Control My Nikon 5.2 Keygen.epub
Everardo Laboy
The cells were lysed with RIPA lysis buffer (Beyotime, China). Protein extracts were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and then incubated with primary antibodies overnight at 4C. After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies for 1h at room temperature. The enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL) and a ChemiDoc MP Imaging System (Bio-Rad) were used to visualize the immunoblots. GAPDH or β-actin was used as a protein loading control. The intensity of the protein bands was quantified using ImageJ software (NIH, Bethesda, MD; ). Protein expression levels were normalized to the levels of GAPDH or CD63. The antibodies to CD63, calreticulin, TSG101, α-SMA, TGF-β1, SOCS3, SMAD3, p-SMAD3, GAPDH, β-actin, and HRP-linked anti-rabbit or anti-mouse IgG were purchased from Abcam (Cambridge, UK). The antibodies to collagen types I and III were purchased from Proteintech (Chicago, USA). The concentrations used for all the antibodies were listed in supplementary S2 Table.
Eight-week-old male BABL/c mice were purchased from the Laboratory Animal Center of Nanjing Medical University (Nanjing, China). All mice were maintained in a specific pathogen-free breeding facility with strictly controlled humidity, temperature, and 12-h light/dark cycles.
(A) Schematic diagram of the transwell co-culture system. Top chambers: RAW264.7 cells. Bottom chambers: JS1 cells. (B) The viability of JS1 cells. (C and D) qRT-PCR (C) and immunoblot analysis (D) of fibrotic markers (α-SMA, Col-I, and Col-III) in JS1 cells. (E-G) JS1 cells were cultured with complete supernatants or EVs-free supernatants from SEA-Mφ mono-cultures. The cell viability was determined (E). qRT-PCR (F) and immunoblot analysis (G) of fibrotic markers (α-SMA, Col-I, and Col-III) in JS1 cells. Numbers below the blots represent the quantification of the band intensities expressed as fold change over the control (same for all the immunoblots). All graph data are expressed as the mean SD of 3 biological replicates per group. *P < 0.05, **P < 0.01, ***P
Chilled lysis buffer supplemented with protease and phosphatase inhibitors (KEYGEN, China) was used to extract protein, after which a BCA assay (KEYGEN, China) was used to quantify protein levels based on provided directions. Lysates (30 μg) were separated via 10% SDS-PAGE and transferred to 0.45 μm PVDF membranes that were subsequently blocked for 2 h with 5% non-fat milk and then probed overnight with rabbit anti-CEBPA (1:500, Cat# YT0551, Immunoway) or mouse anti-BCL2 (1:500, Cat# YM3041, Immunoway) overnight at 4 C. Blots were then probed for 2 h with secondary antibodies, and a Dual Color Infra-red Laser Imaging System (Gene, HK, China) was used to detect protein bands. ImageJ (National Institutes of Health, USA) was used for protein band analyses. GAPDH (1:1000, Proteintech) served as a normalization control.
We have previously employed microarray and qPCR analyses to confirm that CEBPA-DT is upregulated in OSCC in a manner negatively correlated with tumor grading and clinical staging [12]. Given that lncRNAs are known to mediate solid tumor resistance to different chemotherapeutic agents [15, 16], we next prepared chemoresistant strains of the HSC4 and Cal27 OSCC cell lines by treating them with continuous low doses of CDDP, yielding chemoresistant cells with higher IC50 values (Fig. 1A). We then assessed CEBPA-DT levels in these cells via qPCR, revealing significant upregulating of this lncRNA in both Cal27-CisR and HSC4-CisR cells relative to their parental cell lines (Fig. 1B). Moreover, CEBPA and BCL2 mRNA and protein expression levels were also dramatically upregulated in both CisR cell lines compared with control groups (Fig. 1C-E). These results indicated that the upregulation of CEBPA-DT/CEBPA/BCL2 was correlated with OSCC cell CDDP resistance.
We next explored whether knocking down the expression of CEBPA-DT was sufficient to enhance the chemosensitivity of OSCC cells by transfecting them with smart silencer (ss-CEBPA-DT) or negative control (ss-NC) constructs (Fig. 2A). For gain-of-function experiments, this lncRNA was instead overexpressed in Cal27-CisR and HSC4-CisR cells by transfecting them with the pc-CEBPA-DT (pcDNA3.1-CEBPA-DT) or pc-NC vectors (Fig. 2C). A subsequent CCK-8 assay revealed that the knockdown of CEBPA-DT was sufficient to impair Cal27-CisR and HSC4-CisR cell viability, reducing their CDDP IC50 values to 13.361.73 μM and 11.810.39 μM, which were lower than those values for ss-NC cells (26.011.81 μM and 23.242.49 μM) (Fig. 2B). The overexpression of CEBPA-DT, in contrast, enhanced the survival of both tested CDDP-resistant OSCC cell lines relative to controls (Fig. 2D). The apoptotic death of these cells was then evaluated by flow cytometry, revealing that ss-CEBPA-DT transfection was associated with enhanced apoptotic death relative to ss-NC transfection even in the absence of CDDP treatment (data not shown). Upon such treatment, 4.06 0.55% of Cal27-CisR and 4.10 0.56% of HSC4-CisR ss-NC transfected cells exhibited signs of early apoptotic death, whereas these percentages rose to 15.34 2.11% and 15.28 0.75%, respectively, following CEBPA-DT knockdown (Fig. 2E). TUNEL staining further confirmed that the frequency of apoptotic cells was significantly higher in the CDDP-treated ss-CEBPA-DT group relative to the control group for both tested cell lines (Fig. 2F). Together, these results suggested that the downregulation of CEBPA-DT can enhance the sensitivity of OSCC cells to cisplatin.
CEBPA-DT up-regulation correlates to cisplatin resistance in OSCC. (A) CCK-8 assay detected IC50 increase in chemo-resistance HSC4-CisR and Cal-CisR cells compared with normal parental cell lines. (B) qRT-PCR detected up-regulation of CEBPA-DT expression in chemo-resistance cell lines. (C-D) qRT-PCR detected mRNA relative expression of CEBPA and BCL2 in CisR cells relative to untreated control groups. (E) Western blot detected protein expression of CEBPA and BCL2 in CisR cells compared with control OSCC cell lines. N=34 independent experiments, *P < 0.05.
CEBPA-DT downexpression enhances cisplatin sensitivity and facilitates cell apoptosis in cisplatin-resistant OSCC cells. (A) qRT-PCR detected the transfection efficiency of CEBPA-DT smart silencer (ss-CEBPA-DT) and negative control (ss-NC) in Cal27-CisR and HSC4-CisR cells. (B) CCK-8 assay detected the IC50 value decrease in ss-CEBPA-DT transfected chemoresistance cells compared with ss-NC transfected group. (C) qRT-PCR detected CEBPA-DT overexpression (pc-CEBPA-DT) and corresponding control (pc-NC) transfection efficiency in Cal27-CisR and HSC4-CisR cells. (D) CCK-8 assay show IC50 increase in CEBPA-DT overexpression (pc-CEBPA-DT transfected) chemo-resistance cells compared with control (pc-NC transfected) group. (E-F) Cell apoptosis rate was detected in ss-CEBPA-DT and ss-NC transfected chemo-resistance cells through Annexin-V-FITC & PI staining assay (E) and TUNEL staining assay (F). N=35 independent experiments, *P < 0.05.
CEBPA is the potential target of CEBPA-DT in regulating OSCC cisplatin chemosensitivity. (A-B) qRT-PCR detected the CEBPA (A) and BCL2 (B) mRNA expression among ss-CEBPA-DT and ss-NC transfected Cal27-CisR and HSC4-CisR cells. (C) Western blot detected CEBPA and BCL2 protein expression in ss-CEBPA-DT and ss-NC transfected chemo-resistance cells. (D-E) qRT-PCR detected the CEBPA (D) and BCL2 (E) mRNA expression among pc-CEBPA-DT and pc-NC transfected Cal27-CisR and HSC4-CisR cells. (F) Western blot detected CEBPA and BCL2 protein expression in pc-CEBPA-DT and pc-NC transfected chemo-resistance cells. GAPDH was used as reference control. (G) Western blot detected CEBPA expression in cytoplasmic and nuclear protein separated in CisR and control cells, pc-NC and pc-CEBPA-DT transfected CisR cell lines, respectively. N=3 independent experiments, *P < 0.05.
CEBPA-DT regulates cisplatin chemosensitivity through CEBPA/BCL2 signaling pathway. (A) qRT-PCR detected the transfection efficiency of CEBPA overexpression (pc-CEBPA) and corresponding negative control (pc-NC) in ss-CEBPA-DT/ss-NC co-transfected Cal27-CisR and HSC4-CisR cells. (B) CCK-8 assay detected the IC50 value in ss-CEBPA-DT and pc-CEBPA co-transfected chemo-resistance cells. (C) Western blot detected CEBPA and BCL2 protein expression in si-CEBPA and si-NC transfected chemo-resistance cells. (D) CCK-8 assay detected the IC50 value in si-CEBPA and si-NC transfected chemo-resistance cells. (E) Western blot detected BCL2 protein expression in si-BCL2 and si-NC transfected chemo-resistance cells. (F) CCK-8 assay detected the IC50 value in only si-BCL2 transfected, si-BCL2 and pc-CEBPA co-transfected chemo-resistance cells.
To explore the role of the CEBPA/BCL2 axis in the context of CEBPA-DT-mediated OSCC cell chemoresistance, we next overexpressed this transcription factor in OSCC cells transfected with ss-CEBPA-DT (Fig. 4A). Subsequent CCK-8 assays revealed that the overexpression of CEBPA was sufficient to enhance the viability and reduce the CDDP sensitivity of ss-CEBPA-DT co-transfected CDDP-resistant OSCC cells relative to cells transfected with the control pc-NC overexpression construct, increasing IC50 values from 13.470.40 μM and 11.860.36 μM to 21.260.41 μM and 19.530.47 μM (Fig. 4B). We then knocked down CEBPA in these tumor cell lines (Fig. 4C), revealing that this impaired the viability of Cal27-CisR and HSC4-CisR cells (Fig. 4D). Such CEBPA downregulation also significantly impaired BCL2 expression relative to control si-NC transfection in both tested cell lines (Fig. 4C). Moreover, we down-regulated BCL2 expression with siRNA transfection (Fig. 4E) following detected the changes of chemo-sensitivity. The results showed that the IC50 was dramatically decreased in CisR cells co-transfected with pc-CEBPA and BCL2 siRNA relative to those of pc-CEBPA transfected cells, which indicated that CEBPA overexpression induced chemo-sensitivity reduction was greatly rescued by BCL2 knockdown (Fig. 4F).
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