Journal: bioRxiv

Article Title: HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

doi: 10.1101/2021.06.09.447550

Figure Lengend Snippet: (A) Immunoblot analyses of differentiated cell markers TUJ1 (a neuronal marker) and GFAP (an astrocyte marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and harvested for immunoblot analyses with specific antibodies against TUJ1, GFAP, HAUSP, and GAPDH (loading control). (B) Quantitative PCR (RT-PCR) analyses of TUJ1 and GFAP mRNA levels in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for the indicated days, and harvested for RT-PCR analyses. Student t test was used to assess the significance. ***, p<0.001. ( C and D ) Immunofluorescent staining of TUJ1 (a neuronal marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and immunostained with a specific antibody against TUJ1 (in green) and counterstained with DAPI to indicate nuclei (in blue). Scale bar, 30 µm. Quantifications of TUJ1-positive cells were shown (D). Student t test was used to assess the significance. ns, no significant difference; ***, p<0.001. ( E and F ) Immunofluorescent staining of GFAP (an astrocyte marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and immunostained with a specific antibody against GFAP (in green) and counterstained with DAPI to indicate nuclei (in blue). Scale bar, 30 µm. Quantifications of GFAP-positive cells were shown (F). Student t test was used to assess the significance. ns, no significant difference; ***, p<0.001.

Article Snippet: Specific antibodies against HAUSP (Bethyl, A300-033A), SOX2 (Bethyl, A201-741A; or Santa Cruz, SC-365964), OLIG2 (R&D System, AF2418), GFAP (Biolegend, 644702), TUJ1 (Biolegend, 801201), MAP2 (Biolegend, 801801), GAPDH (R&D System, MAB5718), HA (Sigma, 11867423001), α-Tubulin (Sigma, T6199), Flag (Sigma, F1804), Ubiquitin (Biolegend, 646302), Cleaved Caspase 3 (Cell Signaling, 9661), Cleaved PARP (Cell Signaling, 5625), and anti-Flag-agarose (Sigma, A2220) were used for immunoblot and/or immunoprecipitation.

Techniques: Western Blot, Marker, Expressing, Control, Transduction, Infection, Cell Culture, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: bioRxiv

Article Title: The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division

doi: 10.1101/2020.06.04.134734

Figure Lengend Snippet: Myl5 localizes to mitotic spindle poles during mitosis. ( A ) The LAP (GFP-TEV-S-Peptide)-tagged-Myl5 HeLa inducible stable cell line was treated with Dox for 16 hours to express GFP-Myl5 and cells were fixed, stained with Hoechst 33342 DNA dye, and anti-a-Tubulin and anti-GFP antibodies and imaged by immunofluorescence microscopy. Images show the cell cycle subcellular localization of GFP-Myl5 during interphase, prometaphase, metaphase, anaphase and cytokinesis. Note that GFP-Myl5 localizes to the mitotic spindle poles during mitosis. Bar indicates 5μm. ( B-C ) Same as in A , except that cells were also stained with antiNUMA ( B ) or anti-Pericentrin ( C ) antibodies. Note that GFP-Myl5 localizes to the mitotic spindle poles overlapping with the NUMA and Pericentrin localization signal. Bar indicates 5μm. ( D ) Analysis of endogenous Myl5 protein levels throughout the cell cycle. HeLa cells were synchronized in G1/S, released into the cell cycle and cells were harvested at the indicated time points. Protein extracts were prepared, resolved by SDS-PAGE, transferred to a PVDF membrane and immunoblotted with the indicated antibodies. Note that the Myl5 protein levels remain constant throughout the cell cycle, whereas the Cyclin B levels increase as the cells enter mitosis (6-10 hour time points). Gapdh is used a loading control.

Article Snippet: Immunofluorescence, immunoblotting, and immunoprecipitations were carried out using antibodies against: Myl5 and MYO10 (Proteintech: 14249-1-AP and 24565-1-AP); Pericentrin (Novus Biologicals: NB-100-68277); GFP (Abcam: ab290); Gapdh, S-tag (GeneTex: GTX128060 and GTX100118); a-Tubulin (Serotec: MCAP77); Cyclin B (Santa Cruz: sc-245).

Techniques: Stable Transfection, Staining, Immunofluorescence, Microscopy, SDS Page, Membrane, Control