for 24 hrs induced similar intracellular signaling pathway changes as observed in late-stage mSOD1 G93A mouse muscle tissue. Akt phosphorylation decreased following TNFα treatment ( A , E ). Meanwhile, PTEN expression was downregulated, suggesting it may not be a key modulator of Akt phosphorylation ( B , E ). However, CTMP expression significantly increased ( C , E ), as seen in the ALS mouse muscle. FOXO1 phosphorylation decreased ( D , E ), allowing it to permit transcription of atrophy genes such as Atrogin-1 , which was significantly upregulated ( F ). Blot images are cropped from different membranes used for data collection and analysis (See Supplemental Figures). * P < 0.01; *** P < 0.001.

Article Snippet: Then, the membranes were incubated with the following primary antibodies in PBS + 0.1% Tween 20 (PBST) + milk blocking buffer (ThermoPierce) overnight at 4 °C or 1 hr at room temperature: mouse anti-pan Akt, rabbit anti-p-Akt Ser473 , rabbit anti-LAMP1 (1:1,000), a marker for lysosomal activation, TNFα (1:1,000), rabbit anti-CTMP (1:1,000) (Cell Signaling Inc.), rabbit anti-LC3 (1:500; Abcam), a marker of autophagosome flux, rabbit anti-MURF-1, a marker of atrophy (1:1000, EMC Biosciences), and mouse anti-β-tubulin (Cell Signaling Inc.) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich) were used as loading controls.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis

doi: 10.1038/s41598-019-40553-2

Figure Lengend Snippet: Knocking down CTMP in TNFα-treated differentiated C2C12 myotubes using siRNA rescued the decrease in Akt phosphorylation. Incubating differentiated TNFα-treated C2C12 myotubes with siRNA against CTMP for 73 hours significantly decreased CTMP expression and increased Akt phosphorylation, indicating a direct relationship between CTMP expression and Akt phosphorylation in this muscle atrophy model. Blot images are cropped from different membranes used for data collection and analysis (See Supplemental Figures). * P < 0.05.

Article Snippet: Then, the membranes were incubated with the following primary antibodies in PBS + 0.1% Tween 20 (PBST) + milk blocking buffer (ThermoPierce) overnight at 4 °C or 1 hr at room temperature: mouse anti-pan Akt, rabbit anti-p-Akt Ser473 , rabbit anti-LAMP1 (1:1,000), a marker for lysosomal activation, TNFα (1:1,000), rabbit anti-CTMP (1:1,000) (Cell Signaling Inc.), rabbit anti-LC3 (1:500; Abcam), a marker of autophagosome flux, rabbit anti-MURF-1, a marker of atrophy (1:1000, EMC Biosciences), and mouse anti-β-tubulin (Cell Signaling Inc.) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich) were used as loading controls.

Techniques: Expressing

Journal: Oncotarget

Article Title: CTMP, a predictive biomarker for trastuzumab resistance in HER2-enriched breast cancer patient

doi: 10.18632/oncotarget.10719

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis for patients (n=369)

Article Snippet: After blocking, the slides were incubated with primary antibody against CTMP (Cell signaling), AKT-p473 (Cell signaling) and HER2 followed by biotin-conjugated secondary antibody, polymer-HRP and diaminobenzidine tetrahydroxychloride (DAB) solution.

Techniques:

Journal: Oncotarget

Article Title: CTMP, a predictive biomarker for trastuzumab resistance in HER2-enriched breast cancer patient

doi: 10.18632/oncotarget.10719

Figure Lengend Snippet: A . CTMP, HER2 and AKT activity were analyzed in SkBR3 and BT-483 by Western blot. B . After 5 days trastuzumab treatment to SkBR3 and BT-483 cells, proliferation was examined using the MTT assay. Ectopic overexpression of different levels of CTMP in SkBR3 cells C . or knockdown of CTMP in BT-483 cells D . was used to modulate trastuzumab response as measured by the MTT assay. E . Western blots were performed to evaluate AKT, ERK and PDK1 and their activation in SkBR3 cells after transfection with GFP or GFP-CTMP. mean ± SEM; *, P < 0.05; ***, P < 0.001; n.s., no significance.

Article Snippet: After blocking, the slides were incubated with primary antibody against CTMP (Cell signaling), AKT-p473 (Cell signaling) and HER2 followed by biotin-conjugated secondary antibody, polymer-HRP and diaminobenzidine tetrahydroxychloride (DAB) solution.

Techniques: Activity Assay, Western Blot, MTT Assay, Over Expression, Activation Assay, Transfection

Journal: Oncotarget

Article Title: CTMP, a predictive biomarker for trastuzumab resistance in HER2-enriched breast cancer patient

doi: 10.18632/oncotarget.10719

Figure Lengend Snippet: Western-blot was performed to determine AKT activity in CTMP-overexpressing SkBR3 (A) and in CTMP-silenced BT-483 cells (B) after trastuzumab treatment for 24 hours. C . CTMP-overexpressing SkBR3 and BT483 were treated with trastuzumab with or without AKT inhibitor IV, rapamycin and PD98058 for 72 hours and cell proliferation ability was determined by the MTT assay.

Article Snippet: After blocking, the slides were incubated with primary antibody against CTMP (Cell signaling), AKT-p473 (Cell signaling) and HER2 followed by biotin-conjugated secondary antibody, polymer-HRP and diaminobenzidine tetrahydroxychloride (DAB) solution.

Techniques: Western Blot, Activity Assay, MTT Assay

Journal: Oncotarget

Article Title: CTMP, a predictive biomarker for trastuzumab resistance in HER2-enriched breast cancer patient

doi: 10.18632/oncotarget.10719

Figure Lengend Snippet: A . CTMP, p-AKT and HER2 expression level were examined in HER2-enriched patients by IHC. In each sample, 100 cells were scored from 0 to 3 and the sum was taken as the staining index. B . The expression levels were compared between trastuzumab responders and non-responders by student t test. C . Person's correlation was used to analyze the correlation between CTMP and p473-AKT. PD: progressing disease; NED: non-evidence of disease; mean ± SEM; *, P<0.05.

Article Snippet: After blocking, the slides were incubated with primary antibody against CTMP (Cell signaling), AKT-p473 (Cell signaling) and HER2 followed by biotin-conjugated secondary antibody, polymer-HRP and diaminobenzidine tetrahydroxychloride (DAB) solution.

Techniques: Expressing, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting CRMP-4 by lentivirus-mediated RNA interference inhibits SW480 cell proliferation and colorectal cancer growth

doi: 10.3892/etm.2016.3588

Figure Lengend Snippet: Expression of CRMP-4 in colorectal cancer and control tissues. (A) Western blot analysis of the protein expression of CRMP-4 in tissues from three patients and three healthy individuals, with GAPDH used as a loading control. Data obtained from three independent experiments were normalized against GAPDH and are expressed as the mean ± standard deviation, as analyzed with one-way analysis of variance. (B) Immunohistochemical analysis of the expression of CRMP-4 in tissues (stain, hematoxylin; magnification, ×100). *P<0.05 vs. the N group. CRMP-4, collapsin response mediator protein 4; N: normal colon tissue from healthy persons, A: normal adjacent tissues of colorectal cancer, C: colorectal cancer tissue.

Article Snippet: CRMP-4 (cat. no. 4612) and GAPDH (cat. no. 5174) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Standard Deviation, Immunohistochemical staining, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting CRMP-4 by lentivirus-mediated RNA interference inhibits SW480 cell proliferation and colorectal cancer growth

doi: 10.3892/etm.2016.3588

Figure Lengend Snippet: Verification of knockdown CRMP-4 expression by lentivirus. (A) SW480 cells infected with lentivirus. (B) Reverse transcription-polymerase chain reaction and western blot analysis. GAPDH was used as an internal control. CRMP-4, collapsin response mediator protein 4; lv-si-CRMP-4, SW480 cells infected with lv-si-CRMP-4 lentivirus; lv-si-scramble, SW480 cells infected with lv-si-scramble lentivirus.

Article Snippet: CRMP-4 (cat. no. 4612) and GAPDH (cat. no. 5174) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting CRMP-4 by lentivirus-mediated RNA interference inhibits SW480 cell proliferation and colorectal cancer growth

doi: 10.3892/etm.2016.3588

Figure Lengend Snippet: Downregulation of CRMP-4 inhibits the proliferation of SW480 cells. (A) Viability of SW480 cells were evaluated by MTT assay. (B) Proliferation of SW480 cells were evaluated by BrdU incorporation. Data were obtained from three independent experiments and are expressed as the mean ± standard deviation, as analyzed using one-way analysis of variance. *P<0.05 vs. the si-CRMP-4 group at the same time point. CRMP-4, collapsin response mediator protein 4; OD, optical density.

Article Snippet: CRMP-4 (cat. no. 4612) and GAPDH (cat. no. 5174) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: MTT Assay, BrdU Incorporation Assay, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Targeting CRMP-4 by lentivirus-mediated RNA interference inhibits SW480 cell proliferation and colorectal cancer growth

doi: 10.3892/etm.2016.3588

Figure Lengend Snippet: Colon tumor sizes in each group. (A) Colon tumor of mice injected with si-scramble cells or si-CRMP-4 cells after 4 weeks of injection. (B) Colon tumor size in each group after lentivirus injection at 1, 2, 3 and 4 weeks, respectively. Data were obtained from three independent experiments and are expressed as the mean ± standard deviation, as analyzed using one-way analysis of variance. *P<0.05 vs. the si-CRMP-4 group at the same time point. CRMP-4, collapsin response mediator protein 4.

Article Snippet: CRMP-4 (cat. no. 4612) and GAPDH (cat. no. 5174) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Injection, Standard Deviation