Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, SDS Page, Western Blot, Infection, Staining, Software, Construct, Expressing

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3+ signal on or proximal to (i.e. within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with Bap31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in (). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. ( E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from fractions 1-7 (representing disassembled virus) and fraction 8 (representing assembled virus) was quantified and normalized to the Scr control. ** p ≤ 0.01; ns= not significant.

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, Infection, Staining, Membrane, Transport Assay, SDS Page, Western Blot, Virus

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP+Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Construct, Staining, Infection, Expressing, Control, SDS Page, Western Blot

Journal: JCI Insight

Article Title: Multiomic analysis of microRNA-mediated regulation reveals a proliferative axis involving miR-10b in fibrolamellar carcinoma

doi: 10.1172/jci.insight.154743

Figure Lengend Snippet: ( A ) Protein expression of DNAJB1-PRKACA (DP) is detected with a protein kinase A catalytic α subunit (PKA) antibody. WT PKAc, DP major, and DP minor are identified. Lane 1, FLC-H cell line; lane 2, nonmalignant liver; lane 3, FLC patient sample; lane 4, FLC PDX sample. Vinculin expression for loading control is shown in the lower panel. Uncropped immunoblot shown in , A and B. ( B ) qPCR showing the RQV of miR-10b in FLC-H cells 6 days after 500 nM treatment with miR-10b LNA or scrambled sequence compared with mock ( n = 4 each condition). ( C ) Luciferase signal (RLU) in FLC-H cells after 6 days of 500 nM miR-10b LNA treatment is shown as RQV compared with the scrambled negative control (6 trials, n = 6 each condition). ( D ) qPCR showing the RQV of FANCC , KLF11 , SEC14L2 , SIRT5 , SUN2 , and TRIM35 in FLC-H cells after 6 days of 500 nM miR-10b LNA treatment compared with the negative control ( n = 5–7 trials with 3 replicates for each condition, SIRT5 n = 2 trials). ( E ) Soft agar colony formation of FLC-H cells 35 days after 500 nM miR-10b LNA compared with the negative control shown as RQV. ( F ) Representative nitro blue tetrazolium–stained images shown (2 trials, n = 8 each condition). ( G ) EdU incorporation in FLC-H cells 6 days after 500 nM treatment with miR-10b LNA compared with the negative control shown as RQV (2 trials, n = 6 each condition). ( H ) Representative DAPI- and EdU-stained images show total and proliferative cells, respectively. Scale bars: 100 μm. In all assays, each dot represents the average signal across technical replicates for a single biological replicate. P values are calculated by 2-tailed Student’s t test. P values reported in B and D were adjusted for multiple testing correction post hoc by the Benjamini-Hochberg method.

Article Snippet: Individual gene assay IDs include the following: CDH1, hs01023895; DNAJB1-PRKACA, custom; FANCC, hs0098454; KLF11, hs00231614; miR-10b, 002218; miR-21, 000397; miR-182, 002334; PTEN, hs02621230; RNU6, 001973; RPS9, hs02339424; SEC14L2, hs00391446; SIRT5, hs00978331; SUN2, hs00391446; and TRIM35, hs00324633 (Thermo Fisher Scientific).

Techniques: Expressing, Control, Western Blot, Sequencing, Luciferase, Negative Control, Staining

Journal: The Journal of Cell Biology

Article Title: Nuclear envelope rupture is induced by actin-based nucleus confinement

doi: 10.1083/jcb.201603053

Figure Lengend Snippet: The LINC complex is absent from chromatin hernia. Representative images of U2OS GFP-NLS shLmnB1 cells arrested with thymidine, fixed, and labeled with LmnA antibodies (top three rows, rabbit; bottom, mouse) and (from top to bottom) Sun1, Sun2, nesprin1 (Nsp1), and nesprin2 (Nsp2) antibodies. Bars, 10 µm.

Article Snippet: Antibodies used for immunoblotting were as follows: mouse antitubulin (1:5,000; Sigma-Aldrich), mouse anti-GAPDH (1:5,000; Genetex), rabbit anti-Sun1 (1:1,000; Novus Biologicals), and rabbit anti-Sun2 (1:1,000; Genetex).

Techniques: Labeling