Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Successful inactivation of ACTB and ACTG1 genes and altered actin cytoskeleton architecture in CR- ACTB [clones with inactivated ACTB with the help of CRISPR/Cas9(D10A) technique] and CR- ACTG1 [clones with inactivated ACTG1 with the help of CRISPR/Cas9(D10A) technique] clones. ( A ) Western blot analysis of control cells and clones without either β or γ actin. To identify isoactins and total actin, appropriate antibodies were used. Corresponding Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. Green rectangles represent statistically higher level of protein in comparison to control cells. ( B ) Densitometric analysis of β, γ, and total actin level in tested clones done on membranes shown in ( A ) ( n = 3). ( C ) The qRT-PCR analysis of ACTB and ACTG1 expression level in CR- ACTB and CR- ACTG1 clones ( n = 9). ( D ) Clones devoid of β or γ actin were immunostained with antibodies recognizing non-muscle actins. All pictures were taken at the same settings used during microscopic observations. Pictures of two other clones for every condition are shown in . ( E ) Representative plots presenting fluorescence distribution of stained isoactins in tested clones shown in ( D ). Quantitative analysis of area under curve (AUC) from plots ( n = 6). ( F ) Clones were immunostained with anti-β and anti-γ actin antibodies to show cellular distribution of both isoactins. Red arrows point at stress fibers and green ones at actin mesh. ( G ) Western blot analysis of filamentous and globular actin levels in clones without β or γ actin growing on coverslips for 72 h. Nitrocellulose membranes were incubated with mouse anti-total actin antibodies. Quantitative estimation of filamentous ( F ) to globular ( G ) ratio ( n = 8–9). ( H ) Clones were stained with fluorescently labelled phalloidin and DNase I to detect F- and G-actin. White arrows highlight F-actin accumulation. Results are expressed as the mean ±SD ( B , E , G ) or ±SEM ( C ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***).

Article Snippet: Plasmids used for inactivation of ACTB (catalogue number: sc-400000-NIC) and ACTG1 (catalogue number: sc-400006-NIC) genes as well control set of plasmids (sc-437281), phorbol-12-myristate-13-acetate (PMA), lysophosphatidic acid (LPA), and cytochalasin D were bought at Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Clone Assay, CRISPR, Western Blot, Quantitative RT-PCR, Expressing, Fluorescence, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Cells devoid of β or γ actin had impaired invasion abilities, but only CR- ACTG1 cells’ 2-D migration was affected. ( A – D ) Spontaneous 2-D migration of tested cells. The cells of studied clones growing on IncuCyte ® ImageLock plates were recorded over 72 h with IncuCyte ® Live Cell Analysis Imaging System ( n = 30). ( A ) Trajectories of single cells’ migration. ( B ) Calculated covered distances of the cells. ( C ) Estimation of velocity and ( D ) directionality of cells’ migration. ( E ) Wound healing assay was performed on control (CR-CTRL), CR- ACTB, and CR- ACTG1 cells to analyze collective migration over 72 h ( n = 3). ( F ) Analysis of 3-D migration/invasion of control cells and cells without β or γ actin ( n = 16). The cells were seeded onto Matrigel™ gel placed in the upper compartment of a Transwell™ and to the lower compartment a medium enriched in serum was added. ( G ) Gelatin digestion assay. The cells seeded on fluorescently labeled gelatin were 12 h later fixed and stained with Alexa Fluor™ 488 phalloidin. Bright spots represent digested gelatin by cells. Pink arrows point at invadopodia. ( H ) Quantitative analysis of invadopodia number and ( I ) dimensions of digested area ( n = 36). ( J ) Cells after fixation were stained with antibodies recognizing cofilin and cortactin. Across the area rich in invadopodia a line was drawn and intensities of fluorescence for detected cortactin and cofilin were plotted on corresponding graphs. Pink arrows point at invadopodia. ( K ) Western blot analysis of cell lysates. Membranes were probed with antibodies detecting cofilin and cofilin phosphorylated at Ser3 (p-cofilin 3 ). Corresponding densitometric analysis and Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. Green rectangles represent statistically higher level of protein in comparison to control cells. ( L ) Calculated p-cofilin 3 :cofilin ratio ( n = 3). Results are expressed as the mean ± SD ( B – F , H , K ) or median ( I ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.0001 (****).

Article Snippet: Plasmids used for inactivation of ACTB (catalogue number: sc-400000-NIC) and ACTG1 (catalogue number: sc-400006-NIC) genes as well control set of plasmids (sc-437281), phorbol-12-myristate-13-acetate (PMA), lysophosphatidic acid (LPA), and cytochalasin D were bought at Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Migration, Clone Assay, Live Cell Imaging, Wound Healing Assay, Labeling, Staining, Fluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Stress fibers’ formation is disturbed in clones devoid of β or γ actin. ( A ) CR-CTRL, CR- ACTB, and CR- ACTG1 cells upon stimulation with LPA were fixed and stained with Alexa Fluor™ 488 phalloidin. Red arrows point at stress fibers. ( B ) The number of thick stress fibers was calculated for every condition and presented as a bar chart ( n = 61–81). ( C ) Activity of RhoA, Rac1, and Cdc42 was estimated after lysophosphatidic acid (LPA) stimulation ( n = 3). ( D ) F:G actin ratio was evaluated in the cells on the base of Western blot analysis of F- and G-actin levels in studied clones ( n = 9). Membranes were incubated with anti-total actin antibodies. ( E ) The cells were immunostained with antibodies detecting the human FH1/FH2 domain-containing protein 1 (FHOD1) and Myosin IIa. Yellow arrows highlight Myosin IIa accumulation. ( F ) Plots of averaged ( n = 9) and individual fluorescence intensity distribution for FHOD1 and Myosin IIa staining across a cell. ( G ) Western blot analysis of cell lysates. Membranes were probed for FHOD1, Myosin IIa, and myosin light chain phosphorylated at Thr18 and Ser19 (pMLC 18/19 ). Corresponding densitometric analysis and Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. ( H ) The cells were stained with anti-pMLC 19 antibodies and fluorescently labeled phalloidin. Results are expressed as the mean ± SD ( C , D ) or ± SEM ( B ), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.0001 (****).

Article Snippet: Plasmids used for inactivation of ACTB (catalogue number: sc-400000-NIC) and ACTG1 (catalogue number: sc-400006-NIC) genes as well control set of plasmids (sc-437281), phorbol-12-myristate-13-acetate (PMA), lysophosphatidic acid (LPA), and cytochalasin D were bought at Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Clone Assay, Staining, Activity Assay, Western Blot, Incubation, Fluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Changed distribution of actin binding proteins (ABPs) within leading edge upon lamellipodium formation challenge. ( A ) The cells were stained with vasodilator-stimulated phosphoprotein (VASP), actin-related protein 3 (Arp3), and neural Wiskott-Aldrich syndrome protein (N-WASP), recognizing antibodies. Yellow arrows point at circular dorsal ruffles. ( B ) Plots of averaged fluorescence intensity distribution of stained VASP, Arp3, and N-WASP. Lines were drawn from pericentral region of a cell (position 0 μm) towards the border of a cell (10 μm) ( n = 9). Representative white lines are shown on merged microphotographs in ( A ). Black arrow points at the range for which there is a statistically significant difference for CR- ACTG1 cells in comparison to control cells. ( C ) The cells were stained using specific antibodies to detect Wiskott-Aldrich syndrome protein family member 2 (WAVE-2) and Arp3. Yellow arrows point at circular dorsal ruffles. ( D ) Plots of averaged fluorescence intensity distribution for WAVE-2 from the center of a cell (position 0 μm) towards the border of a cell (position 15 μm). Representative white lines are shown on merged microphotographs in ( C ). ( E ) Cell lysates were analyzed by Western blotting. Membranes were incubated with antibodies directed against N-WASP, WAVE-2, and Arp3. Corresponding densitometric analysis and Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. Green and red rectangles represent statistically higher and lower levels of protein, respectively, in comparison to control cells. Results are expressed as the mean only, p ≤ 0.05 (*), p ≤ 0.01 (**).

Article Snippet: Plasmids used for inactivation of ACTB (catalogue number: sc-400000-NIC) and ACTG1 (catalogue number: sc-400006-NIC) genes as well control set of plasmids (sc-437281), phorbol-12-myristate-13-acetate (PMA), lysophosphatidic acid (LPA), and cytochalasin D were bought at Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Binding Assay, Staining, Fluorescence, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Changed FAs’ signaling in CR-CTRL, CR- ACTB, and CR- ACTG1 clones. Western blot analysis of control clones and clones without given isoactin. Corresponding Ponceau S stainings of membranes are shown in . Forty μg of protein was loaded on every lane. Membranes were probed for proteins found within focal adhesions and playing a significant role in FAs’ signaling: VASP, phosphorylated at Ser157 VASP (pVASP 157 ), phosphorylated at Ser239 VASP (pVASP 239 ), α-parvin, phosphorylated at Tyr530 proto-oncogene tyrosine-protein kinase Src (pSrc 530 ), and phosphorylated at Tyr397 focal adhesion kinase 1 (pFAK 397 ). Densitometric analysis of studied proteins levels in tested clones was done and presented as bar charts ( n = 3–6). Green and red rectangles represent statistically higher and lower level of protein, respectively, in comparison to control cells. Results are expressed as the mean ± SD, p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***).

Article Snippet: Plasmids used for inactivation of ACTB (catalogue number: sc-400000-NIC) and ACTG1 (catalogue number: sc-400006-NIC) genes as well control set of plasmids (sc-437281), phorbol-12-myristate-13-acetate (PMA), lysophosphatidic acid (LPA), and cytochalasin D were bought at Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Clone Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

doi: 10.3390/ijms21082746

Figure Lengend Snippet: Proposed mechanism of influence of disturbed actin organization in CR- ACTB and CR- ACTG1 cells on melanoma cells’ motility and adhesion. Cells devoid of β actin that grew in full medium exhibited more nascent (VASP-rich) focal adhesions (nFAs) and mature (VASP- and α-parvin-rich) focal adhesions (FAs) than did CR-CTRL cells. Although the CR- ACTG1 and CR- ACTB cells possess more mature FAs under the same conditions than control cells, FAs of CR- ACTG1 cells are smaller. Moreover, CR- ACTG1 cells have many more nascent FAs than both control and CR- ACTB cells. Alterations in FA dynamics, stress fiber formation, and the distribution of some ABPs at the leading edge results in changes in Rac1 activation, improved 2-D migration, and impaired invasion in CR- ACTB cells. In the case of CR- ACTG1 cells, the impaired FA turnover, increased number of thick stress fibers, and decreased level of Arp3 at the leading edge led to lowered activation status of RhoA and finally to impeded 2-D and 3-D migration. For details, please see the discussion. In summary, the lack of γ actin has more severe effects on melanoma cells than the lack of β actin. We believe that changes in the formation/turnover of FAs might be caused by the different properties of non-muscle actins’ interactions with ABPs.

Article Snippet: Plasmids used for inactivation of ACTB (catalogue number: sc-400000-NIC) and ACTG1 (catalogue number: sc-400006-NIC) genes as well control set of plasmids (sc-437281), phorbol-12-myristate-13-acetate (PMA), lysophosphatidic acid (LPA), and cytochalasin D were bought at Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Activation Assay, Migration

Journal: Oncotarget

Article Title: Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

doi: 10.18632/oncotarget.19016

Figure Lengend Snippet: Time-scheme of cardiomyocyte derivation from A top) HG8 and D top) R1 and R1 HIF-1α−/− mESC. Myh6 and Myh7 relative mRNA levels in ( A ) cardiomyocytes derived from HG8 mESC ( n = 3), ( C ) cardiomyocytes derived from parental HG8 and HG8 HIF-1α−/− mESC ( n = 3), and ( D ) cardiomyocytes derived from R1 and R1 HIF-1α−/− mESC determined by qRT-PCR ( n = 4 (R1), n = 3 ( R1 HIF-1α − / − )). Data are presented as 2 −ΔCq ± SEM (groups were compared using one-way ANOVA with the Tukey (HSD) post hoc test * p < 0.05). Total MHCα and β protein levels ( B ) in cardiomyocytes derived from HG8 mESC ( n = 2) and ( E ) in cardiomyocytes derived from R1 and R1 HIF-1α−/− mESC (cropped representative western blots from total n = 2 are shown).

Article Snippet: To prepare the HG8 cell line deficient in HIF-1α, HG8 cells grown to confluency in ES medium in a 60mm cell culture dish were transfected with 2ug of HIF-1α Double Nickase Plasmid (sc-420856-NIC, Santa Cruz Biotechnology, USA), by means of Lipofectamine 2000 (Invitrogen, USA).

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

doi: 10.18632/oncotarget.19016

Figure Lengend Snippet: Representative western blot showing protein levels of HIF-1α and HIF-2α ( n = 3) in ( A ) cardiomyocytes derived from R1 and R1 HIF-1α−/− mESC exposed to 1% O 2 and ( B ) cardiomyocytes derived from HG8 mESC exposed to 1% O 2 and DMOG. Cropped representative western blots are shown from total of n = 3. mRNA levels of ( C ) Glut1 and ( D ) VEGF in cardiomyocytes derived from HG8 mESC exposed to 1% O 2 or DMOG determined by qRT-PCR ( n = 3). Data are presented as 2 −ΔCq ± SEM (groups were compared using one-way ANOVA with the Tukey (HSD) post hoc test * p < 0.05).

Article Snippet: To prepare the HG8 cell line deficient in HIF-1α, HG8 cells grown to confluency in ES medium in a 60mm cell culture dish were transfected with 2ug of HIF-1α Double Nickase Plasmid (sc-420856-NIC, Santa Cruz Biotechnology, USA), by means of Lipofectamine 2000 (Invitrogen, USA).

Techniques: Western Blot, Derivative Assay, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

doi: 10.18632/oncotarget.19016

Figure Lengend Snippet: ( A ) Graphical representation of predicted HIF binding sites in the locus encoding for mouse MHCα and MHCβ; sites conserved in mouse and rat are marked with arrows. ( B ) ChIP analysis of HIF-1α binding to predicted binding sites performed on samples from 10 day-differentiated R1 mESCs exposed to 1% O 2 hypoxia for 24 h. HIF-1α binding to its target gene Glut1 served as ChIP assay positive control (cropped representative gels are shown from n = 4 (intron 25–26, Glut1) or n = 3 (other clusters)).

Article Snippet: To prepare the HG8 cell line deficient in HIF-1α, HG8 cells grown to confluency in ES medium in a 60mm cell culture dish were transfected with 2ug of HIF-1α Double Nickase Plasmid (sc-420856-NIC, Santa Cruz Biotechnology, USA), by means of Lipofectamine 2000 (Invitrogen, USA).

Techniques: Binding Assay, Positive Control

Journal: Oncotarget

Article Title: Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

doi: 10.18632/oncotarget.19016

Figure Lengend Snippet: ( A ) Scheme showing the derivation of fetal hearts and kinetics of changes in Myh6 and Myh7 mRNA levels in mouse explanted fetal hearts cultured in vitro determined by qRT-PCR; for comparison, Myh6 and Myh7 mRNA levels in an adult heart sample are shown. Data are expressed as mean 2 −ΔCq ± SEM Myh6 and Myh7 ( n = 10 (48 h), n = 7 (0 h, 24 h), n = 4 (3 h, 22 h, 2 h wo FBS), n = 3 (6 h, 12 h, 18 h, 20 h, 26 h)), n = 1 (adult heart). Effect of hypoxia and the pharmacological stabilization of HIF on ( B ) Myh6 and Myh7 relative mRNA levels determined by qRT-PCR ( n = 9 (0 h), n = 7 (normoxia), n = 8 (1% O 2 ), n = 5 (DMOG); same data depicted in Figure are displayed in grey for comparison); ( C ) stabilization of HIF-1α protein after 6 h of in vitro culture in normoxia and 1% O 2 (cropped representative western blots are shown, n = 3), mRNA levels of ( D ) Glut1, ( E ) Vegf, and ( F ) CaIX after 24 h determined by qRT-PCR ( n = 3), data are presented as 2 −ΔCq ± SEM (groups were compared using one-way ANOVA with the Tukey (HSD) post hoc test * p < 0.05; † p < 0.05).

Article Snippet: To prepare the HG8 cell line deficient in HIF-1α, HG8 cells grown to confluency in ES medium in a 60mm cell culture dish were transfected with 2ug of HIF-1α Double Nickase Plasmid (sc-420856-NIC, Santa Cruz Biotechnology, USA), by means of Lipofectamine 2000 (Invitrogen, USA).

Techniques: Cell Culture, In Vitro, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

doi: 10.18632/oncotarget.19016

Figure Lengend Snippet: ( A ) mTOR inhibition using rapamycin validated by western blot detection of its downstream target p70 s6 kinase ( n = 6) and the stabilization of HIF-1α protein by hypoxia ( n = 4) ( B ) Myh6 and Myh7 relative mRNA levels in normoxia and in 1 % O 2 hypoxia determined by qRT-PCR. Data are presented as 2 −ΔCq ± SEM ( n = 5 (normoxia), n = 4 (1% O 2 ), n = 3 (rapamycin treatment); in grey same data depicted in Figure are displayed for comparison; groups were compared using one-way ANOVA with the Fisher's LSD post hoc test * p < 0.05; † p < 0.05).

Article Snippet: To prepare the HG8 cell line deficient in HIF-1α, HG8 cells grown to confluency in ES medium in a 60mm cell culture dish were transfected with 2ug of HIF-1α Double Nickase Plasmid (sc-420856-NIC, Santa Cruz Biotechnology, USA), by means of Lipofectamine 2000 (Invitrogen, USA).

Techniques: Inhibition, Western Blot, Quantitative RT-PCR