Journal: Cell death and differentiation

Article Title: CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway.

doi: 10.1038/s41418-021-00828-6

Figure Lengend Snippet: Fig. 5 PAK4 mediates the oncogenic effect of CDK15 in colorectal cancer. A p-β-catenin(Ser675), c-Myc, p-MEK1/2 (Ser217/221), and p-ERK1/ 2 (Thr202/Tyr204) were detected by western blot after CDK15 knockdown in CCD18-co, SW480 and HCT116 cells. B Cells with PAK4 silencing and CDK15 overexpression were established. PAK4 and CDK15 expression was determined by western blot. C PAK4 knockdown reverses cell proliferation induced by CDK15 in CCD18-co and HCT116 cells. MTT assay was used to detect cell proliferation. D Anchorage-independent growth in CCD18-co and HCT116 cells with PAK4 silencing and CDK15 overexpression. Left panels: representative images (Scale bar: 200 μm). Right panels: Colonies were counted using Image J-Plus (Scale bar: 200 μm) and data represented statistical analysis of colony number ratio. E Anchorage-independent growth in CCD18-co and HCT116 cells treated with PAK4 inhibitor (PF-3758309). Left panel: representative images of colonies (Scale bar: 200 μm). Right panel: statistical analysis of the colony ratio. F Western blot to validate β-catenin and MEK/ERK signaling pathway in HCT116 cells with indicated treatment. Data were presented as mean values ± SD from triplicate experiments. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used in our study: anti-CDK15 (Cat#PA5-28595, Invitrogen), anti-CDK15 (Cat#TA811934, ORIGENE), anti-PAK4 (Cat#sc-390507, Santa Cruz), anti- phospho-β-catenin (Ser675) (Cat#4176, Cell Signaling Technology), anti-β-catenin (Cat # 8480, Cell Signaling Technology), antiphospho-ERK1/2 (Thr202/Tyr204)(Cat# 4370, Cell Signaling Technology), Anti-ERK1/2 (Cat#4695, Cell Signaling Technology), anti-phospho-MEK1/2 (Ser217/221)(Cat# 9154, Cell Signaling Technology), anti-MEK1/2 (Cat# 4694, Cell Signaling Technology), anti-c-Myc antibody (Cat#ab32072, Abcam), anti-Flag (Cat #F1804, Sigma), anti-HA (Cat #3724, Cell Signaling Technology), anti-His (Cat #ab137839, Abcam), anti-GAPDH (#HRP-60004, Proteintech), anti-β-actin (#HRP-60008, Proteintech).

Techniques: Western Blot, Knockdown, Over Expression, Expressing, MTT Assay

Journal: Cell death and differentiation

Article Title: CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway.

doi: 10.1038/s41418-021-00828-6

Figure Lengend Snippet: Fig. 7 Targeting PAK4 delays tumor growth in patient-derived xenografts. A Clinical information for HJG208 and HJG210 from patient’s cancer tissues. B, C 0.9% NaCl as vehicle, 5 mg/kg or 20 mg/kg PF-3758309 were intraperitoneally injected once per day for 20 days, and tumor volume was monitored every 2–5 days (n = 9–10 mice per group). D, E Tumor photographs. F, G Tumor weight and tumor growth inhibition (H, I) normalized to control group. J Levels of p-β-catenin (Ser675), c-Myc, p-MEK1/2 (Ser217/221), and p-ERK1/2 (Thr202/Tyr204) in harvested tumor tissues were assessed by immunohistochemistry. Representative photographs for each antibody in different groups are shown (100×; Scale bar: 50 μm). K Statistical analysis for immunohistochemistry staining. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± SD.

Article Snippet: The following antibodies were used in our study: anti-CDK15 (Cat#PA5-28595, Invitrogen), anti-CDK15 (Cat#TA811934, ORIGENE), anti-PAK4 (Cat#sc-390507, Santa Cruz), anti- phospho-β-catenin (Ser675) (Cat#4176, Cell Signaling Technology), anti-β-catenin (Cat # 8480, Cell Signaling Technology), antiphospho-ERK1/2 (Thr202/Tyr204)(Cat# 4370, Cell Signaling Technology), Anti-ERK1/2 (Cat#4695, Cell Signaling Technology), anti-phospho-MEK1/2 (Ser217/221)(Cat# 9154, Cell Signaling Technology), anti-MEK1/2 (Cat# 4694, Cell Signaling Technology), anti-c-Myc antibody (Cat#ab32072, Abcam), anti-Flag (Cat #F1804, Sigma), anti-HA (Cat #3724, Cell Signaling Technology), anti-His (Cat #ab137839, Abcam), anti-GAPDH (#HRP-60004, Proteintech), anti-β-actin (#HRP-60008, Proteintech).

Techniques: Derivative Assay, Injection, Inhibition, Control, Immunohistochemistry, Staining

Journal: Cell death and differentiation

Article Title: CDK15 promotes colorectal cancer progression via phosphorylating PAK4 and regulating β-catenin/ MEK-ERK signaling pathway.

doi: 10.1038/s41418-021-00828-6

Figure Lengend Snippet: Fig. 8 Lentivirus-mediated CDK15 silencing inhibits colorectal tumor growth in patient-derived xenografts. A Clinical information for HJG86 from patient’s cancer tissues. B Mice received the lentiviruses (shNT, shCDK15-3, shCDK15-7) via intratumoral injection every 3 days for a total of four times. Tumor volume was monitored every 2–5 days for four continuous weeks (n = 8 mice per group). C Tumors photographs. D Tumor weight measured at the end of the study. E Tumor growth inhibition normalized to control group. F Levels of CDK15, p-β-catenin (Ser675), c-Myc, p-MEK1/2(Ser217/221), and p-ERK1/2 (Thr202/Tyr204) in harvested tissues were assessed by immunohistochemistry. Representative photographs in different groups are shown (100×; Scale bar: 50 μm). G Statistical analysis for immunohistochemistry staining. H Schematic model for the findings of this work: Aberrant CDK15 in CRC binds PAK4 and phosphorylates PAK4 at the S291 site. Accumulated phosphorylation of S291 upregulates the β-catenin/c-Myc and MEK/ERK signals, which in turn contribute to CRC tumor growth. Statistical differences were evaluated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean ± SD.

Article Snippet: The following antibodies were used in our study: anti-CDK15 (Cat#PA5-28595, Invitrogen), anti-CDK15 (Cat#TA811934, ORIGENE), anti-PAK4 (Cat#sc-390507, Santa Cruz), anti- phospho-β-catenin (Ser675) (Cat#4176, Cell Signaling Technology), anti-β-catenin (Cat # 8480, Cell Signaling Technology), antiphospho-ERK1/2 (Thr202/Tyr204)(Cat# 4370, Cell Signaling Technology), Anti-ERK1/2 (Cat#4695, Cell Signaling Technology), anti-phospho-MEK1/2 (Ser217/221)(Cat# 9154, Cell Signaling Technology), anti-MEK1/2 (Cat# 4694, Cell Signaling Technology), anti-c-Myc antibody (Cat#ab32072, Abcam), anti-Flag (Cat #F1804, Sigma), anti-HA (Cat #3724, Cell Signaling Technology), anti-His (Cat #ab137839, Abcam), anti-GAPDH (#HRP-60004, Proteintech), anti-β-actin (#HRP-60008, Proteintech).

Techniques: Derivative Assay, Injection, Inhibition, Control, Immunohistochemistry, Staining, Phospho-proteomics

Journal: The Journal of cell biology

Article Title: The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

doi: 10.1083/jcb.201408060

Figure Lengend Snippet: Figure 4. Rpgrip1l deficiency causes impaired proteasomal activity at primary cilia. (A–F and I–L) MEFs were isolated from E12.5 WT and Rpgrip1l−/− embryos. (A and B) Western blot analysis of WT and Rpgrip1l−/− MEF lysates (n = 4 embryos, respectively). (C) Western blot analysis of WT and Rpgrip1l−/− MEF lysates (n = 3 embryos, respectively). (A–C) Actin serves as a loading control. (A) Phospho-(S33/37/T41)-β-Catenin is significantly increased in serum-starved Rpgrip1l−/− MEF lysates (82% of all cells had cilia; in serum-starved Rpgrip1l+/+ MEF lysates, 88.67% of all cells possessed cilia) but not in non–serum-starved Rpgrip1l−/− MEF lysates (4% of all cells displayed cilia; in non–serum-starved Rpgrip1l+/+ MEF lysates, 6.67% of all cells carried cilia; C). (B) Non–phospho-(S33/37/T41)-β-Catenin is unaltered in serum-starved Rpgrip1l−/− MEF lysates. Black lines indicate that interven- ing lanes have been spliced out. (D–F, I, and L) Immunofluorescence on MEFs of E12.5 WT and Rpgrip1l−/− embryos (both genotypes: p-β-Catenin: n = 5; p-β-Catenin (3D-SIM, n = 3; Ubiquitin, n = 4; Gli3-190, n = 6; ZsProSensor-1, n = 3; n refers to the number of embryos, respectively). Per embryo, 15 cilia were quantified for p-β-Catenin, 10 cilia were quantified for p-β-Catenin (3D-SIM) and for Ubiquitin, and 20 cilia were quantified for Gli3-190. (G and H) Immunofluorescence on limbs of E12.5 WT and Rpgrip1l−/− embryos (n = 3 embryos, respectively). Per embryo, 20 cilia were quantified for p-β-Catenin and Ubiquitin. All quantified proteins are shown in red (D–J), the ciliary axoneme is marked by acetylated α-tubulin (green; D–J), and the BB is marked by γ-tubulin (blue; D–F, H, and I) or by Pcnt2 (blue; G). (J and K) Immunofluorescence on MEFs of WT embryos (n = 4). 25 cilia per embryo were used for phospho-(S33/37/T41)-β-Catenin and cilia length quantification. (L) Proteasome activity assay on WT and Rpgrip1l−/− MEFs. Cilia are marked by acetylated α-tubulin (α-Tub), and centrosomes/basal bodies are marked by γ-tubulin. Colored squares mark cilia with basal bodies (yellow squares) as well as centrosomes (red squares), which are presented magnified. The green ZsProSensor-1 protein signal is exclusively detected at the ciliary base in Rpgrip1l−/− MEFs. Error bars show standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 1 µm.

Article Snippet: We used the following primary antibodies: rabbit anti-actin (A2066; Sigma-Aldrich), mouse anti-FLAG (200472; Agilent Technologies), mouse anti-Gapdh (G8795; Sigma-Aldrich), goat anti-Gli3 (AF3690; R&D systems), mouse anti-HA (MMS-101P; Covance), rabbit anti-Hprt (ab10479; Abcam), rabbit anti-Myc (sc-789; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-(S33/37/T41)-β-Catenin (9561; Cell Signaling Technology), rabbit anti–nonphospho-(S33/37/ T41)-β-Catenin (4270; Cell Signaling Technology), rabbit anti-Psma5 (PA1-1962; Thermo Fisher Scientific), mouse anti-Pcnt (611814; BD), goat anti-Pcnt2 (sc-28145; Santa Cruz Biotechnology, Inc.), goat anti-Psmd2 (IMG-3751; Imgenex), rabbit anti-Psmd3 (S2824; Sigma-Aldrich), rabbit anti-Psmd4 (14899-1-AP; Proteintech), rabbit anti-Smo (ab72130; Abcam), mouse anti–acetylated α-tubulin (T6793; Sigma-Aldrich), mouse anti–γ-tubulin (T6557; Sigma-Aldrich), and rabbit anti-Ubiquitin (U5379; Sigma-Aldrich).

Techniques: Activity Assay, Isolation, Western Blot, Control, Immunofluorescence, Ubiquitin Proteomics

Journal: The Journal of cell biology

Article Title: The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

doi: 10.1083/jcb.201408060

Figure Lengend Snippet: Figure 8. Proteasomal activity is unaltered at Rpgrip1l−/− centrosomes. (A–C) Immunofluorescence on MEFs isolated from E12.5 WT and Rpgrip1l−/− embryos (WT: p-β-Catenin (treated with DMSO or MG132): n = 3 embryos; both genotypes: p-β-Catenin and Ubiquitin: n = 3 embryos, respectively). The ciliary axoneme is marked by acetylated α-tubulin (green) and the centrosomes (basal bodies in case of ciliary presence) by γ-tubulin (blue). All quantified proteins are shown in red. An axonemal-like green staining is not visible, demonstrating that the blue staining marks centrosomes. (A) After treatment of WT MEFs with the proteasome inhibitor MG132, the amount of phospho-(S33/37/T41)-β-Catenin is significantly increased at the centrosome. (B and C) The amounts of phospho-(S33/37/T41)-β-Catenin and Ubiquitin are unaltered at the centrosome of Rpgrip1l−/− MEFs. (A–C) Per embryo, 20 cilia were used in the quantifications. Error bars show standard error of the mean. *, P < 0.05. Bars, 0.5 µm.

Article Snippet: We used the following primary antibodies: rabbit anti-actin (A2066; Sigma-Aldrich), mouse anti-FLAG (200472; Agilent Technologies), mouse anti-Gapdh (G8795; Sigma-Aldrich), goat anti-Gli3 (AF3690; R&D systems), mouse anti-HA (MMS-101P; Covance), rabbit anti-Hprt (ab10479; Abcam), rabbit anti-Myc (sc-789; Santa Cruz Biotechnology, Inc.), rabbit anti-phospho-(S33/37/T41)-β-Catenin (9561; Cell Signaling Technology), rabbit anti–nonphospho-(S33/37/ T41)-β-Catenin (4270; Cell Signaling Technology), rabbit anti-Psma5 (PA1-1962; Thermo Fisher Scientific), mouse anti-Pcnt (611814; BD), goat anti-Pcnt2 (sc-28145; Santa Cruz Biotechnology, Inc.), goat anti-Psmd2 (IMG-3751; Imgenex), rabbit anti-Psmd3 (S2824; Sigma-Aldrich), rabbit anti-Psmd4 (14899-1-AP; Proteintech), rabbit anti-Smo (ab72130; Abcam), mouse anti–acetylated α-tubulin (T6793; Sigma-Aldrich), mouse anti–γ-tubulin (T6557; Sigma-Aldrich), and rabbit anti-Ubiquitin (U5379; Sigma-Aldrich).

Techniques: Activity Assay, Immunofluorescence, Isolation, Ubiquitin Proteomics, Staining

Journal: Viruses

Article Title: Rotavirus-Mediated Suppression of miRNA-192 Family and miRNA-181a Activates Wnt/β-Catenin Signaling Pathway: An In Vitro Study

doi: 10.3390/v14030558

Figure Lengend Snippet: RV infection triggers β-Catenin accumulation. Cells infected with RV were harvested at different time points followed by protein expression analysis by Western blotting and Confocal microscopy techniques. ( A ) RV upregulates β-catenin expression during early (3–12 h) hours of infection. Expression of phospho β-catenin Ser37 downregulated in RV infected cells. Expression of phospho β-catenin Ser552 upregulated in RV infected cells; ( B ) Changes in the expression levels of different regulators of the Wnt/β-catenin cellular signaling pathway. Expression of ICAT downregulated in RV infected cells. SMAD4 is activated during RV infection and acts as an attenuator of β-catenin proteasomal degradation. The downstream molecule of β-catenin and SMAD4, TCF1/7 and CCND2 are also activated during RV infection; ( C ) Phosphorylation at Ser552 and destabilization of Ser37 induces β-catenin accumulation in the cytoplasm and nucleus as evident from IF imaging. Scale bars: 50 μm.

Article Snippet: The membranes were then probed with antibodies specific to the proteins phospho-β-catenin ser33/37 (Santa Cruz Biotechnology, Santa Cruz, CA, USA: 57535); phospho-β-catenin ser552 (Cell Signaling Technology, Danvers, MA, USA: 5651); β-catenin (Cell Signaling Technology, Danvers, MA, USA: 8480); ICAT (Santa Cruz Biotechnology: 293489); SMAD4 (Abcam, Waltham, MA, USA: 110175); TCF1/7 (Cell Signaling Technology, Danvers, MA, USA: 2203); CCND2 (Santa Cruz Biotechnology: 56305); Wnt-1 (Santa Cruz Biotechnology: 514531); Wnt-5a (Santa Cruz Biotechnology: 365370); phospho-LRP6 (Cell Signaling Technology, Danvers, MA, USA: 2568); LRP6 (Cell Signaling Technology, Danvers, MA, USA: 3395); phospho-Axin (Merck Millipore, Burlington, MA, USA: ABN1032); Axin (Santa Cruz Biotechnology: 293190); Naked-1 (Cell Signaling Technology, Danvers, MA, USA: 2201), RV-NSP1 (a kind gift from Prof. Koki Taniguchi); RV-NSP4 (prepared according to the standard protocol from Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan); and RV- VP6 (HyTest, Turku, Finland: 3C10).

Techniques: Infection, Expressing, Western Blot, Confocal Microscopy, Phospho-proteomics, Imaging

Journal: Viruses

Article Title: Rotavirus-Mediated Suppression of miRNA-192 Family and miRNA-181a Activates Wnt/β-Catenin Signaling Pathway: An In Vitro Study

doi: 10.3390/v14030558

Figure Lengend Snippet: Wnt-1/Wnt-5a pathway activates during RV infection. ( A ) Expression of Wnt-1 and Wnt-5a have been gradually upregulated during RV infection. LRP6 has also been hyper phosphorylated during RV infection; ( B ) Phosphorylation of Axin and its total component have been reduced in RV infected cells. Expression of Nkd1 is suppressed in RV infected cells; ( C ) Phosphorylation of β-catenin Ser552 significantly inhibited in siRNA-FzD9 treated but RV infected cells. The total β-catenin level is also suppressed. The expressions of Wnt-1 and Wnt-5a are also inhibited in siRNA-FzD9 treated but RV infected cells. This may be the result of negative feedback mechanism of Nkd1 or Axin or may be that some other factors are involved in this pathway. The siRNA-FzD9 restricted the RV infection too, as evident by RV-NSP4 expression.

Article Snippet: The membranes were then probed with antibodies specific to the proteins phospho-β-catenin ser33/37 (Santa Cruz Biotechnology, Santa Cruz, CA, USA: 57535); phospho-β-catenin ser552 (Cell Signaling Technology, Danvers, MA, USA: 5651); β-catenin (Cell Signaling Technology, Danvers, MA, USA: 8480); ICAT (Santa Cruz Biotechnology: 293489); SMAD4 (Abcam, Waltham, MA, USA: 110175); TCF1/7 (Cell Signaling Technology, Danvers, MA, USA: 2203); CCND2 (Santa Cruz Biotechnology: 56305); Wnt-1 (Santa Cruz Biotechnology: 514531); Wnt-5a (Santa Cruz Biotechnology: 365370); phospho-LRP6 (Cell Signaling Technology, Danvers, MA, USA: 2568); LRP6 (Cell Signaling Technology, Danvers, MA, USA: 3395); phospho-Axin (Merck Millipore, Burlington, MA, USA: ABN1032); Axin (Santa Cruz Biotechnology: 293190); Naked-1 (Cell Signaling Technology, Danvers, MA, USA: 2201), RV-NSP1 (a kind gift from Prof. Koki Taniguchi); RV-NSP4 (prepared according to the standard protocol from Department of Virology and Parasitology, Fujita Health University School of Medicine, Aichi, Japan); and RV- VP6 (HyTest, Turku, Finland: 3C10).

Techniques: Infection, Expressing, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling.

doi: 10.1074/jbc.M406467200

Figure Lengend Snippet: FIG. 7. Inhibition of PI3Ks does not dramatically alter -catenin phosphorylation or levels. A, E14tg2a ES cells (left hand panel) and E14p85 ES cells (right hand panel) treated as described in the legend to Fig. 1 (A and B). B, cell lysates were prepared from E14tg2a (left hand panel) incubated in the absence or presence of 5 M LY294002 for 4–6 days or E14p85 ES cells (right hand panel) that had been induced to express p85 by the removal of tetracycline (Tet) for the times shown or maintained in Tet as a control. 20 g of each protein sample were separated through either 7.5% (for anti--catenin blots) or 10% (for anti-pPKBsub blotting) acrylamide gels by SDS-PAGE. Immunoblotting was carried out first with antibody specific for -catenin phosphorylated at serines 33 and 37 and threonine 41. Blots were stripped and reprobed with anti-pan -catenin antibody and then stripped again and probed with either SHP-2 or p85 antibodies to assess equivalence of loading and expression of p85. Immunoblotting of the same samples shown in B (left hand panel) with the pPKBsub antibody was used to the demonstrate the sustained inhibitory effect of LY294002 over the time course.

Article Snippet: Primary antibodies were used at the following dilutions for immunoblotting: 0.1 g/ml mouse monoclonal antibody recognizing phosphotyrosine 4G10 (Upstate Biotechnology, 05–321); 1:1000 for rabbit polyclonal antibodies recognizing dual phosphorylation of ERK1 and 2 at Thr202/Tyr204 (anti-pERK, Cell Signaling Technology 9101), phosphotyrosine 705 of STAT3 (anti-pSTAT3, CST 9131), phosphoserine 473 of PKB (anti-pPKB, CST 9271), phosphoserines 21 or 9 of GSK-3 / (anti-pGSK3 / , CST 9331), anti-phospho(Ser/ Thr)PKB substrate (anti-pPKBSub, CST 9611, which was used for detection of phosphorylated S6 protein), phosphoserine 33/37 phosphothreonine 41 -catenin (anti-p -catenin, CST 9561), anti-pan -catenin (CST 9562), anti-pan PKB (CST 9272); 1:2000 anti-total ERK (panERK, Santa Cruz Biotechnology, sc-93), anti-STAT3 (panSTAT3, sc-482), anti-STAT5 (sc-835), anti-SHP-1 (sc-287), anti-SHP-2 (sc 293), anti-Oct4 (sc-9081), and anti-p85 (Upstate Biotechnology, 06–195).

Techniques: Inhibition, Phospho-proteomics, Incubation, Control, SDS Page, Western Blot, Expressing