Journal: Molecular Oncology

Article Title: MAP4K4 and WT1 mediate SOX6‐induced cellular senescence by synergistically activating the ATF2–TGFβ2–Smad2/3 signaling pathway in cervical cancer

doi: 10.1002/1878-0261.13613

Figure Lengend Snippet: SOX6 induces the senescence of CC cells. (A) The expression levels of SOX6 mRNA in cervical cancer (CC) tissues and normal cervical tissues were analyzed in GEO datasets. (B) HeLa‐HA‐SOX6‐tet and HeLa‐HA‐SOX6ΔHMG‐tet cells were plated into six‐well plates and were treated with Dox (2 μg·mL −1 ) or solvent control. After incubating for 2 weeks, the cell colonies were stained with 1% crystal violet and observed under natural light and (C) the levels of SOX6 and LaminB1 proteins were detected by western blot. (D) HeLa and CaSki cells were transfected with plex‐HA‐SOX6, plex‐HA‐SOX6ΔHMG, or vector control (CTRL). Four days later, the senescent cells were analyzed by SA‐β‐gal staining. (E) HeLa‐HA‐SOX6‐tet and HeLa‐HA‐SOX6ΔHMG‐tet cells were treated with Dox (2 μg·mL −1 ) or solvent control. Four days later, the senescent cells were analyzed by SA‐β‐gal staining, (F) and the SAHF formation was assessed by H3K9me3 staining. (G) HeLa‐HA‐SOX6‐tet and HeLa‐HA‐SOX6ΔHMG‐tet cells were treated with Dox (2 μg·mL −1 ) or solvent control, and the level of IL‐8 protein in the culture supernatant were detected by ELISA. (H) During the 6 days of Dox treatment, the percentage of senescent HeLa‐HA‐SOX6‐tet cells was analyzed by SA‐β‐gal staining at each day. At the same time, the levels of LC3B‐I and LC3B‐II proteins were analyzed by western blot. (I) HeLa‐HA‐SOX6‐tet cells were transfected with MAP4K4 ‐specific siRNA (siMAP4K4) or control siRNA (siCTRL), and were treated with Dox (2 μg·mL −1 ) or solvent control for 4 days. The senescent cells were analyzed by SA‐β‐gal staining. α‐tubulin was used as the internal control in western blot analysis. The percentage of SA‐β‐gal‐positive cells was analyzed at three fields. Data were shown as mean ± SEM of three independent experiments. ** P < 0.01, *** P < 0.001, ns, nonsignificant, Student's t ‐test. CC, cervical cancer; Dox, doxycycline; SAHF, senescence‐associated heterochromatin foci.

Article Snippet: The human CC cell lines HeLa (RRID: CVCL_0030), CaSki (RRID: CVCL_1100) and SiHa (RRID: CVCL_0032) used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were authenticated by short tandem repeat profiling.

Techniques: Expressing, Solvent, Control, Staining, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: MAP4K4 and WT1 mediate SOX6‐induced cellular senescence by synergistically activating the ATF2–TGFβ2–Smad2/3 signaling pathway in cervical cancer

doi: 10.1002/1878-0261.13613

Figure Lengend Snippet: TGFβ2–Smad–p53–p21 WAF1/CIP1 pathway mediates the SOX6‐induced senescence of CC cells. (A) HeLa and CaSki cells were transfected with plex‐HA‐SOX6, plex‐HA‐SOX6ΔHMG, or vector control (CTRL), (B) HeLa‐HA‐SOX6 cells were treated with Dox (2 μg·mL −1 ) or solvent control, (C) and HeLa‐HA‐SOX6‐tet cells were transfected with TGFB2 ‐specific siRNA‐1 (siTGFB2‐1), TGFB2 ‐specific siRNA‐2 (siTGFB2‐2), or control siRNA (siCTRL). The levels of TGFβ2–Smad signal pathway‐related proteins and senescence‐related proteins were analyzed by western blot. (D) HeLa‐HA‐SOX6‐tet cells transfected with siTGFB2‐1, siTGFB2‐2, or siCTRL were treated with Dox (2 μg·mL −1 ) or solvent control for 4 days, and the senescent cells were analyzed by SA‐β‐gal staining. (E) The cell viability was analyzed by CCK‐8 assays. (F) HeLa‐HA‐SOX6‐tet cells were transfected with CDKN1A ‐specific siRNA‐1 (si‐CDKN1A‐1), CDKN1A ‐specific siRNA‐2 (si‐CDKN1A‐2), or control siRNA (siCTRL) and were treated with Dox (2 μg·mL −1 ) or solvent control for 4 days. The protein levels of HA‐SOX6 and p21 WAF1/CIP1 were measured by western blot, (G) and the senescent cells were analyzed by SA‐β‐gal staining. The percentage of SA‐β‐gal‐positive cells was analyzed at three fields. ACTB mRNA and β‐Actin or α‐tubulin protein was used as the internal controls for RT‐qPCR and western blot, respectively. Data were shown as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, nonsignificant; Student's t ‐test. CC, cervical cancer; Dox, doxycycline.

Article Snippet: The human CC cell lines HeLa (RRID: CVCL_0030), CaSki (RRID: CVCL_1100) and SiHa (RRID: CVCL_0032) used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were authenticated by short tandem repeat profiling.

Techniques: Transfection, Plasmid Preparation, Control, Solvent, Western Blot, Staining, CCK-8 Assay, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: MAP4K4 and WT1 mediate SOX6‐induced cellular senescence by synergistically activating the ATF2–TGFβ2–Smad2/3 signaling pathway in cervical cancer

doi: 10.1002/1878-0261.13613

Figure Lengend Snippet: ABT‐263 and ABT‐199 overcome the SOX6‐mediated cisplatin resistance. (A) HeLa‐HA‐SOX6‐tet and (B) CaSki‐HA‐SOX6‐tet cells were treated with gradient concentration of cisplatin, and the cell viability was assessed by CCK‐8 assays. (C) HeLa‐HA‐SOX6‐tet cells were pretreated with Dox (2 μg·mL −1 ) or solvent control, and then were treated with cisplatin (10 μM) and ABT‐263 (5 μM) or solvent control. The levels of apoptosis‐related proteins were detected by western blot. α‐tubulin was used as the internal control. (D) HeLa‐HA‐SOX6‐tet cells were pretreated with Dox (2 μg·mL −1 ) or solvent control, and then were treated with cisplatin (10 μM) and ABT‐263 (10 μM), ABT‐199 (10 μM), or solvent control. The levels of apoptosis‐related proteins were detected by western blot, β‐Actin was used as the internal control, (E) and the cell viability was assessed by CCK‐8 assays. (F) HeLa‐HA‐SOX6‐tet cells were subcutaneously injected into the left flank of 15 BALB/c nude mice. After 1 week of injection, the mice were intraperitoneally injected with Dox (20 mg·kg −1 ) for 1 week, and then the mice were divided into three groups ( n = 5/group). The mice of group I were intraperitoneally injected with Dox and the solvent control of cisplatin, and were orally fed with the solvent control of ABT‐263 every other day; The mice of group II were intraperitoneally injected with Dox and cisplatin (3 mg·kg −1 ), and were orally fed with the solvent control of ABT‐263 every other day; The mice of group III were intraperitoneally injected with Dox and cisplatin (3 mg·kg −1 ), and were orally fed with ABT‐263 (100 mg·kg −1 ) every other day. After 3 weeks of the combined treatment with cisplatin and ABT‐263, the mice were sacrificed under anesthesia. (G) The volumes of xenograft tumors were measured every week. (H) The weights of tumor blocks were measured. Data were shown as mean ± SEM of three independent experiments. ** P < 0.01; *** P < 0.001; ns, nonsignificant; Student's t ‐test. Dox, doxycycline; i.p., intraperitoneal injection; o.g., oral gavage.

Article Snippet: The human CC cell lines HeLa (RRID: CVCL_0030), CaSki (RRID: CVCL_1100) and SiHa (RRID: CVCL_0032) used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were authenticated by short tandem repeat profiling.

Techniques: Concentration Assay, CCK-8 Assay, Solvent, Control, Western Blot, Injection