Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: cAMP increases Na+/K+-ATPase activity, protein abundance at the plasma membrane and the distance traveled by the Na+/K+-ATPase-containing vesicles in A549-GFPα1 cells. (A) A549-GFPα1 cells were incubated in the absence (CT) or presence of 50 μM forskolin (FSK) for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three experiments. (B) A549-GFPα1 cells were incubated as in A, and the Na+/K+-ATPase abundance at the basolateral plasma membrane was determined by western blot of the BLM fraction using a specific antibody against GFP. E-cadherin was used as a loading control. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (C) The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Vesicles were randomly selected from those that showed plus-end-directed displacement. Left panel shows a representative image of A549-GFPα1 cells. Arrowhead indicates the vesicle whose trajectory is shown in the right panel before (CT) and after FSK treatment (FSK). (D) Average contour length traveled by the vesicles as a function of time. The black line represents control vesicles; at 60 seconds, upon addition of FSK (red line), the vesicles move at a faster rate. The average contour length is determined by averaging over many trajectories as described in the Materials and Methods. **P<0.01; ***P<0.001. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Activity Assay, Incubation, Western Blot, Labeling, Single-particle Tracking, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The three isoforms of myosin-V are expressed in A549 cells. (A) RT-PCR using mRNA obtained from A549 and HeLa cells. Primers used for the amplification are described in supplementary material Table S6. (B) Cell lysates from A549 and HeLa cells were obtained and analyzed by western blot with specific antibodies against the three myosin-V isoforms. A representative western blot is shown. (C) The particulate fraction (100,000 g pellet) of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. Rab5 and Rab7 are used as markers of early and late endosomes, respectively. (D) Gradients obtained in C were scanned and the marker content was digitally quantified as indicated. Results are expressed as percentage of the total amount of protein.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Marker

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and myosin-Vc colocalize with Na+/K+-ATPase. (A) A549-GFPα1 cells were incubated in the absence or presence of 50 μM FSK for 10 minutes, basolateral membranes (BLM) and intracellular compartments (IC) were isolated and the Na+/K+-ATPase abundance was determined by western blot using a specific antibody against GFP. E-cadherin and actin were used as loading controls for the BLM and IC fractions, respectively. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (B) The IC fraction of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. C+, positive control.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Incubation, Isolation, Western Blot, Positive Control

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a myosin-Va stalk-tail. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Va that has a m-cherry-tag (red) (m-cherry-DN-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (B) Average contour length traveled by the vesicles in A as a function of time. The black line represents the control vesicles; FSK was added at time 60 seconds and is represented as a red line. (C) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Vc that has a m-cherry-tag (red) (m-cherry-DN-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (D) Average contour length traveled by the vesicles in C as a function of time. The black line represents control vesicles; FSK was added at 60 seconds and is represented as the red line. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, Imaging, Transfection, Dominant Negative Mutation, Labeling

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a shRNA against myosin-Va. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Va that has a m-cherry-tag (red) (m-cherry-sh-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions. (B) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Vc that has a m-cherry-tag (red) (m-cherry-shRNA-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions (C). Graph represents the average contour length traveled by the vesicles as a function of time, calculated as described in methods. The black line represents the m-cherry-sh-Va vesicles and the red line, the m-cherry-sh-Vc vesicles. (D) A549-GFPα1 cells were transfected with a shRNA against myosin-Va or myosin-Vc, cell lysates were isolated and the myosin-Va (left panel) or myosin-Vc (right panel) abundance was determined by western blot using specific antibodies. E-cadherin and tubulin were used as loading controls. Scale bars: 10 μm and 4 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, shRNA, Imaging, Transfection, Labeling, Isolation, Western Blot

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Dominant-negative myosin-Va mimics cAMP-mediated Na+/K+-ATPase increased activity and recruitment to the plasma membrane in A549-GFPα1 cells. (A) Stable clones expressing myosin-Va tail (DN-Va) and myosin-Vc tail (DN-Vc) were generated as described. Expression of the constructs in the permanent clones was analyzed by western blotting using and antibody against the V5 tag. A representative western blot is shown. (B) A549-GFPα1 cells (CT) and A549-GFPα1 cells permanently transfected with DN-Va and DN-Vc were incubated in the absence or presence of 50 μM FSK for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three different experiments. (C) Control (CT), DN-Va and DN-Vc cells were incubated in the absence or presence of 50 μM FSK for 10 minutes and western blots of the basolateral membrane fraction were performed using a specific antibody against GFP. E-cadherin was used as loading control. A representative western blot is shown. *P<0.05; **P<0.01; n.s., not significant; u.s., unstimulated.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Dominant Negative Mutation, Activity Assay, Clone Assay, Expressing, Generated, Construct, Western Blot, Transfection, Incubation

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and the Na+/K+-ATPase-containing vesicles colocalize. A549-GFPα1 cells were fixed, permeabilized and blocked. Myosin-Va was visualized by using an anti-myosin-Va antibody and a secondary antibody labeled with Alexa Fluor 568. GFP was directly visualized. Cellular distribution of Na+/K+-ATPase-GFPα1 and myosin-Va was analyzed using a Zeiss LSM 510 laser-scanning confocal microscope and colocalization (blue) was determined using the LSM 510 Meta software.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Labeling, Microscopy, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Microtubules and actin filaments are involved in Na+/K+-ATPase traffic. (A) Live imaging of A549 cells incubated with 10 μM nocodazole for 3 hours. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the microtubule cytoskeleton in control (left) and nocodazole (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and nocodazole (right) conditions. (B) Live imaging of A549 cells incubated with 5 μM cytochalasin D (Cyto D) for 1 hour. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the actin cytoskeleton under control (left) and cytochalasin D (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and cytochalasin D (right) conditions. (C) Average contour length traveled by the vesicles as a function of time. The blue line represents the control vesicles; the black line, cells treated with cytochalasin D and the red line, cells treated with nocodazole. Scale bars: 10 μm and 2 μm (inset images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Imaging, Incubation, Labeling, Single-particle Tracking, Software, Immunofluorescence

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 3. Doxorubicin-induced apoptosis in leukemic cells is dependent on Rac1. (A,B) The Rac1 inhibitor reduces doxorubicin-induced apoptosis in U937 and Jurkat cells. The cells were pretreated with the specific Rac1 inhibitor (NSC23766, 10 μM, 1 h), before their treatment with doxorubicin (Dox) for 24 h. Apoptosis was determined by annexin V staining and flow cytometry analysis. (C,D) Overexpression of the Rac1 dominant negative form N17Rac1 inhibits doxorubicin-induced apoptosis. The cells were transfected with pcDNA3.1, N17Rac1 + pcDNA3.1 or with N17Rac1 + WT-Rac1 plasmids. Viable cells were recovered after 24 h by ficoll gradient. The cells were then treated with doxorubicin for 24 h. Apoptosis was determined by annexin V staining and flow cytometry analysis. The results represent mean values ± SD from three independent experiments. *P < 0.05, **P < 0.01. (E) Rac1 inhibition blocks caspase-9 and -3 activation by doxorubicin. Jurkat cells were treated as indicated and after 12 h of doxorubicin treatment, the levels of native and active caspase-9 and -3 were determined by western blot analysis. β-actin was used as a loading control. The illustrated blots are representative of three independent experiments.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Staining, Flow Cytometry, Over Expression, Dominant Negative Mutation, Transfection, Inhibition, Activation Assay, Western Blot, Control

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 4. Collagen inhibits doxorubicin-induced Rac1 activation via α2β1 integrin. U937 (A) and Jurkat (B) cells were cultured on BSA (-) or on collagen (Col) and then treated or not with doxorubicin (Dox) for 3 h. The cells were harvested, lysed and Rac1 activation was determined by Rac1-GLISA assay. (C) The effect of collagen is mediated via α2β1 integrin. Jurkat cells were treated as above except that before their culture on collagen they were pretreated with 10 μg/ml of control IgG or with the blocking anti-α2 integrin mAb (P1E6). The results represent mean values ± SD from three independent experiments. **P < 0.01.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Cell Culture, Control, Blocking Assay

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 5. Collagen inhibits Rac1 activation independently from ABCC1. (A) ABCC1 protein levels in Jurkat T cells transfected with control and specific ABCC1 siRNA levels. The immunoblot is representative of three independent experiments. (B) ABCC1 silencing does not affect collagen-mediated Rac1 inhibition. Control and ABCC1 siRNA-transfected Jurkat T cells were cultured on BSA or on collagen and then treated with doxorubicin (Dox) for 3 h. The cells were harvested, lysed and Rac1 activation was determined by Rac1-GLISA assay. (C) The ABCC1 inhibitor MK571 does not affect collagen-mediated Rac1 inhibition in U937 cells. The cells were pretreated with the vehicle or MK571 (10 μM) for 1 h after which, the cells were cultured on BSA or collagen and then treated with Dox. Rac1 activation was determined by Rac1-GLISA assay. The results represent mean values ± SD from three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Transfection, Control, Western Blot, Inhibition, Cell Culture

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 6. Rac1 inhibition reduces DNA damage intensity and H2AX phosphorylation induced by doxorubicin. (A–C) The cells were treated or not with doxorubicin (Dox) for 6 h in the presence or absence of the Rac1 inhibitor NSC23766 (NSC). Alkaline comet assay was performed and stained nucleoids were visualized by epifluorescence microscopy using FITC filter. (A) Representative fields corresponding to each treatment were photographed. (B,C) The intensity of DNA strand breaks in U937 and Jurkat cells was quantified using visual scoring as described under “Experimental procedures section”. The results represent mean values ± SD obtained from three independent experiments. **P < 0.01. (D,E) The cells were treated with doxorubicin in the presence or absence of NSC23766 as described above and the levels of phosphorylated H2AX (γ-H2AX) were determined by immunoblot analysis. The β-Actin blot was used as a loading control. Blots are representative of three independent experiments.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Inhibition, Phospho-proteomics, Alkaline Single Cell Gel Electrophoresis, Staining, Epifluorescence Microscopy, Western Blot, Control

Journal: Scientific reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Figure 8. Rac1 is involved in doxorubicin-induced JNK activation and Mcl-1 downregulation. U937 and Jurkat cells were treated or not with doxorubicin (Dox) in the presence or absence of the Rac1 inhibitor NSC23766. After 8 h of treatment, the cells were lysed and the levels of phospho-JNK1/2 (A&B) and Mcl-1 (C&D) were determined by immunoblot analysis. The blots were stripped and reprobed with anti-β-actin antibody for equal loading. The blots are representative of three independent experiments.

Article Snippet: The Rac1 inhibitor (NSC23766) was obtained from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: The differentiation potential of ESCs was altered by culture conditions. ( A ) KhES-1 ESCs in single-cell suspensions were seeded on VNT-N-coated dishes and cultured with Essential 8 (Es8) for 5 passages. The cells were then collected for embryoid body (EB) formation or transferred to Repro FF2 culture medium (RFF2). KhES-1 cells were cultured for 5 passages and collected for EB formation or transferred to Es8 again. KhES-1 cells were cultured for 5 passages, followed by EB formation assays. Photographs of KhES-1 cultures with Es8 or RFF2 medium (upper) at day 1 of culture; EBs at day 14 (lower) are shown. Gene expression profiles of cells in the indicated culture conditions were determined by a qRT-PCR scorecard panel and appended below the relevant photograph. Scale bar: 1.0 mm. ( B ) List of candidate genes and short description related to differentiation potential of PSCs. Value of methylation status and corresponding its gene expression in PSCs cultured with RFF2, S-P and Es8 and shown as in the order of RFF2/S-P/Es8. ( C ) Schematics of CHD7 isoforms, location of PCR primers and antibody (upper), and CHD7 mRNA transcripts used in this study (lower). ( D ) Gene expression of CHD7 in KhES-1 cultures with Es8 (P5 and P15) or RFF2 medium (P5 and P15) determined by qRT-PCR. P: passage numbers. (n = 3 analytical replicates). ( E ) Copy numbers of CHD7 isoform 1 or isoform X4 determined by digital-PCR. Five ng of total RNA obtained from KhES-1 cells cultured with Es8 or RFF2 medium were used as the template, and the copy numbers of the isoforms in each RNA sample were calculated using primer set listed in Fig. 1C. ( F ) CHD7 isoforms detection by Western blotting. Total cell lysates (5.3 μg) from KhES-1 cells cultured with Es8 (P11) or RFF2 medium (P21) were applied to the indicated lanes. CHD7 isoform 1 (expected mass 336 kDa), isoform 2 (101 kDa), and isoform X4 (183 kDa) were detected with antibodies for human CHD7. The signal was visualized by a secondary antibody linked to horseradish peroxidase.

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Cell Culture, Gene Expression, Quantitative RT-PCR, Methylation, Digital PCR, Western Blot

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: Downregulation of CHD7 disrupted differentiation in EB formation assays. ( A ) Protocol for EB formation and siCHD7 transfection. Cells were transfected with small double-stranded interfering RNA targeting CHD7 ( siCHD7 ) or nonspecific control siRNA (mock) on day 0. Cells were transferred to low-attachment plates 24 h after transfection with siRNA and cultured with Es6 supplemented with ROCK inhibitor (RI) for 24 h. The medium was then exchanged with fresh Es6 medium and cultured for another 72 h. The morphology of EBs and their gene expression profiles on days 4, 5 and 14 after transfection with siRNA were determined using a qRT-PCR scorecard panel. ( B ) Expression of CHD7 in KhES-1 cells transfected with siCHD7 or control siRNA (mock) or in non-transfected cells determined by qRT-PCR (time course sampling). Gene expression of CHD7 was standardized according to the average CHD7 expression in KhES-1 cells cultured with Es8 medium, which was independently measured 3 times. A representative result from 3 biological replicates. (n = 3 analytical replicates). ( C ) Expression of CHD7 in KhES-1 cells transfected with siCHD7 or control siRNA (mock) by Western blotting (sampling at day 2). ( D ) Photographs of non-transfected EBs (upper panels) and siCHD7 -transfected (middle panels) or control siRNA-transfected (mock, lower panels) KhES-1 cells on days 4, 5, and 14 are shown. Gene expression profiles of KhES-1 cells under the designated conditions, as determined by qRT-PCR scorecard panel, are shown below the relevant photographs. Scale bar: 1 mm. The representative datasets from 3 independent experiments are shown.

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Transfection, Control, Cell Culture, Gene Expression, Quantitative RT-PCR, Expressing, Sampling, Western Blot

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: Upregulafx1tion of CHD7 isoform 2 mRNA induced “spontaneous” differentiation in ESCs. ( A ) Protocol for cell culture and transfection with mCHD7 . mCHD7 or control mRNA (mock) was transfected into KhES-1 cells on day 0 and day 1 (two times). Cells were passaged on day 2, reseeded at 3 × 10 5 cells/well in 6-well plates, and cultured another 24 h. ( B ) Expression of CHD7 after mCHD7 or control mRNA ( GFP -transfected; mock) transfection (day 0) was determined by qRT-PCR (time course sampling). CHD7 gene expression by qRT-PCR was standardized according to the average CHD7 expression in KhES-1 cells cultured with RFF2 medium measured independently 3 times. A representative result from 3 biological replicates. (n = 3 analytical replicates). ( C ) Western blotting (sampling at day 3) of CHD7 in KhES-1 after mCHD7 or control mRNA ( GFP -transfected; mock) was transfected at day 0. NT: non-transfected control. ( D ) The growth of mock-transfected KhES-1 cells (green line) was comparable to that in non-transfected control cells (blue line), whereas that of mCHD7 -transfected KhES-1 cells (red line) was dramatically suppressed on days 2 and 3. ( E ) Photographs of non-transfected (upper panels), mCHD7 -transfected (middle panels), and mock mRNA-transfected (lower panels) KhES-1 cells cultured with RFF2 medium on days 1, 2 and 3. The gene expression profiles obtained by qRT-PCR scorecard panel are presented below the relevant photo. Scale bar: 1 mm. The representative datasets from 3 independent experiments are shown.

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Cell Culture, Transfection, Control, Expressing, Quantitative RT-PCR, Sampling, Gene Expression, Western Blot

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: Transfection with CHD7 dominant-negative (DN) mRNA transcripts disrupted differentiation potential and cell proliferation of ESCs. ( A ) Protocol for transfection with CHD7 DN mRNA transcripts and EB formation assay. CHD7 DN1: transcript of chromodomain mRNA; CHD7 DN2: transcript of SANT-SLIDE domain mRNA (Figure ). Transcripts were transfected into KhES-1 cells on day 0, and the cells were then transferred to low-attachment plates for 24 h, followed by culture in Es6 medium with Rock Inhibitor (RI) for 24 h for EB formation. Microscopic observation of EBs and gene expression profiles by qRT-PCR scorecard panel on day 3 after transfection with DN mRNA. ( B ) CHD7 DN 1 and CHD7 DN2 expression levels were determined by qRT-PCR. A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of day 3-EBs from non-transfected, CHD7 DN1-, CHD7 DN2-, CHD7 -DN1 + CHD7 -DN2-transfected, and mock mRNA-transfected KhES-1 3 days after transfection. Gene expression profiles were determined using qRT-PCR scorecard panels and are shown below the relevant photograph. The cell number on day 3 of culture in one well of a 6-well plate was scored and appended at the top right corner of the relevant image. Non-transfected KhES-1 cultured with Es8 on day 0 (left panel) was used as a control. Representative results of 3 independent experiments are shown. Scale bar: 1 mm.

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Transfection, Dominant Negative Mutation, Tube Formation Assay, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: Downregulation of CHD7 disrupted the proliferation of ESCs cultured with Es8 medium. ( A ) Protocol for siCHD7 transfection. siCHD7 or nonspecific control siRNA (mock) was transfected into KhES-1 cells on day 0 and day 1. Medium was changed (CM) every day. On days 0–3, cells were harvested for cell counting, and CHD7 expression was determined by qRT-PCR. ( B ) CHD7 gene expression in KhES-1 cells transfected with siCHD7 or control siRNA (mock) or in non-transfected cells was determined by qRT-PCR. CHD7 gene expression was standardized according to the average CHD7 expression in KhES-1 cells cultured with Es8 medium (independently measured 3 times). A representative result from 3 biological replicates is shown. (n = 3 analytical replicates). ( C ) Photographs of KhES-1 cells without transfection (upper panels) or with siCHD7 (middle panels) or control siRNA transfection (mock, lower panels) on day 3. The cell number scored is appended at the upper right corner of the respective photograph. ( D ) The cell number was scored for the indicated conditions at the designated day.

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Cell Culture, Transfection, Control, Cell Counting, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: Human CHD7 co-localized with suppressive chromatin remodelers. ( A ) The binding sites for 17 factors in the map were obtained from a public ESC H1 ChIP-seq database. The odds ratio representing the correlation between binding sites for each pair of factors was calculated. Green indicates high homology between factors, and red indicates no high homology between factors. ( B ) ChIP-qPCR with anti-CHD7 antibody for mock - or si CHD7 -transfected H9 cells. The mock - or si CHD7 -transfected H9 cells in undifferentiated state were immune-precipitated (IP) either with anti-CHD7 antibody or control IgG. qPCR for the putative promoter region of POU5F1 , NANOG , EP300 , EZH2 , SUZ12 or BRG1 were conducted for IP samples. Respective bar shows as fold enrichment of quantity of amplified target gene of interest by IP with anti-CHD7 antibody against that with control IgG in mock - or si CHD7 H9 transfectants. (n = 3 analytical replicates).

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Binding Assay, ChIP-sequencing, ChIP-qPCR, Transfection, Control, Amplification

Journal: Scientific Reports

Article Title: Differentiation potential of Pluripotent Stem Cells correlates to the level of CHD7

doi: 10.1038/s41598-017-18439-y

Figure Lengend Snippet: CHD7 levels mediated the differentiation potential of PSCs. ( A ) The copy numbers of CHD7 isoform 1 in PSCs (ESC: H9, KhES-1, iPSC: PFX#9, 201B7, SHh#2) and their passage numbers (P) are listed in the table. Cells were culture either on feeder with iPSC medium in cell clumps or with Es8, S-P, S-P ACF or RFF2 on VNT-N in single cells or in cell clumps (S-P ACF) ND: Not Determined. Cells cultured with RFF2 on VNT-N failed to demonstrate differentiation potential in EB formation assay (gray columns). ( B ) PSCs (ESC:H9, KhES-1, iPSC:PFX#9, 201B7, SHh#2) were cultured either on feeder with iPSC medium in cell clumps (on feeder cell clumps) or on VNT-N with Es8 in single cells (Es8/N single cells) or S-P ACF in cell clumps (S-P ACF/N clumps) or in single cells (S-P ACF/N single cells), and their differentiation potential was examined by the gene expression profiles of day 14-EBs derived them. The copy numbers of CHD7 prior to EB formation assay was determined and appended beneath the relevant graph.

Article Snippet: Briefly, cDNA was generated from 5 ng total RNA extracted from KhES-1 cells cultured with Es8 or RFF2 medium using TaqMan Gene Expression Assays (Hs00215010_m1; Thermo Fisher).

Techniques: Cell Culture, Tube Formation Assay, Gene Expression, Derivative Assay

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: MC1R expression is upregulated in human melanoma cells upon treatment with BRAFi and HDACi. (A) qRT-PCR analysis of MC1R mRNA expression in BRAFV600E A2058 cells after 24 h of incubation with BRAFi dabrafenib (Dabr) and HDACi 4-phenylbutyrate (PBA) and in BRAFWT MEWO cells after 24 h treatment with HDACi PBA and vorinostat (Vor) (n = 3). Data are presented as normalized mean MC1R mRNA ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs controls; (B) flow cytometry histograms and protein expression of MC1R in A2058 cells after 24 h treatment with BRAFi: Vem (5 μM) and Dabr (2 μM); HDACi: PBA (2 mM) and Vor (2 μM); and in MEWO cells after incubation with PBA (2 mM) and Vor (2 μM). Experiments were conducted in triplicate. Data are expressed as relative expression of MC1R vs isotype control (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs controls); (C) MC1R binding with [125I]NDP-α-MSH in A2058 and MEWO cells following incubation with Dabr (1−10 μM), Vor (0.5−10 μM), and PBA (0.5−10 mM) for 12−24 h (n = 4). Data are expressed as MC1R-ligand binding vs dimethyl sulfoxide (DMSO)-treated cells (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs controls); all experiments were performed in duplicate (n = 2).

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Flow Cytometry, Control, Binding Assay, Ligand Binding Assay

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: Upregulation of MC1R in melanoma cells is mediated by the transcription factor MITF. Immunoblotting analysis of MC1R and MITF expressions in (A) BRAFV600E cells (A2058) following 24 h incubation with dabrafenib (Dabr) (1−10 μM) and vorinostat (Vor) (1−10 μM) and in (B) BRAFWT cells (MEWO) following 24 h exposure to HDACi vorinostat (Vor) (1−10 μM) and 4-phenylbutyrate (PBA) (1−10 mM); immunoblotting analysis of MC1R in (C) A2058 and (D) MEWO cells in response to 24 h incubation with Dabr (1 μM), Vor (1 μM), or PBA (1 mM), with normal MITF expression (negative scrambled Dsi-RNA) and attenuated MITF expression (MITF Dsi-RNA). All experiments were conducted in duplicate (n = 2).

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Western Blot, Incubation, Expressing

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: Combination of BRAF, HDAC inhibitors, and MC1R-targeted 212Pb α-particle therapy significantly impairs BRAFV600E melanoma A2058 tumor growth and improves survival. (A) Representative IHC staining of MC1R in A2058 melanoma-bearing animals that were treated with HDACi PBA (90 mg kg−1, i.p., q.d.), BRAFi vemubrafenib (10 mg kg−1, p.o., b.i.d.) (n = 2); (B) 2 h postinjection SPECT/CT imaging of A2058 melanoma in athymic nu/nu mice treated with vemurafenib (Vem 10 mg kg−1, p.o.) and 4-phenylbutyrate (PBA 90 mg kg−1, i.p.) using [203Pb]DOTA-MC1L as the imaging tracer. Organs of interest are indicated as T (tumor), K (kidney), and B (bladder); (C) average tumor volume for each group of animals after treatments were initiated; data are expressed as mean ± SD; statistical analysis: *p < 0.05, **p < 0.01; (D) overall fractional survival in each treatment cohort over 100 days (n = 9−10 per group): vemurafenib (Vem) (10 mg kg−1, p.o., b.i.d.), 4-phenylbutyrate (PBA) (90 mg kg−1, i.p. q.d.), and 212Pb α-particle therapy (212Pb) (single dose of 5.2 MBq [212Pb]DOTA-MC1L). Statistical analysis: ****p <0.0001 212Pb versus control; ****p < 0.0001 212Pb versus Vem; **p < 0.01 212Pb/Vem vs 212Pb; **p < 0.01 212Pb/Vem/PBA vs 212Pb/Vem.

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Immunohistochemistry, Single Photon Emission Computed Tomography, Imaging, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Polar interactions formed by Arg 249 with neighbouring residues in p53 WT . b Change in polar contacts with neighbouring residues due to mutation of Arg 249 to serine in p53 R249S . b Superimposed view of the mutation site in p53 WT and p53 R249S . Residues of p53 WT are shown in green and for p53 R249S they are shown in pink. Change in the structure at 249th position is highlighted by a circle. Residues that acquired significantly different conformations are shown in stick representation. c Root mean square deviations of residues in p53 WT and p53 R249S . (a) RMSD of Arg 248 during the course of MD simulation in p53 WT (green) and in p53 R249S (red). (b) RMSD of residues from 249 to 271 in p53 WT (green) and p53 R249S (red)

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: a Difference in the molecular surface near Y220 position in p53 WT and p53 Y220C mutant. a Size of cavity in p53 WT . b Mutation from Tyr to Cys at 220th position rearranges the conformation of surrounding residues, especially located in the loop region. Widening of the loop near 220th position and removal of the Tyr side chain creates a larger cavity in p53 Y220C protein. c Superimposition of the two molecular surfaces reveals the stringency of the p53 WT (green color) as compared to the p53 Y220C (red color). b Water network near 220th residue in p53 WT and p53 Y220C protein. a Tyr 220 stabilizes the residues of surrounding loops with the help of water molecules. b Cys 220 stabilizes the cavity by solvating it with water molecules. Binding of the withanolides with p53 WT near Tyr 220. Wi-A c and Wi-N d were found to interact with the surface residues near Tyr 220 as no deep cavity was present in p53 WT

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Interactions of Wi-A with p53 Y220C near Cys 220. a Binding pose of Wi-A within the binding site near Cys 220 b 2D representation of the interactions of Wi-A with p53 Y220C . Interactions of Wi-N with p53 Y220C protein structure. c Binding pose of Wi-N within the binding cavity of p53 Y220C near Cys 220 d 2D representation of the interactions of Wi-N with surrounding residues within the cavity of p53 Y220C . Charge complementarity of Wi-A and Wi-N with the binding cavity. Wi-A e and Wi-N f both were fitting inside the cavity according to the charge distribution within it. White region represents the hydrophobic region whereas blue and red represents the hydrophilic region

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A furnished wild type p53 function in mutant p53 (p53 Y220C ) horboring hepatoma cells. a Viability assay of human hepatocarcinoma with wild type p53 (HuH-6), mutant p53 (HuH-7), and telomerized human cells bearing p53 mutants (p53 V143A , p53 R249S and p53 R273H ). b Comparison of response of HuH-6 and HuH-7 cells to Wi-A. Dose response was observed for both the cell lines. HuH-7 showed stronger cytotoxicity to Wi-A. c Western blot showed reduction in mortalin and increase in p53 in cells treated with 1 μM Wi-A in the p53 mutants, p53 V143A and p53 R273H ; p53 R249S cells possessed low expression that remained undetected on these blots. In contrast to increase in the expression of p53 V143A and p53 R273H , mutant p53 Y220C protein expression was decreased in Wi-A treated cells. d Dose dependent decrease in mutant p53 Y220C protein expression in Wi-A treated cells. e Immunostaining of mortalin and p53 (40 x magnification) in control and Wi-A (0.5 μM) showing increase in nuclear p53 V143A and p53 R273H . HuH-6 (p53 WT ) cells showed increase in nuclear p53 staining. In contrast, HuH-7 (p53 Y220C ) cells exhibited decrease in p53 nuclear staining

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Mutagenesis, Viability Assay, Comparison, Western Blot, Expressing, Immunostaining, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced restoration of wild type p53 and induction of senescence in HuH-7 cells. a Wild type p53 reporter activity in mock (untransfected), control (transfected and untreated) and Wi-A (transfected and Wi-A treated) cells. Luciferase reporter assays driven either by p53 consensus binding sites (PG13-Luc) or by a p21 WAF-1 promoter (WWP) showed an increase in Wi-A treated cells. b Flow cytometry analysis revealed G 2 cell cycle phase arrest in HuH-7 cells. c Immunostaining of p21 WAF-1 in HuH-6 and HuH-7 control and Wi-A treated cells showing increase in p21 WAF-1 expression in the latter. d Senescence-associated β-galactosidase staining was observed in Wi-A treated HuH-6 and HuH-7 cells (10 x phase magnification). e Wi-A treated HuH-6 and HuH-7 cells showed enhanced staining for nuclear heterochromatin protein HP1γ

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Activity Assay, Control, Transfection, Luciferase, Binding Assay, Flow Cytometry, Immunostaining, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Wild type p53 function in p53 Y220C mutant harboring cells by treatment with Ashwagandha derived anticancer withanolides: bioinformatics and experimental evidence

doi: 10.1186/s13046-019-1099-x

Figure Lengend Snippet: Wi-A induced apoptosis of HuH-7 cells. a Annexin-V staining revealed induction of early apoptosis in Wi-A treated cells. b Apoptosis in Wi-A treated HuH-7 cells was marked by cleavage of caspase3, increase in p21 WAF-1 and phosphorylated p53. c Wi-A treated cells showed Single Strand Breaks as determined by a comet assay, 40 X magnification. d Wi-A rich extract (AL-βCD) treated HuH-6 (p53 WT ) and HuH-7 (p53 Y220C ) cells showed increase and decrease in nuclear p53, respectively. e Increase in the expression of p21 WAF-1 was observed in AL-βCD treated HuH-6 and HuH-7 cells. f HuH-7 cells showed strong cytotoxicity to AL-βCD

Article Snippet: Rabbit anti-PARP-1 (H-250), anti-caspase-3 (H-277), goat anti-PML (N-19) (Santa Cruz), mouse anti-p53 (DO-1 and Fl-393; pan-p53 antibodies recognizing wild type as well as mutant p53 epitopes) (sc-126, Santa Cruz) [ ], Y5-detecting mutant p53 only (ab32049, Abcam); rabbit anti-PARP-9, anti-p21 WAF-1 (12D1) (Cell Signaling Technology) antibodies were used.

Techniques: Staining, Single Cell Gel Electrophoresis, Expressing

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, illustration of the HCMV AC (red) and nuclear (blue) rotation phase, highlighting primary imaging windows. b-c, Western blot and immunofluorescence showing αTAT1 depletion suppresses microtubule acetylation. Fluorescence intensity of acetylated microtubules was quantified; n = 303 cells total, ****p≤0.0001, two-tailed student’s t-test. All data points are shown within violin plots. Similar results yielded from 3 independent experiments. d-e, αTAT1 depletion suppresses nuclear rotation. Representative stills from and rotational analyses are shown. Rotation frequency above or below 180° is shown in e; bars represent mean ± SEM; n = 309 cells total,***p≤0.001, two-tailed student’s t-test. f-g, Spatial distribution and intensity of DNA (hoescht), AC marker (gB), acetylated microtubules (Ac-K40-MT) and SUN1 using CNN (g) or DNA, gB and SUN1 using MASK-RCNN (h) analyses. Lines represent mean ± SEM; n = 34,712 cells total in dataset from 3 independent biological replicates for f; n = 2,214 cells total for g. h, αTAT1 depletion suppresses SUN1 polarization. For quantification, SUN1 was classed as polarized (green), intermediate (pink) or non-polarized (orange). n = 583 cells total. Similar results yielded from 3 independent experiments.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Imaging, Western Blot, Immunofluorescence, Fluorescence, Two Tailed Test, Marker

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a-b, Representative stills from time lapse imaging and measurements of rotation frequency above or below 180° in uninfected or infected NHDFs expressing GFP-Histone nanobody. Bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 281 cells total from 3 independent biological replicates; ****p≤0.0001. Note that nuclear rotation above 180° occurs in approximately 80% of infected cells imaged, while lower levels of rotation occur in the remaining population. Such extensive rotation is extremely rare in uninfected cells. c-d, Expression of a K40R mutant form of tubulin suppresses the formation of acetylated microtubule filaments. Fluorescence intensity of acetylated tubulin is shown in b; All data points are shown within violin plots, statistics use two-tailed student’s t-test, n = 250 cells total, ****p ≤ 0.0001. Data shown is representative of 3 independent biological replicates. e-f, Expression of a K40R mutant form of tubulin suppresses nuclear rotation. Representative stills from are shown in e and rotational analyses are shown in f. Rotation frequency above or below 180° is shown in d; bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 157 cells total, **p ≤ 0.01 g, schematic of CNN-based classification and analysis pipeline measuring fluorescence intensities across individual cells in different channels. Output for the AC (red) next to the nucleus (blue) is illustrated. h, Representative confocal z-section and deconvolved z-section image of SUN1 polarization in HCMV-infected cell. Acetylated microtubules and the AC (stained with the viral protein gB) are also shown. Data shown is representative of 3 independent biological replicates. i , Spatial distribution and intensity of DNA, gB and SUN2 using CNN. Lines represent mean ± SEM; n ≥ 17,484 cells total from 3 independent biological replicates.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Imaging, Infection, Expressing, Two Tailed Test, Mutagenesis, Fluorescence, Staining

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, WB analysis of SUN1 levels over the course of infection with HCMV at MOI 1. Early (IE1/2), intermediate (UL44) and late (pp65, pp28) proteins demonstrate stages of infection at each timepoint, representative of 3 independent biological replicates. b-d, Neural network-based single cell analysis of SUN1 expression during HCMV infection. b , Illustration of CNN analysis pipeline that classifies cells by the predominant infectious cycle stage identified at each timepoint. c , Representative examples of uninfected NHDFs or NHDFs at various stages of infection, stained for SUN1, IE1/2 and TGN46. Expression of IE1/2 and gradual remodeling of the Golgi network serve as markers of infection stage. Polarization of SUN1 is seen between 24–72 h.p.i. d , CNN-based classification of cells based on IE1/2 expression levels, filtering out uninfected cells, reveals a gradual expansion of the nucleus and Golgi, characteristic of HCMV infection, occurs concomitantly with a gradual increase in expression and polarization of SUN1 toward the AC (i-v). Discrete populations of cells are filtered for inclusion in each timepoint (vii), with cells from other kinetic classes removed from analysis marked in grey (viii-x).Comparing unfiltered (lighter colored violin plots, left segment) versus filtered (dark colored violin plots, right segment) cell populations reveals the power of trained networks to more precisely analyze only infected cells within the population, more clearly revealing the increase in nuclear volume and SUN1 abundance, which peaks at approximately 2-fold (xi-xiii). Lines represent mean ± SEM; n = 37,800 cells total from 3 independent biological replicates. Violins as in Fig. 4a . e , Mask-RCNN analysis pipeline uses manually annotated masks of the AC, nucleus and combined (HCMV) to train a Mask-RCNN architecture to classify and segment microscopy images of HCMV infection. Once trained, whole cover-slip scanning datasets can be run through the model to perform instantaneous single cell quantification on high-confidence infected cells. This quantification has high spatial awareness and can be used to perform linescans between two specific subcellular compartments (e.g. the AC and nucleus) or to rotate and align nuclei to perform average projections (as in ).

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Infection, Single-cell Analysis, Expressing, Staining, Microscopy

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a-c, SUN1 depletion suppresses nuclear rotation. Representative stills from and rotational analyses are shown. Western blot is representative of 3 independent experiments. Rotation frequency above or below 180° is shown in c; n = 162 cells total from 3 independent biological replicates, ***p≤0.001. d-e, Effects of Nesprin-2G constructs on nuclear rotation. Representative stills and rotation analyses from are in f. Frequency of rotations above or below 180° are in g; bars represent mean ± SEM, n = 127 cells total from 5 independent biological replicates, **p≤0.01, ***p≤0.001, ****p≤0.0001, two-tailed student’s t-test.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Western Blot, Construct, Two Tailed Test

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a-b, Expression of a SUN1 mutant that does not engage Nesprin-2G impairs nuclear rotation in HCMV-infected cells. a, Representative stills from time lapse recordings of NHDFs expressing Tag-GFP2 forms of SUN1 Full Length (FL) or SUN1 lacking the lumenal domain (SUN1ΔLu) that mediates interactions with Nesprin-2G, infected with HCMV-UL99mCherry. Rotation traces from this imaging are shown to the right. Analyses focused on cells expressing intermediate levels of SUN1-GFP constructs as high levels of expression can result in retention of Nesprin-2G in the endoplasmic reticulum (ER). b, Quantification of rotation frequencies above or below 180°; bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 138 cells total from 3 independent biological replicates, ***p≤0.001. This data further confirms that interactions with Nesprin-2G are necessary for nuclear rotation to occur. c-f, RNAi-mediated depletion of BICD2 using either of two independent siRNAs suppresses nuclear rotation and SUN1 polarization. c, Illustration of SUN1:Nesprin-2G interactions with microtubule motors through AD regions, or SUN2:Nesprin-2G interactions with myosin through CH domains to control nuclear movement. d, Illustration of GFP-Nesprin-2G constructs with CH and/or AD domains, along with the LEWD>LEAA kinesin-binding mutant. e, Western blot analysis of BICD2 expression representative of 3 independent replicates. Arrow points to BICD2, specifically depleted by two independent siRNAs. f, Representative stills and rotational analyses from Video 6 showing effects of BICD2 depletion on nuclear rotation. g, Rotation frequency above or below 180° in control or BICD2 depleted cells, bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 144 cells total cells from n = 3–4 independent biological replicates, ***p ≤ 0.001. h, Depletion of BICD2 impairs SUN1 polarization. Spatial distribution and intensity of DNA (hoescht), AC marker (gB), and SUN1 are shown for control and BICD2 depleted cells and are representative of 3 independent biological replicates. For quantification, SUN1 was classed as polarized (green), intermediate (pink) or non-polarized (orange). n = 235 cells total. i-k , Expression of a dominant-negative fragment of BICD2 reduces nuclear rotation and SUN1 polarization. NHDFs expressing TagGFP2 control or TagGFP2-BICD2 N-terminus (NT) were infected with HCMV UL99-mCherry. i , Representative still images from time lapse recordings and rotation traces are shown. j, Quantification of rotation frequencies above or below 180°; bars represent mean ± SEM, n = 91 cells total from 2 independent biological replicates. k , Representative images of SUN1 localization in NHDFs expressing TagGFP2 control or TagGFP2-BICD2-NT NHDFs are shown, consistent with 3 independent biological replicates. Quantification of SUN1 polarity categorized as fully polarized, intermediate polarity or not polarized is shown; n = 149 cells total.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Expressing, Mutagenesis, Infection, Imaging, Construct, Two Tailed Test, Control, Binding Assay, Western Blot, Marker, Dominant Negative Mutation

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Lamin A/C is downregulated and lacks polarity in HCMV-infected cells. Lines represent mean ± SEM; n = 10,934 cells total from 3 independent biological replicates. b, Depletion of αTAT1, SUN1 or BICD2 inhibits Emerin polarization and causes aberrant F-actin networks. Representative images are shown for each condition, similar to data from 3 independent replicates. c-d , Emerin depletion blocks nuclear F-actin formation. c, WB analysis demonstrating the efficacy of Emerin siRNAs. d, Representative images and quantification of nuclear F-actin (nAC) frequency are shown for each condition, bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 401 cells total from 3 independent biological replicates, ***p≤0.001. Fluorescence intensity shows Emerin depletion in cells. e , Emerin depletion does not affect SUN1 polarization. Representative images and quantification of SUN1 polarization is shown for each condition; n = 321 cells. SUN1 was characterized as polarized, intermediate polarity or not polarized. f , Expression of actin-binding mutants of Emerin blocks nuclear F-actin formation but not nuclear rotation. NHDFs expressing nAC-TagGFP and mCherry-Emerin wildtype or actin-binding mutants (m151, m175) were infected with HCMV UL99-mCherry. Representative still images and rotation traces from time lapse imaging are shown. Quantification of nuclear rotation frequencies above or below 180° are shown for each condition; the presence of nuclear F-actin was also quantified in the same time lapse images, n = 72 cells total (upper) and n = 79 cells total (lower). Note that in order to image nAC-TagGFP cells were infected with HCMV UL99-mCherry. As such, mCherry signal in these images originates from both mCherry-Emerin and the viral UL99-mCherry, showing the cytoplasmic AC and nuclear rotation in infected cells under all conditions. Data shown is representative of 3 independent replicates.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Infection, Two Tailed Test, Fluorescence, Expressing, Binding Assay, Imaging

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Distribution of histone H3 forms in HCMV-infected cells. Lines represent mean ± SEM; Total H3 (n = 13,774 cells total), H3K4me3 (n = 31,886 cells total), H3K27me3 (n = 30,874 cells total), H3K9me2 (n = 34,342 cells total), H3K9me3 (n = 13,790 cells total). Violin plots represent median (white point), interquartile range (IQR, box), and maximum/minimum values 1.5 x IQR outside the IQR (whiskers). b, Representative image of viral genomic DNA (gDNA) and H3K9me3 foci. White line delineates the peak of H3K9me3 foci in overlay images. c, Depletion of αTAT1, BICD2 or SUN1 reduces the polarity and abundance of viral gDNA. Fluorescence as a function of nuclear area was used to measure the extent of gDNA polarity, and mean fluorescence intensity was used to measure gDNA levels; All data points are shown within violin plots, n = 840 cells total, *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed student’s t-test. d-e , Actin-binding mutants of Emerin inhibit H3K9me3 polarization without affecting SUN1 polarization. d, Representative images are shown. e, Quantification of H3K9me3 (n = 220 cells total) and SUN1 (n = 300 cells total) polarity, categorized as polarized, intermediate (int.) or not polarized. f , Nuclear-localized Arpin inhibits nuclear F-actin formation (bars represent mean ± SEM, n = 1,237 cells total from 3 independent biological replicates; **p≤0.01, two-tailed student’s t-test). g - h , Nuclear-localized Arpin inhibits H3K9me3 (n = 368 cells total) but not SUN1 (n = 236 cells total) polarization. i , Expression of nuclear-localized polymerization-deficient actin inhibits nuclear F-actin formation (bars represent mean ± SEM, n = 1,157 cells total from 3 independent biological replicates; **p≤0.01, two-tailed student’s t-test). j - k , Nuclear-localized polymerization-deficient actin inhibits H3K9me3 (n = 234 cells total) but not SUN1 (n = 185 cells total) polarization. All data representative of 3 independent biological replicates.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Infection, Fluorescence, Two Tailed Test, Binding Assay, Expressing

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Depletion of αTAT1, BICD2 or SUN1 reduces H3K9me3 polarization. Representative images are shown. b , Expression of BICD2 dominant-negative (BICD2-NT) or SUN1 lacking its lumenal domain (SUN-ΔLu) that mediates interactions with Nesprin-2G inhibits the polarization of H3K9me3 in HCMV-infected cells. Representative images and quantification of H3K9me3 polarization are shown; n = 171 cells total. c , Depletion of Emerin impairs the polarization of H3K9me3 in HCMV-infected cells. Representative images and quantification of H3K9me3 polarization are shown; n = 490 cells total. For all experiments, data shown is representative of 3 independent biological replicates.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Expressing, Dominant Negative Mutation, Infection

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: Top, In uninfected cells chromatin and silenced domains are heterogeneously distributed throughout the nucleus, as discussed in the main text. Middle , Upon HCMV infection, nuclear F-actin is induced and reorganized through the action of acetylated microtubules that exert mechanotransductive pulling forces on Nesprin-2G:SUN1-containing LINC complexes, polarizing them towards the AC. In doing so, this creates extreme polarity in inner-nuclear Emerin, directing nuclear F-actin organization; enriched red regions represent polarized LINC-Emerin complexes in the nuclear membrane. This extreme polarity draws silenced (H3K9me3) histones and associated host DNA towards this region of the nucleus, through the action of nuclear F-actin networks. As viruses employ a wide range of strategies to prevent chromatinization and silencing of their own DNA, viral gDNA is not drawn to the AC-proximal sites of H3K9me3 polarization. The polarization of inactive histones and host DNA likely pushes viral DNA to the opposing side of the nucleus, through space-filling. This segregation of viral and host DNA creates an optimal environment for viral DNA replication and production of infectious virus particles. Lower , Polarization of the nucleus fails to occur if key components driving the process are inhibited; if microtubules are not mechanically strengthened through acetylation, if connections between microtubules and nuclear membrane complexes are lost, or if nuclear F-actin is not organized by Emerin. Notably, nuclear F-actin and Emerin do not control nuclear rotation, but cytoplasmic microtubule-derived forces that cause nuclear rotation control Emerin localization, F-actin formation and intranuclear polarity. As such, cytoplasmic forces on the nuclear surface organize nuclear factors to control genetic polarity. Failure to create this polarity results in a suboptimal environment for viral DNA replication.

Article Snippet: Primary antibodies used for immunofluorescence were: HCMV glycoprotein B (gB; USBiological: C9100–21N); SUN1 (Novus Biologicals: NBP1–87396); SUN2 (Thermo Fisher Scientific: PA5–51539); HCMV IE1/2 (Abcam: ab53495); Emerin (Proteintech: 10351–1-AP); Lamin A/C (Santacruz: sc-7292); γH2AX (phospho S139; Abcam: ab11174); H3K9me3 (Abcam: ab8898); Histone 3 (Abcam: ab195277); Ac-K40 tubulin (Sigma: T6793); mCherry/RFP[5F8] (Chromotek: 60706002AB); H3K9me2 (Cell Signalling: 4658); H3K27me3 (Cell Signalling: 9733); H3K4me3 (Cell Signalling: 9751) Quantification of Ac-K40 MTs was performed using ImageJ to extract >100 regions of interest (ROIs; 500×500 pixels) surrounding HCMV ACs stained with gB.

Techniques: Infection, Membrane, Virus, Control, Derivative Assay

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: Cells (A549 or GFP-Rab11a-WT) were infected at a multiplicity of infection (MOI) of 3 with PR8 and, at the indicated time points, were fixed and stained for NP by immunofluorescence ( a. – i., k. – p. ). For live imaging (j.), cells (A549) were co-transfected with a plasmid encoding Rab11a-WT and mCherry-NP and simultaneously infected with PR8 virus at an MOI of 10 and were live imaged at 12hpi (n = –15 – 33). Above each boxplot, same letters indicate no significant difference between conditions, while different letters indicate a statistical significance at α = 0.05. Abbreviations: AU, arbitrary unit; WT, Rab11a Wild type; CM, complete; Ncz, nucleozin. a. Boxplot depicting the fold change in cytoplasmic to nuclear vRNP (NP, as proxy) concentration at different temperatures (°C). P = 0.0362; one-way ANOVA followed by Tukey multiple comparisons of means. b. Scatter plot of nucleation density (ρ) versus degree of supersaturation (S), as a measure of propensity to remain dispersed in the cytoplasm, at different temperatures (°C). c. Scatter plot of vRNP concentration within inclusions (C dense , AU) versus surrounding cytoplasm (C dilute , AU) at different temperatures (°C). d. Scatter plot of C dilute (AU) versus total cytoplasmic vRNP concentration C cytoplasm (AU) at different temperatures (°C). e. Scatter plot of fold change in free energy of partition (ΔΔG), cal.mol -1 , at the indicated temperatures (°C). f. Boxplot of fold change in free energy of partition (ΔΔG, cal.mol -1 ) at different temperatures (°C). P < 8.01e-16; one-way ANOVA followed by Tukey multiple comparisons of means. g. Boxplot depicting the fold change in the ratio of cytoplasmic to nuclear vRNPs concentration at different times of infection or when overexpressing Rab11a (WT) at 8hpi. P = 0; Kruskal Wallis Bonferroni treatment. h. Scatter plot of vRNP concentration within inclusions (C dense , AU) versus surrounding cytosol (C dilute , AU) at different times of infection (hpi) or Rab11a (WT) overexpression. i. Boxplot of fold change in free energy of partition (ΔΔG, cal.mol -1 ) at different times of infection or with Rab11a (WT) overexpression at 8hpi. P = 0; Kruskal Wallis Bonferroni treatment. j. Time lapse images showing fission (blue arrow) and fusion (yellow arrow) events of inclusions (NP, as proxy) at 16hpi in cells expressing endogenous and overexpressed Rab11a (WT) (extracted from Supplementary Videos 1,2). Scale bar = 2 µm. k. Boxplot depicting the fold change in the ratio of cytoplasmic to nuclear vRNPs concentration before and after Ncz (5 mM) treatment at 8hpi. P = 6.16e-14; Kruskal Wallis Bonferroni treatment. l. Scatter plot of nucleation density (ρ, µm -2 ) versus degree of supersaturation (S) changes before and after nucleozin (Ncz) treatment at 8hpi. m. Scatter plot of vRNP concentration outside inclusions (C dilute , AU) versus total cytoplasmic concentration (C cytoplasm , AU). Coloured lines are non-linear fitted models of grouped data points in the graph. n. Scatter plot of fold change in free energy of partition (ΔΔG, cal.mol -1 ) versus surrounding cytoplasmic vRNP concentration (C dilute , AU) before and after nucleozin (Ncz) treatment at 8hpi. o. Boxplot of fold change in free energy of partition (ΔΔG, cal.mol -1 ) before and after nucleozin (Ncz) treatment at 8hpi. P = 0; Kruskal Wallis Bonferroni treatment. p. Scatter plot of vRNP concentration within inclusions (C dense , AU) versus surrounding cytosol (C dilute , AU) before and after nucleozin (Ncz) treatment at 8hpi. All the values calculated for the thermodynamics have been included as supplementary Table 1, distributed in different excel sheets for each parameter.

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Infection, Staining, Immunofluorescence, Imaging, Transfection, Plasmid Preparation, Virus, Concentration Assay, Over Expression, Expressing

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: Cells (A549 or GFP-Rab11-WT) were infected at a multiplicity of infection (MOI) of 3 with PR8 virus and, at the indicated time points, were fixed and stained for NP by immunofluorescence ( a. - m. ). For live imaging ( n. ), A549 cells were co-transfected with a plasmid encoding Rab11a-WT and mCherry-NP and simultaneously infected with PR8 virus at an MOI of 10 and were live imaged at 12hpi. The cytoplasmic vRNP concentration increases with time of infection (hpi) and was used as a proxy for cytoplasmic vRNP concentration changes. ( b. – m. ) We adapted the method published by to determine concentration Cdense as the mean fluorescence intensity of vRNPs in the segmented IAV inclusions, while concentration C dilute was extrapolated from the cytoplasmic vRNP intensity outside the inclusions. Also, size and shape of inclusion was extracted from the segmented inclusions. Each dot is the average value of measured parameter within or outside IAV inclusions per cell, while the continuous black lines are non-linear fitted models for all data. Conditions were normalized to an infection state without IAV inclusions (3 hpi) that is indicated by the dashed black line. Above each boxplot, same letters indicate no significant difference between them, while different letters indicate a statistical significance at α = 0.05 using one-way ANOVA, followed by Tukey multiple comparisons of means for parametric analysis, or Kruskal-Wallis Bonferroni treatment for non- parametric analysis. Abbreviations: AU, arbitrary unit, and WT, Rab11a-WT. a. Immunofluorescence images of infected and fixed A549 cells at different times post-infection (hpi, a proxy for changing cytoplasmic vRNP concentration); NP (green), Rab11 (red), and nucleus (blue). Scale bar = 10 µm. b. Scaterr plot of vRNP concentration withing inclusions (C dense - AU) and its total cytoplasmic vRNP concentration (C cytoplasm , AU). c. Scatter plot of vRNP concentration outside inclusions (C dilute , AU) versus total cytoplasmic vRNPs concentration (C cytoplasm , AU) with time of infection. Coloured lines are non-linear fitted models of the data points in the graph. d. Scatter plot of (C dense , AU) in free energy or partition (ΔΔG) versus area of inclusion. e. Scatter plot showing nucleation density ( , µm -2 ) versus degree of supersaturation at different time of infection. f. Dot plot and model depicting nucleation density (ρ) over time of infection (hpi). g. Boxplot of inclusion area per cell at different post- infection time (hpi). P = 0; Kruskal Wallis Bonferroni treatment. h. Boxplot of inclusion aspect ratio at different hours post-infection (hpi). P < 2e-16; one-way ANOVA, followed by Tukey multiple comparisons of means. i. Scatter plot of inclusion circularity versus roundness at different time post infection (hpi). j. Scatter plot of fold change in free energy of partition (ΔΔG) relative to 3hpi versus area of inclusion. k. Scatter plot of fold change in free energy of partition (ΔΔG, where ΔG = -RT In K, and )versus C dilute (AU) with time of IAV infection. To validate the method for calculating partition coefficient and degree of supersaturation relative to its application in other manuscripts, we compared different methods to calculate the C dilute , Cdense, S, and Snominal ( l. – m. ). l. Scatter plot of partition coefficient (K) derived from averaged vRNP concentration in all inclusion per cell (C dense total, AU) versus partition coefficient calculated from vRNP concentration from individual inclusion (C dense , AU) in each cell at different time post infection (hpi). m. Scatter plot of nominal degree of supersaturation versus degree of supersaturation at different time post infection (hpi). n. Time lapse image of fission (blue arrow) and fusion (yellow arrow) dynamics of viral inclusions at 16hpi in cells expressing endogenous and overexpressed Rab11a (Supplementary Video 9,10).

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Infection, Virus, Staining, Immunofluorescence, Imaging, Transfection, Plasmid Preparation, Concentration Assay, Fluorescence, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: a. Experimental schematics and data to assess if Ncz treated IAV inclusions dissolve in response to shocks. A549 cells were infected at an MOI of 3 with PR8 virus and treated with Ncz or DMSO at 7hpi. An hour later, cells were treated for 30 min with 80% water (hypotonic shock, Hyp), with 1,6-hexanediol (Hex) or complete media (CM) as control. Cells were fixed at 8.5h, stained for NP by immunofluorescence and the percentage of cells with IAV inclusions was scored manually. P = 0; Kruskal Wallis Bonferroni treatment. b. – j. A549 cells were transfected with plasmids encoding either GFP-NP ( b. – f. , i. – j. ) or photoactivatable GFP-NP ( g. – h. ) and simultaneously infected with PR8 virus at an MOI of 10, being live imaged from 12hpi onwards. b. Scheme showing how IAV inclusions were tracked over time and plot depicting the resulting mean square displacement (µm 2 ) of IAV inclusions in the presence or absence of Ncz. P = 0; Kruskal Wallis Bonferroni treatment. c. Schematics of the coarsening assay model, in which liquid and hardened IAV inclusions are represented by orange and blue dots, respectively. Unlike hardened inclusions, native liquid inclusions to fuse and relax to a spherical droplet. d. Pseudo-colored time-lapse images of coalescing viral inclusions (GFP-NP used as proxy; extracted from Supplementary Videos 3,4) in the presence or absence of Ncz. e. Aspect ratio (AR) was used as a measure of IAV inclusion coalescence into a sphere. Mean AR per time was fitted to a linear model (bold coloured lines). Horizontal grey dash lines depict a perfect sphere (aspect ratio = 1). f. Boxplot of the fusion time (sec) of IAV liquid inclusions. Dots represent fusion time of individual fusion event. g. Schematic of a fluorescence loss after photoactivation (FLAPh) experiment. h. Time lapse pseudo-colour images showing the fluorescence loss in photoactivated IAV inclusions (photoactivatable GFP-NP used as proxy) upon treatment with nucleozin or DMSO (extracted from Supplementary Videos 5,6). i. Fluorescence intensity decay of photoactivated (PhotoGFP-NP) normalised to the corresponding IAV inclusions expressing cherryNP. Coloured lines are single exponential model fitting (y0 = (1-a) + ae -kt ) of the data point, dots are the mean of the data per second, and vertical lines denote the standard deviation (SD) per time (sec). j. Half-life of liquid and hardened IAV inclusions decay post-activation (sec). P = 1.386e-6; Kruskal Wallis Bonferroni treatment. k. A549 cells were transfected with plasmids encoding mcherry-NP and co-infected with PR8 virus at an MOI of 10. At 12hpi, cells were treated with nocodazole (10 μg/mL) for 2h to reduce the highly stochastic motion of liquid IAV inclusions and subsequently treated with DMSO or Ncz. Small regions inside IAV inclusions were photobleached to assess internal rearrangement of vRNPs (mCherry-NP as proxy). Time lapse pseudocolor images shows the fluorescence recovery after photobleaching (FRAP, extracted from Supplementary Videos 7,8). Bar = 2 μm l. Mice were intranasally infected with 4000 plaque forming units (PFU) of X31 virus, and after 2 days were treated with PBS or 69 µg of nucleozin at 30 min, 1h or 2h before the collection of the lungs. Immunofluorescence images show sections of lung tissue stained for NP (red) and nucleus (blue) after PBS or Ncz treatment. m.-p. A549 cells stably expressing a dominant negative version of Rab11a fused to GFP (Rab11a-DN) were transfected with cherry-NP ( n., o.) or later stained for NP (p.) and infected (PR8, MOI=3) before treating with Ncz or DMSO at 12hpi. m. Schematic depicting the possible outcomes when Rab11a-DN cell lines are treated with Ncz n. Time lapse pseudocolor images show fusion of IAV inclusions in a coarsening assay of PR8 infected Rab11a-DN cell line treated with Ncz or DMSO (extracted from Supplementary Videos 9,10). o. Plot depicting the aspect ratio of fusing inclusions over time in infected Rab11a-DN cell line treated with Ncz. p. Boxplot comparing the fold change in free energy of partition (ΔΔG, cal.mol -1 ) of IAV inclusions, normalized to 3hpi, in PR8 infected cells overexpressing Rab11a (DN and WT, red box) at 12 hpi. P < 0.01; Kruskal Wallis Bonferroni treatment. Cells were treated with DMSO or nucleozin and values compared to those obtained in control cells expressing endogenous Rab11a (at 8 and 12 hpi). Above each boxplot, same letters indicate no significant difference between them, while different letters indicate a statistical significance at α = 0.05.

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Infection, Virus, Control, Staining, Immunofluorescence, Transfection, Fluorescence, Expressing, Standard Deviation, Activation Assay, Stable Transfection, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: (a. - k.) A549 cell were infected at a multiplicity of infection (MOI) of 3 with PR8 virus for 8 hrs, then incubated with nucleozin (Ncz) for 5mins - 2hrs before fixing and staining for NP and Rab11 by immunofluorescence. ( b. – j. ) We adapted the method published by to determine concentration Cdense as the mean fluorescence intensity of vRNPs in the segmented IAV inclusions, while concentration C dilute was extrapolated from the cytoplasmic vRNP intensity outside the inclusions. Also, size and shape of inclusion was extracted from the segmented inclusions. Each dot is the average value of measured parameter within or outside IAV inclusions per cell, while the continuous black lines are non-linear fitted models for all data. In ( i. – j. ), conditions were normalized to an infection state without IAV inclusions (3 hpi) that is indicated by the dashed black line. Above each boxplot, same letters indicate no significant difference between them, while different letters indicate a statistical significance at α = 0.05 using one-way ANOVA, followed by Tukey multiple comparisons of means for parametric analysis, or Kruskal-Wallis Bonferroni treatment for non-parametric analysis. Abbreviations: AU, arbitrary unit, and WT, Rab11a-WT, CM, complete media and Ncz, nucleozin. a. Immunofluorescence images of A549 cells infected and fixed before and after Ncz treatment. NP (green), Rab11a (red). Scale bar = 10µm. b. Boxplot of inclusion area per cell before and after Ncz treatment. P = 0; Kruskal Wallis Bonferroni treatment. c. Boxplot of inclusion aspect ratio before and after nucleozin (Ncz) treatment. P < 2e-16; Kruskal Wallis Bonferroni treatment. d. Scatter plot of inclusion circularity versus roundness before and after Ncz treatment. e. Boxplot showing the number of inclusions per cell before before and after Ncz treatment. P = 0; Kruskal Wallis Bonferroni treatment. f. Scatter plot of vRNP concentration within inclusions (C dense , AU) and its total cytoplasmic vRNP concentration (C cytoplasm , AU) before and after Ncz treatment. g. Boxplot showing inclusion C dilute (AU) drop till 20min of Ncz resident time after which it plateaus. P = 0; Kruskal Wallis Bonferroni treatment. h. Boxplot showing increasing inclusion Cdense (AU) with increasing Ncz residence time. P = 0; Kruskal Wallis Bonferroni treatment. To validate the method for calculating partition coefficient and degree of supersaturation relative to its application in other manuscripts, we compared different methods to calculate the C dilute , Cdense, S, and Snominal ( i. – j. ). i. Scatter plot of partition coefficient (K) derived from average vRNP concentration in all inclusions (C dense total, AU) per cell vs partition coefficient calculated from individual vRNP concentration of each inclusion (C dense , AU) per cell before and after Ncz treatment. j. Scatter plot of norminal degree of supersaturation versus degree of supersaturation before and after Ncz treatment. k. Graphical abstract depicting the hardening effects of Ncz on vRNPs and mesoscale inclusions. (l.- m.) A549 cell were infected at a multiplicity of infection (MOI) of 3 with PR8 virus or mock-infected for 12 hrs, incubated with 5µM Ncz or vehicle (DMSO) for 30mins, then subjected to heat ranging from 37°C to 62°C (for 7mins) before collecting lysates in Laemllís buffer. l. Western blot showing the PB1, NP and actin bands. m. Band quantification of PB1 normalised to control.

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Infection, Virus, Incubation, Staining, Immunofluorescence, Concentration Assay, Fluorescence, Derivative Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: ( a. – c. ) Mice were intranasally infected with 4000 plaque forming units (PFU) of X31 virus, and after 2 days were intraperitoneally injected with PBS or 69 µg of nucleozin (Ncz) at 30 min, 1h or 2h before the collection of the lungs. a. Bar chart of viral titre (PFU g/ Lung) of mice after infection with X31 and treatment with PBS or Ncz. Data in ( b. - c. ) were extracted from segmented inclusions (NP, as proxy) from fixed immunofluorescence images of lung tissues. b. Boxplot showing the number of inclusions per cell in lung sections; P = 0.01399; Kruskal Wallis Bonferroni treatment. c. Boxplot showing the area (µm 2 ) of inclusions from cells in lung sections. P = 3.378e-8; Kruskal Wallis Bonferroni treatment. ( d. – f. ) A549 cells constitutively expressing GFP-Rab11a-WT and GFP-Rab11a-DN were mock- or PR8- infected for 12h and thereafter, treated with Ncz or sham vehicle-DMSO. d. Immunofluorescence images of inclusions (NP - magenta, Rab11 – green) and ER (Calnexin - cyan). Nuclei and cell periphery delimited by yellow and white dash line respectively, and white boxed are insets showing presence or absence of inclusions. Scale bar = 10 µm. e. Scatter plot of circularity versus roundness of inclusions. Above each boxplot, same letters indicate no significant difference between groups while different letters indicate a statistical significance at α < 0.05. f. Bar chart showing viral titre (PFU/ mL). Experiments were done in twice with triplicate samples.

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Infection, Virus, Injection, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: ( a. – b., e. – h. ) Cell lines constitutively expressing GFP-Rab11a-WT and GFP-Rab11a-DN were infected for 12 h with PR8 at a multiplicity of infection (MOI) of 5 and treated with nucleozin (Ncz) or sham vehicle (DMSO) ( e. – h. ). Thereafter, cells were lysed in mild (NP40) or strong detergent (SDS), while NP40 lysate was ultracentrifuged (100,000 g) to pellet materials in condensates from the soluble fraction in the supernatant. Soluble and total host and viral proteome were identified by LC-MS/MS and solubility was determined as the ratio of soluble NP40- to SDS-derived total proteome abundances at the indicated hour post infection (hpi). a. Schematic of experimental workflow for solubility proteome profiling (SPP) of infected GFP-(Rab11a-WT and Rab11a-DN) cell lines ( see methods ). b. Volcano plot of host proteome solubility in a 12-hr infected Rab11a-DN cell line relative to Rab11a-WT line. Red and blue dots signify insoluble (mitochondria) and soluble proteins respectively. c. Immunofluorescence images showing vRNPs associate with mitochondria. At 8hpi, HeLa cells infected with PR8 at an MOI of 3 were fixed and stained for vRNPs (using NP as proxy, red), mitochondria (Tom20, grey) and Drp1 or Rab11 (green). Insets show areas of vRNP/ mitochondria/Drp1 and vRNP/ mitochondria/Rab11a aggregation. Note that Drp1 may serve as a bridge between mitochondria and vRNPs. d. Violin plot of single cell analysis of mitochondria distributions during infection ( see methods ). ns, not significant; P < 0.0001; Kruskal Wallis Bonferroni treatment. e. Volcano plot representing relative host protein abundance in Rab11a-WT and Rab11a-DN infected cell lines (at 12 hpi) after treatment with nucleozin or DMSO. Differentially upregulated proteins in these conditions (statistical significance – see methods ) are indicated in blue dots. f. Volcano plot representing relative solubility of host and viral proteins in Rab11a-WT and Rab11a-DN infected cell lines (at 12 hpi) after treatment with nucleozin. Differentially soluble proteins in these conditions (statistical significance – see methods ) are indicated in pink and green dots. g. Bar graph comparing viral proteins abundances (in log 2 scale) in Rab11a-WT and Rab11a-DN cell lines PR8-infected (12 hpi) and treated with either nucleozin or DMSO. h. Bar graph comparing solubility (in log 2 scale) of viral proteins when PR8 infected (12 hpi) Rab11a-WT and Rab11a-DN cell lines were treated with either nucleozin (Ncz) or sham vehicle (DMSO). . i. Heat map showing the log 2 relative solubility of all influenza proteins (y-axis) in Rab11a-DN mutant cell line in comparison to wild type Rab11a-WT cells at 12 hours post infection (hpi). Asterisk (*) represents proteins changing in a statistically significant manner ( see methods ).

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Expressing, Infection, Liquid Chromatography with Mass Spectroscopy, Solubility, Derivative Assay, Immunofluorescence, Staining, Single-cell Analysis, Quantitative Proteomics, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Influenza A virus liquid condensates can undergo pharmacological hardening

doi: 10.1101/2022.08.03.502602

Figure Lengend Snippet: Cell lines constitutively expressing GFP-Rab11a-WT and GFP-Rab11a-DN; were infected for 12 hrs with PR8 at a multiplicity of infection (MOI) of 5. Thereafter, cells were lysed in mild (NP40) or strong detergent (SDS), while NP40 lysate was ultracentrifuged (100,000 g) to pellet materials in condensates from the soluble fraction in the supernatant. Soluble and total host and viral proteome were identified by LC-MS/MS and solubility was determined as the ratio of soluble NP40- to SDS- derived total proteome abundances at the indicated hour post infection (hpi). a. Volcano plot representing the differential abundance of host proteins in 12hpi infected Rab11a-DN cell line compared to Rab11a-WT line. High and low abundance proteins are represented in green and magenta dots respectively. b. Heat map showing changes in median relative abundance (in log 2 scale) of the influenza proteome at 12hpi in Rab11a-DN cell line compared to Rab11a-WT line. High abundance and low abundance proteins are represented in green and magenta colours respectively. c. Dot plots representing the gene ontology (GO) with terms of biological processes overrepresented among significant hits from proteome-wide upregulated and downregulated proteins at 12hpi in Rab11a-DN cell line compared to Rab11a-WT line. The grey fill represents the adjusted p-value. d. Dot plots representing the gene ontology (GO) with terms of biological processes overrepresented among significant hits from proteome-wide differentially soluble proteins at 12hpi in Rab11a-DN cell line compared to Rab11a-WT line. The grey fill shows the adjusted p-value. e. Scatter plot comparing the relative changes in host protein abundance in 12 h infected Rab11a-DN with respect to Rab11a-WT (y-axis) versus changes in host protein abundance in 12 h infected A549 with respect to mock infection in log 2 scale. Blue dots represent proteins with significant infection response in Rab11a-DN versus Rab11a-WT cell lines at 12 hpi. f. Scatter plot comparing the relative changes in host protein solubility in infected Rab11a-DN with respect to Rab11a-WT (y-axis) versus changes in host protein solubility in infected A549 with respect to mock infection, scaled to log 2 . Blue dots represent proteins with significant solubility response at 12hpi infection in Rab11a-DN versus Rab11a-WT cell lines.

Article Snippet: Antibodies used were rabbit polyclonal against Rab11a (1:100; Proteintech, 15903-1-AP), calnexin (1:1000, Abcam, 22595), TRIM25 (1: 100, Abcam, ab167154), and NP (1:1000; gift from Prof Paul Digard), mouse polyclonal against NS1 (Neat, in-house from hybridoma made at the IGC antibody facility), mouse monoclonal against NP (1:1000; Abcam, 20343), Tom20 (1:200; Sigma-Aldrich, WH0009804M1) and Drp1 (1:200; Abcam, ab56788).

Techniques: Expressing, Infection, Liquid Chromatography with Mass Spectroscopy, Solubility, Derivative Assay, Quantitative Proteomics

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, illustration of the HCMV AC (red) and nuclear (blue) rotation phase, highlighting primary imaging windows. b-c, Western blot and immunofluorescence showing αTAT1 depletion suppresses microtubule acetylation. Fluorescence intensity of acetylated microtubules was quantified; n = 303 cells total, ****p≤0.0001, two-tailed student’s t-test. All data points are shown within violin plots. Similar results yielded from 3 independent experiments. d-e, αTAT1 depletion suppresses nuclear rotation. Representative stills from and rotational analyses are shown. Rotation frequency above or below 180° is shown in e; bars represent mean ± SEM; n = 309 cells total,***p≤0.001, two-tailed student’s t-test. f-g, Spatial distribution and intensity of DNA (hoescht), AC marker (gB), acetylated microtubules (Ac-K40-MT) and SUN1 using CNN (g) or DNA, gB and SUN1 using MASK-RCNN (h) analyses. Lines represent mean ± SEM; n = 34,712 cells total in dataset from 3 independent biological replicates for f; n = 2,214 cells total for g. h, αTAT1 depletion suppresses SUN1 polarization. For quantification, SUN1 was classed as polarized (green), intermediate (pink) or non-polarized (orange). n = 583 cells total. Similar results yielded from 3 independent experiments.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Imaging, Western Blot, Immunofluorescence, Fluorescence, Two Tailed Test, Marker

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a-b, Representative stills from time lapse imaging and measurements of rotation frequency above or below 180° in uninfected or infected NHDFs expressing GFP-Histone nanobody. Bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 281 cells total from 3 independent biological replicates; ****p≤0.0001. Note that nuclear rotation above 180° occurs in approximately 80% of infected cells imaged, while lower levels of rotation occur in the remaining population. Such extensive rotation is extremely rare in uninfected cells. c-d, Expression of a K40R mutant form of tubulin suppresses the formation of acetylated microtubule filaments. Fluorescence intensity of acetylated tubulin is shown in b; All data points are shown within violin plots, statistics use two-tailed student’s t-test, n = 250 cells total, ****p ≤ 0.0001. Data shown is representative of 3 independent biological replicates. e-f, Expression of a K40R mutant form of tubulin suppresses nuclear rotation. Representative stills from are shown in e and rotational analyses are shown in f. Rotation frequency above or below 180° is shown in d; bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 157 cells total, **p ≤ 0.01 g, schematic of CNN-based classification and analysis pipeline measuring fluorescence intensities across individual cells in different channels. Output for the AC (red) next to the nucleus (blue) is illustrated. h, Representative confocal z-section and deconvolved z-section image of SUN1 polarization in HCMV-infected cell. Acetylated microtubules and the AC (stained with the viral protein gB) are also shown. Data shown is representative of 3 independent biological replicates. i , Spatial distribution and intensity of DNA, gB and SUN2 using CNN. Lines represent mean ± SEM; n ≥ 17,484 cells total from 3 independent biological replicates.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Imaging, Infection, Expressing, Two Tailed Test, Mutagenesis, Fluorescence, Staining

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, WB analysis of SUN1 levels over the course of infection with HCMV at MOI 1. Early (IE1/2), intermediate (UL44) and late (pp65, pp28) proteins demonstrate stages of infection at each timepoint, representative of 3 independent biological replicates. b-d, Neural network-based single cell analysis of SUN1 expression during HCMV infection. b , Illustration of CNN analysis pipeline that classifies cells by the predominant infectious cycle stage identified at each timepoint. c , Representative examples of uninfected NHDFs or NHDFs at various stages of infection, stained for SUN1, IE1/2 and TGN46. Expression of IE1/2 and gradual remodeling of the Golgi network serve as markers of infection stage. Polarization of SUN1 is seen between 24–72 h.p.i. d , CNN-based classification of cells based on IE1/2 expression levels, filtering out uninfected cells, reveals a gradual expansion of the nucleus and Golgi, characteristic of HCMV infection, occurs concomitantly with a gradual increase in expression and polarization of SUN1 toward the AC (i-v). Discrete populations of cells are filtered for inclusion in each timepoint (vii), with cells from other kinetic classes removed from analysis marked in grey (viii-x).Comparing unfiltered (lighter colored violin plots, left segment) versus filtered (dark colored violin plots, right segment) cell populations reveals the power of trained networks to more precisely analyze only infected cells within the population, more clearly revealing the increase in nuclear volume and SUN1 abundance, which peaks at approximately 2-fold (xi-xiii). Lines represent mean ± SEM; n = 37,800 cells total from 3 independent biological replicates. Violins as in Fig. 4a . e , Mask-RCNN analysis pipeline uses manually annotated masks of the AC, nucleus and combined (HCMV) to train a Mask-RCNN architecture to classify and segment microscopy images of HCMV infection. Once trained, whole cover-slip scanning datasets can be run through the model to perform instantaneous single cell quantification on high-confidence infected cells. This quantification has high spatial awareness and can be used to perform linescans between two specific subcellular compartments (e.g. the AC and nucleus) or to rotate and align nuclei to perform average projections (as in ).

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Infection, Single-cell Analysis, Expressing, Staining, Microscopy

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a-c, SUN1 depletion suppresses nuclear rotation. Representative stills from and rotational analyses are shown. Western blot is representative of 3 independent experiments. Rotation frequency above or below 180° is shown in c; n = 162 cells total from 3 independent biological replicates, ***p≤0.001. d-e, Effects of Nesprin-2G constructs on nuclear rotation. Representative stills and rotation analyses from are in f. Frequency of rotations above or below 180° are in g; bars represent mean ± SEM, n = 127 cells total from 5 independent biological replicates, **p≤0.01, ***p≤0.001, ****p≤0.0001, two-tailed student’s t-test.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Western Blot, Construct, Two Tailed Test

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a-b, Expression of a SUN1 mutant that does not engage Nesprin-2G impairs nuclear rotation in HCMV-infected cells. a, Representative stills from time lapse recordings of NHDFs expressing Tag-GFP2 forms of SUN1 Full Length (FL) or SUN1 lacking the lumenal domain (SUN1ΔLu) that mediates interactions with Nesprin-2G, infected with HCMV-UL99mCherry. Rotation traces from this imaging are shown to the right. Analyses focused on cells expressing intermediate levels of SUN1-GFP constructs as high levels of expression can result in retention of Nesprin-2G in the endoplasmic reticulum (ER). b, Quantification of rotation frequencies above or below 180°; bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 138 cells total from 3 independent biological replicates, ***p≤0.001. This data further confirms that interactions with Nesprin-2G are necessary for nuclear rotation to occur. c-f, RNAi-mediated depletion of BICD2 using either of two independent siRNAs suppresses nuclear rotation and SUN1 polarization. c, Illustration of SUN1:Nesprin-2G interactions with microtubule motors through AD regions, or SUN2:Nesprin-2G interactions with myosin through CH domains to control nuclear movement. d, Illustration of GFP-Nesprin-2G constructs with CH and/or AD domains, along with the LEWD>LEAA kinesin-binding mutant. e, Western blot analysis of BICD2 expression representative of 3 independent replicates. Arrow points to BICD2, specifically depleted by two independent siRNAs. f, Representative stills and rotational analyses from Video 6 showing effects of BICD2 depletion on nuclear rotation. g, Rotation frequency above or below 180° in control or BICD2 depleted cells, bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 144 cells total cells from n = 3–4 independent biological replicates, ***p ≤ 0.001. h, Depletion of BICD2 impairs SUN1 polarization. Spatial distribution and intensity of DNA (hoescht), AC marker (gB), and SUN1 are shown for control and BICD2 depleted cells and are representative of 3 independent biological replicates. For quantification, SUN1 was classed as polarized (green), intermediate (pink) or non-polarized (orange). n = 235 cells total. i-k , Expression of a dominant-negative fragment of BICD2 reduces nuclear rotation and SUN1 polarization. NHDFs expressing TagGFP2 control or TagGFP2-BICD2 N-terminus (NT) were infected with HCMV UL99-mCherry. i , Representative still images from time lapse recordings and rotation traces are shown. j, Quantification of rotation frequencies above or below 180°; bars represent mean ± SEM, n = 91 cells total from 2 independent biological replicates. k , Representative images of SUN1 localization in NHDFs expressing TagGFP2 control or TagGFP2-BICD2-NT NHDFs are shown, consistent with 3 independent biological replicates. Quantification of SUN1 polarity categorized as fully polarized, intermediate polarity or not polarized is shown; n = 149 cells total.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Expressing, Mutagenesis, Infection, Imaging, Construct, Two Tailed Test, Binding Assay, Western Blot, Marker, Dominant Negative Mutation

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Lamin A/C is downregulated and lacks polarity in HCMV-infected cells. Lines represent mean ± SEM; n = 10,934 cells total from 3 independent biological replicates. b, Depletion of αTAT1, SUN1 or BICD2 inhibits Emerin polarization and causes aberrant F-actin networks. Representative images are shown for each condition, similar to data from 3 independent replicates. c-d , Emerin depletion blocks nuclear F-actin formation. c, WB analysis demonstrating the efficacy of Emerin siRNAs. d, Representative images and quantification of nuclear F-actin (nAC) frequency are shown for each condition, bars represent mean ± SEM, statistics use two-tailed student’s t-test, n = 401 cells total from 3 independent biological replicates, ***p≤0.001. Fluorescence intensity shows Emerin depletion in cells. e , Emerin depletion does not affect SUN1 polarization. Representative images and quantification of SUN1 polarization is shown for each condition; n = 321 cells. SUN1 was characterized as polarized, intermediate polarity or not polarized. f , Expression of actin-binding mutants of Emerin blocks nuclear F-actin formation but not nuclear rotation. NHDFs expressing nAC-TagGFP and mCherry-Emerin wildtype or actin-binding mutants (m151, m175) were infected with HCMV UL99-mCherry. Representative still images and rotation traces from time lapse imaging are shown. Quantification of nuclear rotation frequencies above or below 180° are shown for each condition; the presence of nuclear F-actin was also quantified in the same time lapse images, n = 72 cells total (upper) and n = 79 cells total (lower). Note that in order to image nAC-TagGFP cells were infected with HCMV UL99-mCherry. As such, mCherry signal in these images originates from both mCherry-Emerin and the viral UL99-mCherry, showing the cytoplasmic AC and nuclear rotation in infected cells under all conditions. Data shown is representative of 3 independent replicates.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Infection, Two Tailed Test, Fluorescence, Expressing, Binding Assay, Imaging

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Distribution of histone H3 forms in HCMV-infected cells. Lines represent mean ± SEM; Total H3 (n = 13,774 cells total), H3K4me3 (n = 31,886 cells total), H3K27me3 (n = 30,874 cells total), H3K9me2 (n = 34,342 cells total), H3K9me3 (n = 13,790 cells total). Violin plots represent median (white point), interquartile range (IQR, box), and maximum/minimum values 1.5 x IQR outside the IQR (whiskers). b, Representative image of viral genomic DNA (gDNA) and H3K9me3 foci. White line delineates the peak of H3K9me3 foci in overlay images. c, Depletion of αTAT1, BICD2 or SUN1 reduces the polarity and abundance of viral gDNA. Fluorescence as a function of nuclear area was used to measure the extent of gDNA polarity, and mean fluorescence intensity was used to measure gDNA levels; All data points are shown within violin plots, n = 840 cells total, *p≤0.05, **p≤0.01, ***p≤0.001, two-tailed student’s t-test. d-e , Actin-binding mutants of Emerin inhibit H3K9me3 polarization without affecting SUN1 polarization. d, Representative images are shown. e, Quantification of H3K9me3 (n = 220 cells total) and SUN1 (n = 300 cells total) polarity, categorized as polarized, intermediate (int.) or not polarized. f , Nuclear-localized Arpin inhibits nuclear F-actin formation (bars represent mean ± SEM, n = 1,237 cells total from 3 independent biological replicates; **p≤0.01, two-tailed student’s t-test). g - h , Nuclear-localized Arpin inhibits H3K9me3 (n = 368 cells total) but not SUN1 (n = 236 cells total) polarization. i , Expression of nuclear-localized polymerization-deficient actin inhibits nuclear F-actin formation (bars represent mean ± SEM, n = 1,157 cells total from 3 independent biological replicates; **p≤0.01, two-tailed student’s t-test). j - k , Nuclear-localized polymerization-deficient actin inhibits H3K9me3 (n = 234 cells total) but not SUN1 (n = 185 cells total) polarization. All data representative of 3 independent biological replicates.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Infection, Fluorescence, Two Tailed Test, Binding Assay, Expressing

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: a, Depletion of αTAT1, BICD2 or SUN1 reduces H3K9me3 polarization. Representative images are shown. b , Expression of BICD2 dominant-negative (BICD2-NT) or SUN1 lacking its lumenal domain (SUN-ΔLu) that mediates interactions with Nesprin-2G inhibits the polarization of H3K9me3 in HCMV-infected cells. Representative images and quantification of H3K9me3 polarization are shown; n = 171 cells total. c , Depletion of Emerin impairs the polarization of H3K9me3 in HCMV-infected cells. Representative images and quantification of H3K9me3 polarization are shown; n = 490 cells total. For all experiments, data shown is representative of 3 independent biological replicates.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Expressing, Dominant Negative Mutation, Infection

Journal: Nature

Article Title: Cytoplasmic control of intranuclear polarity by human cytomegalovirus

doi: 10.1038/s41586-020-2714-x

Figure Lengend Snippet: Top, In uninfected cells chromatin and silenced domains are heterogeneously distributed throughout the nucleus, as discussed in the main text. Middle , Upon HCMV infection, nuclear F-actin is induced and reorganized through the action of acetylated microtubules that exert mechanotransductive pulling forces on Nesprin-2G:SUN1-containing LINC complexes, polarizing them towards the AC. In doing so, this creates extreme polarity in inner-nuclear Emerin, directing nuclear F-actin organization; enriched red regions represent polarized LINC-Emerin complexes in the nuclear membrane. This extreme polarity draws silenced (H3K9me3) histones and associated host DNA towards this region of the nucleus, through the action of nuclear F-actin networks. As viruses employ a wide range of strategies to prevent chromatinization and silencing of their own DNA, viral gDNA is not drawn to the AC-proximal sites of H3K9me3 polarization. The polarization of inactive histones and host DNA likely pushes viral DNA to the opposing side of the nucleus, through space-filling. This segregation of viral and host DNA creates an optimal environment for viral DNA replication and production of infectious virus particles. Lower , Polarization of the nucleus fails to occur if key components driving the process are inhibited; if microtubules are not mechanically strengthened through acetylation, if connections between microtubules and nuclear membrane complexes are lost, or if nuclear F-actin is not organized by Emerin. Notably, nuclear F-actin and Emerin do not control nuclear rotation, but cytoplasmic microtubule-derived forces that cause nuclear rotation control Emerin localization, F-actin formation and intranuclear polarity. As such, cytoplasmic forces on the nuclear surface organize nuclear factors to control genetic polarity. Failure to create this polarity results in a suboptimal environment for viral DNA replication.

Article Snippet: Then, using SUN1-Isoform-9 (pEGFP-SUN1_916 plasmid; Addgene: #125850) as template, the Sun1-FL and Sun1-ΔLU was cloned into pWPXL-TagGFP2-LINK-MCS using the following primers: SUN1-FL-for (5’-TATTTCGAATTCGATTTTTCTCGGCTTCACATG-3’) SUN1-FL-rev (5’-TTTCATATGACTAGTTTATTACTTGACAGGTTCGCCATGAAC-3’) SUN1-ΔLU-rev (5’-TTTCATATGACTAGTTTATTAGACCGAAGCTGACGGCCCGGC-3’) The truncation made is analogous to the previously described mutant .

Techniques: Infection, Derivative Assay

Journal: Scientific Reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Doxorubicin-induced apoptosis in leukemic cells is dependent on Rac1. ( A , B ) The Rac1 inhibitor reduces doxorubicin-induced apoptosis in U937 and Jurkat cells. The cells were pretreated with the specific Rac1 inhibitor (NSC23766, 10 μM, 1 h), before their treatment with doxorubicin (Dox) for 24 h. Apoptosis was determined by annexin V staining and flow cytometry analysis. ( C , D ) Overexpression of the Rac1 dominant negative form N17Rac1 inhibits doxorubicin-induced apoptosis. The cells were transfected with pcDNA3.1, N17Rac1 + pcDNA3.1 or with N17Rac1 + WT-Rac1 plasmids. Viable cells were recovered after 24 h by ficoll gradient. The cells were then treated with doxorubicin for 24 h. Apoptosis was determined by annexin V staining and flow cytometry analysis. The results represent mean values ± SD from three independent experiments. * P < 0.05, ** P < 0.01. (E) Rac1 inhibition blocks caspase-9 and -3 activation by doxorubicin. Jurkat cells were treated as indicated and after 12 h of doxorubicin treatment, the levels of native and active caspase-9 and -3 were determined by western blot analysis. β-actin was used as a loading control. The illustrated blots are representative of three independent experiments.

Article Snippet: The anti-caspase-3 (E-8) that detects the native and the active fragments of caspase-3, anti-Mcl-1 (22) and anti-β-actin (C-2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, Flow Cytometry, Over Expression, Dominant Negative Mutation, Transfection, Inhibition, Activation Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Rac1 inhibition reduces DNA damage intensity and H2AX phosphorylation induced by doxorubicin. ( A – C ) The cells were treated or not with doxorubicin (Dox) for 6 h in the presence or absence of the Rac1 inhibitor NSC23766 (NSC). Alkaline comet assay was performed and stained nucleoids were visualized by epifluorescence microscopy using FITC filter. ( A ) Representative fields corresponding to each treatment were photographed. ( B , C ) The intensity of DNA strand breaks in U937 and Jurkat cells was quantified using visual scoring as described under “Experimental procedures section”. The results represent mean values ± SD obtained from three independent experiments. ** P < 0.01. ( D , E ) The cells were treated with doxorubicin in the presence or absence of NSC23766 as described above and the levels of phosphorylated H2AX (γ-H2AX) were determined by immunoblot analysis. The β-Actin blot was used as a loading control. Blots are representative of three independent experiments.

Article Snippet: The anti-caspase-3 (E-8) that detects the native and the active fragments of caspase-3, anti-Mcl-1 (22) and anti-β-actin (C-2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Phospho-proteomics, Alkaline Single Cell Gel Electrophoresis, Staining, Epifluorescence Microscopy, Western Blot, Control

Journal: Scientific Reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Collagen/α2β1 integrin inhibits doxorubicin-induced DNA damage and H2AX phosphorylation. ( A – C ) Collagen inhibits doxorubicin-induced DNA damage in U937 and Jurkat leukemic cell lines. The cells were pretreated for 1 h with 10 μg/ml of control IgG or with the blocking anti-α2 integrin antibody (P1E6) and then cultured on BSA or on collagen (Col) for 2 h. After removing cells in suspension, adherent cells were treated with doxorubicin (Dox) for 6 h. At the end, cells were harvested and the alkaline comet assay was performed and stained nucleoids were visualized by epifluorescence microscopy using FITC filter. (A) Representative fields corresponding to each treatment were photographed. ( B , C ) The intensity of DNA strand breaks was quantified using visual scoring as described under “Experimental procedures section”. The results represent mean values ± SD obtained from three independent experiments. * P < 0.05. ( D , E ) The cells were treated as indicated, and the levels of phosphorylated H2AX (γ-H2AX) were determined by immunoblot analysis. The β-Actin blot was used as a loading control. Blots are representative of three independent experiments.

Article Snippet: The anti-caspase-3 (E-8) that detects the native and the active fragments of caspase-3, anti-Mcl-1 (22) and anti-β-actin (C-2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Control, Blocking Assay, Cell Culture, Suspension, Alkaline Single Cell Gel Electrophoresis, Staining, Epifluorescence Microscopy, Western Blot

Journal: Scientific Reports

Article Title: Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation

doi: 10.1038/s41598-019-55934-w

Figure Lengend Snippet: Rac1 is involved in doxorubicin-induced JNK activation and Mcl-1 downregulation. U937 and Jurkat cells were treated or not with doxorubicin (Dox) in the presence or absence of the Rac1 inhibitor NSC23766. After 8 h of treatment, the cells were lysed and the levels of phospho-JNK1/2 (A&B) and Mcl-1 (C&D) were determined by immunoblot analysis. The blots were stripped and reprobed with anti-β-actin antibody for equal loading. The blots are representative of three independent experiments.

Article Snippet: The anti-caspase-3 (E-8) that detects the native and the active fragments of caspase-3, anti-Mcl-1 (22) and anti-β-actin (C-2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot