hela tet on 3g cells  (TaKaRa)

Journal: bioRxiv

Article Title: Chemical control of 2’-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

doi: 10.64898/2026.01.26.701763

Figure Lengend Snippet: ( A ) Schematic of the crRNA sequence used for targeting an EGFP reporter gene and chemical modifications used to probe 2’-OH contacts. In vitro cleavage activity (orange) and cell-based EGFP editing (blue) is shown on the right. 2’-OH contacts with SpCas9 are indicated with red asterisks below. n = 3 or more experimental replicates. Error bars are S.E.M. ( B ) Time-course of in vitro cleavage activity using select crRNAs from panel A. Curves were fitted to an exponential two-phase decay equation. Error is reported as S.E.M. ( C ) Thermal denaturation of Cas9 RNP complexes assayed by absorbance at 280 nm to measure melting temperature ( T m ). n = 2 experimental replicates. Error bars are S.E.M. ( D ) Target DNA binding by dCas9 RNP measured by dot blot filter binding of radiolabeled target DNA. Curves were fit to a one-site binding curve. n = 2 experimental replicates. Error bars are S.E.M. ( E ) CRISPRa-based assay to measure dCas9-VPR binding guided by crTREa crRNA to a Tet-On 3G promoter driving EGFP in HeLa cells. Unmodified crTREa is shown as a control. EGFP expression was quantified by flow cytometry. N = 3 experimental replicates, Error is S.E.M.

Article Snippet: HeLa Tet-On 3G cells (Takara, Cat. #631183) were transduced according to the manufacturer’s recommended protocol.

Techniques: Sequencing, In Vitro, Activity Assay, Binding Assay, Dot Blot, Control, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a Schematic illustration of the identification of RNAs destabilized by MTR4. (left) To identify transcript variant repertoire upregulated upon MTR4 depletion, HeLa cells transfected with siCont or siMTR4 were subjected to short- and long-read sequencing, as presented in Supplementary Fig. . Pale-colored boxes are transcripts identified in this study. (right) To identify the transcript variant stabilized by MTR4 depletion, HeLa cells transfected with siCont or siMTR4 were treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a transcription inhibitor, for the indicated times and then subjected to 3′-sequencing to estimate stability. By combining these data, MTR4-target transcripts were determined. b Schematic representation of two types of 3′ e X tended T ranscript (3XT) and 3′ e X tended R egions (3XRs). The middle exon is the exon other than the first or last exon in annotated transcripts. 3XTs are transcripts that have an extended last exon with (multi-exon 3XT) or without (mono-exon 3XT) splicing events. c A violin plot from NanoBlot results displaying the distribution of sequenced read lengths of a representative 3XT. ATP23 (left, blue) and HECTD2 (right, red) are examples of genes with mono- and multi- exon 3XTs, respectively. Arrowheads indicate the lengths of ATP23 (blue) and HECTD2 (red) 3XT. d qRT-PCR analysis of mono- (blue) and multi-exon (red) 3XT expression in HeLa cells transfected with siRNA targeting MTR4 . Results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. The exact p -values are ATP23 3XT: p = 0.00916 (siCont vs siMTR4#1), p = 0.00074 (siCont vs siMTR4#2), TP53TG1 3XT: p = 0.00077 (siCont vs siMTR4#1), p = 0.00227 (siCont vs siMTR4#2), USP45 3XT: p = 0.00792 (siCont vs siMTR4#1), p = 0.01991 (siCont vs siMTR4#2), HECTD2 3XT: p = 0.00441 (siCont vs siMTR4#1), p = 0.00667 (siCont vs siMTR4#2), SPRED2 3XT: p = 0.00794 (siCont vs siMTR4#1), p = 0.00122 (siCont vs siMTR4#2), KCTD13 3XT: p = 0.02597 (siCont vs siMTR4#1), p = 0.02677 (siCont vs siMTR4#2). Source data are provided as a Source Data file.

Article Snippet: HeLa cells (631183, Takara) were cultured in Dulbecco’s modified Eagle’s medium (043-30085, Wako) supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Variant Assay, Transfection, Sequencing, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a – d (left) qRT-PCR analysis of mono- (blue) and multi-exon (red) 3XT expression in HeLa cells transfected with siRNA targeting EXOSC5 ( a ), PABPN1 ( b ), RBM7 ( c ) or ZCCHC7 ( d ). qRT-PCR results are expressed as the mean ± s.d. ( n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. (middle) qRT-PCR analysis of EXOSC5 , PABPN1 or RBM7 expression in HeLa cells transfected with siRNA targeting EXOSC5 ( a ), PABPN1 ( b ), RBM7 ( c ), or ZCCHC7 ( d ). qRT-PCR results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. (right) Cell lysates from HeLa cells transfected with siRNA targeting the indicated genes were subjected to immunoblotting analysis with anti-EXOSC5 ( a ), anti-PABPN1 ( b ), a nti-RBM7 ( c ), anti-ZCCHC7 ( d ), or anti-GAPDH antibodies. GAPDH was used as a loading control. The exact p -values are a ATP23 3XT: p = 0.00833 (siCont vs siEXOSC5#1), p = 0.00927 (siCont vs siEXOSC5#2), TP53TG1 3XT: p = 0.00048 (siCont vs siEXOSC5#1), p = 0.00067 (siCont vs siEXOSC5#2), USP45 3XT: p = 0.00187 (siCont vs siEXOSC5#1), p = 0.00312 (siCont vs siEXOSC5#2), HECTD2 3XT: p = 0.00187 (siCont vs siEXOSC5#1), p = 0.00312 (siCont vs siEXOSC5#2), SPRED2 3XT: p = 0.00977 (siCont vs siEXOSC5#1), p = 0.02162 (siCont vs siEXOSC5#2), KCTD13 3XT: p = 0.00443 (siCont vs siEXOSC5#1), p = 0.00792 (siCont vs siEXOSC5#2), EXOSC5 : p = 0.00010 (siCont vs siEXOSC5#1), p = 0.00007 (siCont vs siEXOSC5#2); b ATP23 3XT: p = 0.00054 (siCont vs siPABPN1#1), p = 0.01888 (siCont vs siPABPN1#2), TP53TG1 3XT: p = 0.02140 (siCont vs siPABPN1#1), p = 0.04520 (siCont vs siPABPN1#2), USP45 3XT: p = 0.00101 (siCont vs siPABPN1#1), p = 0.00695 (siCont vs siPABPN1#2), HECTD2 3XT: p = 0.00956 (siCont vs siPABPN1#1), p = 0.03642 (siCont vs siPABPN1#2), SPRED2 3XT: p = 0.01983 (siCont vs siPABPN1#1), p = 0.01966 (siCont vs siPABPN1#2), KCTD13 3XT: p = 0.01838 (siCont vs siPABPN1#1), p = 0.03199 (siCont vs siPABPN1#2), PABPN1 : p = 0.00088 (siCont vs siPABPN1#1), p = 0.00021 (siCont vs siPABPN1#2); c RBM7 : p = 0.00033 (siCont vs siRBM7#1), p = 0.00006 (siCont vs siRBM7#2); d ZCCHC7 : p = 7.8721E-07 (siCont vs si ZCCHC7 #1), p = 0.00009 (siCont vs si ZCCHC7 #2). Source data are provided as a Source Data file.

Article Snippet: HeLa cells (631183, Takara) were cultured in Dulbecco’s modified Eagle’s medium (043-30085, Wako) supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing, Transfection, Western Blot, Control

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a The C-rich motif is enriched in MTR4-target multi-exon 3XRs. The table shows the results of MEME motif enrichment analysis. E-value; the expected number of motifs with the given log likelihood ratio (or higher), and with the same width and site count that one would find in a similarly sized set of random sequences. Sites; the number of sites contributing to the construction of the motif. b Venn diagram showing the CCWSCC-matched RNA-binding proteins speculated by Tomtom using different distance metrics. Pearson; Pearson correlation coefficient. Euclidean; Euclidean distance. Sandelin; Sandelin-Wasserman similarity. W; A or T. S; G or C. c qRT-PCR analysis of mono- (blue) and multi-exon (red) 3XT expression in HeLa cells transfected with si-hnRNPK. Results are expressed as the mean ± s.d. ( n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. d Lysates from HeLa cells were subjected to immunoprecipitation with an anti-hnRNPK antibody or rabbit IgG, followed by qRT-PCR analysis to detect the indicated RNAs. SNHG9 and SNHG19 RNA were used as negative controls. Results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. e hnRNPK associates with MTR4 in vivo. Lysates from HeLa cells were subjected to immunoprecipitation with an anti-MTR4 antibody, an anti-hnRNPK antibody or rabbit IgG followed by immunoblotting analysis with anti-MTR4, anti-hnRNPK or anti-GAPDH antibodies. GAPDH was used as a negative control. This experiment was repeated independently two times with similar results. The exact p -values are c HECTD2 3XT: p = 0.03689 (siCont vs si-hnRNPK#1), p = 0.04965 (siCont vs si-hnRNPK2), SPRED2 3XT: p = 0.01871 (siCont vs si-hnRNPK#1), p = 0.02218 (siCont vs si-hnRNPK#2), KCTD13 3XT: p = 0.01953 (siCont vs si-hnRNPK#1), p = 0.04751 (siCont vs si-hnRNPK#2); d HECTD2 3XT: p = 0.01229 (rabbit IgG vs hnRNPK IP), SPRED2 3XT: p = 0.00157 (rabbit IgG vs hnRNPK IP), KCTD13 3XT: p = 0.01079 (rabbit IgG vs hnRNPK IP). Source data are provided as a Source Data file.

Article Snippet: HeLa cells (631183, Takara) were cultured in Dulbecco’s modified Eagle’s medium (043-30085, Wako) supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: RNA Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Immunoprecipitation, In Vivo, Western Blot, Negative Control

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a HeLa cells were transfected with a 3XR-fused rabbit β-globin gene and then subjected to qRT-PCR analysis. Results are expressed as the mean ± s.d. ( n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. b Lysates from HeLa cells transfected with a 3XR-fused rabbit β-globin gene and Flag-tagged hnRNPK were subjected to immunoprecipitation with an anti-Flag antibody followed by qRT-PCR analysis to detect the indicated RNAs. SNHG19 RNA was used as a negative control. Results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. c Schematic representation of wild-type (WT) and deletion mutant (delKH1 ~ 3) hnRNPK proteins. KH; K homology. d Lysates from HCT116 cells transfected with a KCTD13 3XR-fused rabbit β-globin gene along with wild-type or mutant Flag-tagged hnRNPK were subjected to immunoprecipitation with an anti-Flag antibody followed by qRT-PCR analysis to detect β-globin mRNA and SNHG19 RNA. SNHG19 RNA was used as a negative control. Results are expressed as the mean ± s.d. (n = 3 biologically independent replicates). Paired two-sided Student’s t-test; * p < 0.05. The exact p -values are a β-globin : p = 0.00002 (Mock vs HECTD2 3XR), p = 0.00147 (Mock vs SPRED2 3XR), p = 0.00036 (Mock vs KCTD13 3XR); b β-globin : p = 0.01727 (Mock vs HECTD2 3XR), p = 0.03705 (Mock vs SPRED2 3XR), p = 0.00146 (Mock vs KCTD13 3XR); d β-globin : p = 0.01415 (WT vs delKH1), p = 0.01123 (WT vs delKH3). Source data are provided as a Source Data file.

Article Snippet: HeLa cells (631183, Takara) were cultured in Dulbecco’s modified Eagle’s medium (043-30085, Wako) supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Transfection, Quantitative RT-PCR, Immunoprecipitation, Negative Control, Mutagenesis

Journal: Nature Communications

Article Title: The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

doi: 10.1038/s41467-024-51981-8

Figure Lengend Snippet: a The structure of the protein derived from KCTD13 3XT is predicted by AlphaFold and the PSR region (pink) translated from the KCTD13 CDS contains a disorder region. b Detection of KeXT bodies in HeLa cells transfected with siMTR4#2. Representative maximum projection immunofluorescence image of HeLa cells transfected with siMTR4#2 and immunolabeled with KCTD13 3XT protein (orange). Nuclei were stained with Hoechst33258 (blue). Scale bar, 10 µm. This experiment was repeated independently three times with similar results. c Detection of KeXT bodies in HeLa cells transfected with Flag-tagged KCTD13 3XT protein. Immunofluorescence images of HeLa cells transfected with Flag-tagged KCTD13 3XT protein (WT) or Flag-control (Mock) and immunostained with anti-Flag antibody (orange). Nuclei were stained with Hoechst33258 (blue). Scale bar, 10 µm. This experiment was repeated independently three times with similar results. d The 3XR is required for KeXT body assembly. Immunofluorescence images of HeLa cells transfected with PSR-deletion mutant Flag-tagged KCTD13 3XT protein (del-PSR) or Flag-control (Mock) and immunostained with anti-Flag antibody (orange). Nuclei were stained with DAPI (blue). Scale bar, 10 µm. This experiment was repeated independently three times with similar results. e KeXT bodies in HeLa cells transfected with siMTR4#2 in the presence of 1,6-hexandiol or 2,5-hexandiol. Representative maximum projection immunofluorescence image of HeLa cells transfected with siMTR4#2, treated with or without 1,6-hexandiol or 2,5-hexandiol, and immunolabeled with KCTD13 3XT protein (orange). Nuclei were stained with Hoechst33258 (blue). Scale bar, 10 µm. This experiment was repeated independently three times with similar results.

Article Snippet: HeLa cells (631183, Takara) were cultured in Dulbecco’s modified Eagle’s medium (043-30085, Wako) supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Derivative Assay, Transfection, Immunofluorescence, Immunolabeling, Staining, Control, Mutagenesis

Journal: Nature Communications

Article Title: The DIAPH3 linker specifies a β-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow

doi: 10.1038/s41467-024-49427-2

Figure Lengend Snippet: a Schematics of the domain organization of DIAPH1 and 3. The domain boundaries are denoted as the amino acid number in the sequence. DAD diaphanous autoinhibitory domain, DID-DAD interacting domain, FH1 formin homology domain 1, FH2 formin homology domain 2, and CT carboxy-terminal domain that is used in subsequent experiments (amino acids 583–1272 and 540–1171 for DIAPH1 and 3 respectively). b Micrographs of HeLa cells expressing GFP targeted to the surface of mitochondria by a fragment of Tom20 (green), fixed and stained for β- or γ-actin (magenta). Scale bars represent 10 μm. The presented micrographs are representative of three independent experiments. c Relative colocalization, determined by a Manders’ overlap coefficient (tM1) of β- and γ-actin to mitochondria in HeLa cells expressing Tom20-GFP fused to the different CT domains of wildtype DIAPH1 (DIA1), wildtype DIAPH3 (DIA3), DIAPH1 with the DIAPH3 FH2 domain (DIA1 3F) and DIAPH3 with the DIAPH1 FH2 domain (DIA3 1F). Data were presented as mean (solid bar) ± SD (error bars). n = 20 cells analyzed per condition across three independent experiments, * p = 7.25 × 10 −11 , ** p = 1.45 × 10 −11 as calculated by two-sided Mann–Whitney non-parametric tests. d Micrographs of HeLa cells expressing either a Tom20-GFP-DIAPH3-CT fusion protein or a Tom20-GFP-DIAPH3-1F CT (green), fixed and stained for β- or γ-actin (magenta). Scale bars represent 10 μm. The presented micrographs are representative of three independent experiments. e Micrographs of HeLa cells expressing either a Tom20-GFP-DIAPH1-CT fusion protein or a Tom20-GFP-DIAPH1-3F CT (green), fixed and stained for β- or γ-actin (magenta). Scale bars represent 10 μm. White arrowheads point to regions of actin colocalizing to mitochondria. The region boxed is yellow and is magnified in the zoom panel. The presented micrographs are representative of three independent experiments.

Article Snippet: HeLa cell lines inducibly expressing GFP-DIAPH1 and GFP-DIAPH3 were described previously , . cDNAs encoding GFP-DIAPH1-3F, GFP-DIAPH3-1F, GFP-DIAPH1-3L, and GFP-DIAPH3-1L were cloned into a modified pcDNA5 FRT/TO plasmid downstream and in frame with an inserted GFP, using the In-Fusion Cloning Kit (Takara Bio USA, Inc.).

Techniques: Sequencing, Expressing, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: The DIAPH3 linker specifies a β-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow

doi: 10.1038/s41467-024-49427-2

Figure Lengend Snippet: a View of the co-crystal structure of a dimer of FMNL3 FH2 domains (dark blue and green) with two actin subunits (yellow and gray). Positions of the missing linker region and actin N-terminus are inferred in red and with a blue star, respectively. b Close-up view of the area the two missing regions are inferred to occupy. c Sequence alignment of the linker regions of DIAPH1 and 3 . d Top ranked Alphafold2 model of the complex between DIAPH3 FH2 domain (blue) and β-actin (yellow). An ensemble of 10 Local Disordered Region Sampling (LDRS)-generated conformers of the β-actin N-terminus are shown in a color spectrum, a single conformer of the FH2 linker is shown. e Micrographs of HeLa cells expressing a Tom20-GFP-DIAPH1-CT fusion protein that contains the linker sub-domain of DIAPH3 (green), fixed and stained for β- or γ-actin (magenta). f Micrographs of HeLa cells expressing a Tom20-GFP-DIAPH3-CT fusion protein containing the linker sub-domain of DIAPH1 (green), fixed and stained for β- or γ-actin (magenta). For panels e and f: scale bars represent 10 μm; yellow boxed regions are enlarged in adjoining zoom panels; white arrowheads indicate instances of actin-mitochondria colocalization; micrographs are representative of three independent experiments. g Relative colocalization determined by Manders’ overlap coefficient (tM1) of β- and γ-actin to mitochondria in HeLa cells expressing Tom20-GFP fused to the CT domains of DIAPH1 and DIAPH3 containing the linker sub-domain of either DIAPH1 (1 L) or DIAPH3 (3 L). Data were presented as mean (solid bar) ± SD (error bars). n = 20 cells analyzed per condition across three independent experiments. * p = 4.18 × 10 −8 , ** p = 1.74 × 10 −10 , *** p = 1.45 × 10 −11 as determined by two-sided Mann–Whitney non-parametric tests. h The CT of DIAPH1 and 3 containing the linker sub-domain of either DIAPH1 (1 L) or DIAPH3 (3 L) fused to GST were incubated with actin purified from platelets (β-actin rich) or gizzard (γ-actin rich) and polymerized actin (P) separated from unpolymerized (S) by centrifugation. Fractions were analyzed by immunoblotting using actin isoform-specific antibodies. The blots presented are representative of three independent experiments.

Article Snippet: HeLa cell lines inducibly expressing GFP-DIAPH1 and GFP-DIAPH3 were described previously , . cDNAs encoding GFP-DIAPH1-3F, GFP-DIAPH3-1F, GFP-DIAPH1-3L, and GFP-DIAPH3-1L were cloned into a modified pcDNA5 FRT/TO plasmid downstream and in frame with an inserted GFP, using the In-Fusion Cloning Kit (Takara Bio USA, Inc.).

Techniques: Sequencing, Sampling, Generated, Expressing, Staining, MANN-WHITNEY, Incubation, Purification, Centrifugation, Western Blot

Journal: Nature Communications

Article Title: The DIAPH3 linker specifies a β-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow

doi: 10.1038/s41467-024-49427-2

Figure Lengend Snippet: a Micrographs of HeLa cells expressing GFP-DIAPH1, GFP-DIAPH1 containing the DIAPH3 linker (GFP-DIAPH1-3L), GFP-DIAPH3, and GFP-DIAPH3 containing the DIAPH1 linker (GFP-DIAPH3-1L). The presented micrographs are representative of three independent experiments. b Micrographs of HeLa cells expressing GFP-DIAPH1-3L in the presence or absence of endogenous DIAPH1, stained with β- or γ-actin isoform-specific antibodies. The presented micrographs are representative of three independent experiments. c Micrographs of HeLa cells expressing GFP-DIAPH3-1L in the presence or absence of endogenous DIAPH3, stained with β- or γ-actin isoform-specific antibodies. The presented micrographs are representative of three independent experiments. d Top: Cartoon illustrating the subdivision of dividing cells into furrow and polar regions. Bottom: Quantitation of the intensity of the indicated actin isoform staining within the furrow and polar regions of anaphase cells under different conditions. n = 10 cells analyzed across three independent experiments. Data were presented as mean (red bars) ± SD. * p = 1.083 × 10 −5 as determined by two-sided Mann–Whitney non-parametric tests.

Article Snippet: HeLa cell lines inducibly expressing GFP-DIAPH1 and GFP-DIAPH3 were described previously , . cDNAs encoding GFP-DIAPH1-3F, GFP-DIAPH3-1F, GFP-DIAPH1-3L, and GFP-DIAPH3-1L were cloned into a modified pcDNA5 FRT/TO plasmid downstream and in frame with an inserted GFP, using the In-Fusion Cloning Kit (Takara Bio USA, Inc.).

Techniques: Expressing, Staining, Quantitation Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: The DIAPH3 linker specifies a β-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow

doi: 10.1038/s41467-024-49427-2

Figure Lengend Snippet: a Micrographs of HeLa cells expressing DIAPH1 and 3 linker mutants in the absence of either endogenous DIAPH1 or DIAPH3, fixed and stained for phospho-myosin light chain (black) and DNA (red). Scale bars represent 10 μm. The micrographs presented are representative of three independent experiments. b Top: Cartoon illustrating the region of phospho-myosin light chain fluorescence intensity measurement (yellow). Bottom: Quantitation of the fluorescence intensity of phospho-myosin staining within the furrow and polar regions of anaphase cells described in panel a . n = 5 cells analyzed across three independent experiments. Data were presented as mean (red bars) ± SD. “ns” = 0.42, * p = 7.93 × 10 −3 as determined by two-sided Mann–Whitney non-parametric tests. c Micrographs of HeLa cells depleted of endogenous DIAPH3 and expressing either DIAPH3, DIAPH3 1 F, or DIAPH3 1 L stained for phospho-myosin light chain (black) and alpha-tubulin (magenta) to visualize microtubules. Orthogonal views are presented along the green dashed lines in whole cell images. Scale bars represent 10 μM for whole cell images and 2 μM for orthogonal views. The micrographs presented are representative of three independent experiments. d Micrographs of HeLa cells expressing DIAPH1 and 3 linker mutants in the absence of either endogenous DIAPH1 or DIAPH3, fixed and stained for RhoA and DNA (blue). Scale bars represent 10 μm. The micrographs presented are representative of three independent experiments. e Micrographs of HeLa cells expressing DIAPH1 and 3 linker mutants in the absence of either endogenous DIAPH1 or DIAPH3, fixed and stained for Ect2 and DNA (blue). Scale bars represent 10 μm. The micrographs presented are representative of three independent experiments. f Schematic outlining a possible pathway whereby β-actin can feedback to influence RhoA.

Article Snippet: HeLa cell lines inducibly expressing GFP-DIAPH1 and GFP-DIAPH3 were described previously , . cDNAs encoding GFP-DIAPH1-3F, GFP-DIAPH3-1F, GFP-DIAPH1-3L, and GFP-DIAPH3-1L were cloned into a modified pcDNA5 FRT/TO plasmid downstream and in frame with an inserted GFP, using the In-Fusion Cloning Kit (Takara Bio USA, Inc.).

Techniques: Expressing, Staining, Fluorescence, Quantitation Assay, MANN-WHITNEY