Journal: Oncotarget

Article Title: Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells

doi: 10.18632/oncotarget.12236

Figure Lengend Snippet: HaCaT A. - D. or MCF-7 (E) cells were plated for either 6 (A, B, C) or 16 hours (D, E) and stained for β-actin, γ-actin and α-tubulin. Images represent single X/Y sections (A, C, D) and Z section (D, bottom image). Panel B and E represent galleries of optical sections taken with 0.5 μm (B) or 0.3 μm E. step from the ventral (close to the substrate, first image) to the dorsal (last image) side of the HaCaT (B) cell shown in Fig or MCF7 cell (E). Microtubules are distributed in close proximity to the γ-actin network, but not codistributed with the β-actin bundles. Bars, 5 μm (C) and 10 μm.

Article Snippet: Human breast adenocarcinoma MCF7 cell line (ATCC ® HTB22TM) and human immortalized keratinocytes HaCaT (CLS # 300493) were used. pLKO.1 lentiviral DNA constructs together with the pΔR8.2 (#12263, Addgene) and pVSV-G (#8454, Addgene) packaging plasmids were transfected into 293FT packaging cells (R70007, ThermoFisher) using TurboFect Transfection Reagent (R0531, Thermo Scientific).

Techniques: Staining

Journal: bioRxiv

Article Title: IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events

doi: 10.1101/2023.03.05.531184

Figure Lengend Snippet: (A) Cryo-EM structure (PDB: 7PKS) of the human Integrator complex, highlighting the positions of the RNA endonuclease IntS11 (green), IntS6 (orange), and PP2A subunits (teal and pink). There are direct contacts between IntS6 and the PP2A subunits. (B) DL1 cells were treated for 3 d with control (β-gal), Pp2A-29B, or mts dsRNAs and RNA-seq data generated. The sets of endogenous genes up-regulated upon Pp2A-29B or mts depletion (fold change > 1.5 and adjusted P value < 0.001) were compared to the set of 107 genes that were up-regulated upon over-expression of IntS6. (C) Parental DL1 cells (Control) and DL1 cells stably maintaining inducible FLAG-tagged IntS6 or IntS12 transgenes were treated with 500 μM CuSO 4 for 24 h to induce transgene expression. Immunoprecipitation (IP) using anti-FLAG resin was then performed. Western blots of input nuclear extracts (left) and IP (right) are shown. (D) DL1 cells were co-transfected with 300 ng of eGFP reporter plasmid and 100 ng of the indicated IntS6/PP2A subunit over-expression plasmids (driven by the Ubi-p63e promoter). Empty vector (pUb 3xFLAG MCS) was added as needed so that 500 ng DNA was transfected in all samples. CuSO 4 was added for the last 14 h only when measuring eGFP production from the MtnA promoter. Northern blots (20 μg/lane) were used to quantify expression of each eGFP reporter mRNA. Representative blots are shown and RpL32 mRNA was used as a loading control. Data are shown as mean ± SD, N = 3. (*) P < 0.05; n.s. , not significant.

Article Snippet: The C-terminal region of Drosophila IntS11 (amino acids 300-597) was cloned into pRSFDuet-1 using the SalI and HindIII restriction enzyme sites to generate pRSFDuet-1 IntS11 AA 300-597 (Addgene #199329).

Techniques: Cryo-EM Sample Prep, Control, RNA Sequencing, Generated, Over Expression, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Northern Blot

Journal: bioRxiv

Article Title: IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events

doi: 10.1101/2023.03.05.531184

Figure Lengend Snippet: (A) In the absence of Integrator, Pol II is able to productively elongate. (B) Integrator recruitment facilitates transcription termination. (Left) At some protein-coding genes, the Integrator phosphatase module must act prior to or, at minimum, simultaneously with the IntS11 endonuclease to enable cleavage of the nascent RNA, which is subsequently degraded by the RNA exosome. (Right) In contrast, the phosphatase module is dispensable for Integrator function at snRNA and many other protein-coding gene loci. Cleavage by IntS11 enables stable snRNAs to be produced and prematurely terminated mRNAs to be rapidly degraded.

Article Snippet: The C-terminal region of Drosophila IntS11 (amino acids 300-597) was cloned into pRSFDuet-1 using the SalI and HindIII restriction enzyme sites to generate pRSFDuet-1 IntS11 AA 300-597 (Addgene #199329).

Techniques: Produced