Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: F/G‐actin binds nuclear proteins. A) Upper, HEK 293T cells were cultured in 0.5 µ m Jasp, 1 µ m LatB, or 10 µ m CytD for 1 h, lysed with lysis and F‐actin stabilization buffer 2 (LAS2), and subjected to F/G‐actin fractionation. Western blot showed that Jasp induced actin polymerization, while LatB and CytD induced actin depolymerization. Middle, The above cells were also resuspended in the fractionation buffer, and subjected to nuclear fractionation and F/G‐actin fractionation. Western blot showed that Jasp induced nuclear actin polymerization, while LatB and CytD treatment induced nuclear actin depolymerization. Lower, After cultured in 0.25 µ m Jasp, 0.5 µ m LatB, or 5 µ m CytD for 4 h, HEK 293T cells were subjected to nuclear fractionation. Nuclear extracts were lysed with LAS2, and subjected to immunoprecipitation with antibody against actin. The pulldown products were subjected to mass spectrometry analysis, showing that precipitation of nuclear actin pulled down α‐catenin, β‐catenin, and filamin A (FLNA) in Jasp treated cells, and MYBBP1A, NKRF, and profilin 1 (PFN1) in LatB and CytD treated cells. B) The cells were transfected with α‐catenin siRNAs. Western blot showed that silencing α‐catenin decreased α‐catenin expression, but did not affect β‐catenin expression. Silencing α‐catenin decreased both α‐catenin and β‐catenin in the nuclei. The nuclear extracts were incubated with 25 µL biotin‐X Phalloidin, and subjected to biotin‐labeled Phalloidin pulldown assay. Precipitation of F‐actin pulled down α‐catenin and β‐catenin, and pulled down less α‐catenin (23.4%) and β‐catenin (39.8%) by silencing α‐catenin. C) Western blot showed that silencing FLNA decreased its expression, but did not change SMAD2/SMAD3 levels. Silencing FLNA decreased FLNA, SMAD2, and SMAD3 in the nuclei. Precipitation of nuclear F‐actin pulled down FLNA, SMAD2, and SMAD3, but less FLNA (25.7%), SMAD2 (27.8%) and SMAD3 (10.5%) by silencing FLNA. D) Silencing PFN1 decreased PFN1 and MYPOP levels in cell lysate and nuclei. Precipitation of G‐actin pulled down PFN1 and MYPOP, but less of them by silencing PFN1. E) The nuclear fractions of the above cells were lysed with LAS2 and subjected to F/G‐actin fractionation and immunoprecipitation with antibody against actin. Precipitation of nuclear F‐actin pulled down β‐catenin, SMAD2, SMAD3, and precipitation of nuclear G‐actin pulled down MYBBP1A, NKRF, and MYPOP. F, HEK 293T cells were cultured in 0.1 µ m Jasp, 0.2 µ m LatB, and 1 µ m CytD for 16 h, lysed with lysis buffer, and subjected to Western blot. Jasp treatment repressed E‐cadherin, enhanced N‐cadherin and vimentin proteins. LatB and CytD treatment increased E‐cadherin, decreased N‐cadherin and vimentin levels.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Cell Culture, Lysis, Fractionation, Western Blot, Immunoprecipitation, Mass Spectrometry, Transfection, Expressing, Incubation, Labeling

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: F/G‐actin binds EMT‐related functional transcription factors in the nuclei. A) HEK 293T cells were transfected with MYBBP1A, NKRF, or MYPOP, and processed to ELISA. Overexpression of MYBBP1A, NKRF, or MYPOP increased E‐cadherin, and decreased N‐cadherin and vimentin levels. ** p < 0.01 versus vector ( n = 6). B) Left, HEK 293T, MDA‐MB231, and MDA‐MB468 cells were transfected with MYBBP1A, NKRF, or MYPOP, and processed to chamber migration assays for indicated time points, showing that expression of MYBBP1A, NKRF, or MYPOP suppressed cell migration. ** p < 0.01 versus vector ( n = 6). Right, The transfected cells were cultured in basal medium with 700 µ m H 2 O 2 for 24 h. Expression of MYBBP1A, NKRF, or MYPOP suppressed cell survival. ** p < 0.01 versus vector ( n = 6). C) Left, HEK 293T, HaCaT, and MCF‐7 cells were transfected with MYBBP1A, NKRF, or MYPOP siRNAs, and processed to chamber migration assays for indicated time points, showing that silencing MYBBP1A, NKRF, or MYPOP enhanced cell migration. ** p < 0.01 versus oligo ( n = 6). Right, The cells were cultured in basal medium with 650 µ m H 2 O 2 for 24 h, showing that silencing MYBBP1A, NKRF, or MYPOP enhanced cell survival. ** p < 0.01 versus oligo ( n = 6). D) The transfected cell lysates were subjected to ELISA analysis, showing that silencing MYBBP1A, NKRF or MYPOP repressed E‐cadherin, and increased N‐cadherin and vimentin expression. ** p < 0.01 versus oligo ( n = 6). E) The nuclear extracts of the above transfected cells were lysed with LAS2, and subjected to F/G‐actin fractionation and immunoprecipitation with antibody against actin. Western blot showed that precipitation of nuclear G‐actin pulled down MYBBP1A, NKRF, and MYPOP in the nuclei, but precipitation of F‐actin did not pull down these proteins. Western blot showed that precipitation of MYBBP1A, NKRF, or MYPOP pulled down actin in the nuclei. F) Western blot showed that precipitation of nuclear G‐actin pulled down MYBBP1A, NKRF, and MYPOP, which pulled down less MYBBP1A (25.5%), NKRF (12.8%), and MYPOP (12.1%) in the siRNA knock‐down samples, but precipitation of F‐actin did not pull down MYBBP1A, NKRF, and MYPOP in the nuclear fraction. Precipitation of nuclear MYBBP1A, NKRF, and MYPOP also pulled down actin.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Functional Assay, Transfection, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Migration, Expressing, Cell Culture, Fractionation, Immunoprecipitation, Western Blot

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Silencing XPO6/IPO9 alters both F‐actin and G‐actin levels in the nuclei. A) HEK 293T cells were transfected with Exportin 6 (XPO6) or Importin 9 (IPO9) siRNAs, and subjected to nuclear fractionation. Western blot showed that silencing XPO6 with siRNAs (si‐XPO6) increased actin, β‐catenin, SMAD2, SMAD3, MYBBP1A, NKRF, and MYPOP in the nuclei, while silencing IPO9 (si‐IPO9) decreased actin, β‐catenin, SMAD2, SMAD3, MYBBP1A, NKRF, and MYPOP in the nuclei. B) Western blot showed that silencing XPO6/ IPO9 did not affect total actin, E‐cadherin, N‐cadherin, and vimentin expression levels in the cells.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Transfection, Fractionation, Western Blot, Expressing

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Effect of silencing XPO6/IPO9 on levels of nuclear F/G‐actin in the actin polymerization/depolymerization models. A) ELISA showed that medium/si‐XPO6 increased both F‐actin and G‐actin in the nuclei compared to the medium/oligo cells. Jasp/si‐XPO6 cells showed same cell actin dynamics and nuclear G‐actin levels as Jasp/oligo, and increased F‐actin in the nuclei. LatB/si‐XPO6 cells showed the same cell actin dynamics and nuclear F‐actin levels as LatB/oligo, and increased G‐actin in the nuclei. ** p < 0.01 versus oligo ( n = 6). B) The cells were lysed and subjected to Western blotting. Jasp/si‐XPO6 cells showed decreased E‐cadherin, but increased N‐cadherin and vimentin compared to Jasp/oligo, while LatB/si‐XPO6 cells presented increased E‐cadherin, but decreased N‐cadherin and vimentin compared to the LatB/oligo cells. Jasp/si‐XPO6 cells showed enhanced β‐catenin, SMAD2, and SMAD3 expression in the nuclei, while LatB/si‐XPO6 showed increased MYBBP1A, NKRF, and MYPOP expression in the nuclei compared to LatB/oligo treated cells. C) Nuclear F‐actin/G‐actin fraction from the above cells was subjected to immunoprecipitation with antibody against actin. Precipitation of F‐actin pulled down β‐catenin, SMAD2, and SMAD3, which was more evident in Jasp/si‐XPO6 cells. Precipitation of G‐actin pulled down MYBBP1A, NKRF, and MYPOP, which was more evident in LatB/si‐XPO6 cells. D) ELISA confirmed a decrease in both F‐actin and G‐actin in the nuclei of the medium/si‐IPO9 cells compared to the medium/oligo cells. Jasp/si‐IPO9 cells showed similar actin dynamics and nuclear G‐actin levels as Jasp/oligo, but decrease in F‐actin in the nuclei. LatB/si‐IPO9 cells showed similar actin dynamics and nuclear F‐actin levels as LatB/oligo, but decrease in G‐actin in the nuclei. ** p < 0.01 versus oligo ( n = 6). E) Nuclear F‐actin/G‐actin fraction was subjected to immunoprecipitation with antibody against actin. Precipitation of F‐actin pulled down β‐catenin, SMAD2 and SMAD3, while precipitation of G‐actin pulled down MYBBP1A, NKRF, and MYPOP.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunoprecipitation

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Nuclear F/G‐actin regulates EMT via binding and modulating β‐catenin, SMAD2, SMAD3, MYBBP1A, NKRF, and MYPOP expression in the nuclei. A) Upper, HEK293T cells were transfected with mDia2 with or without XPO6 siRNAs. The cells and nuclear extracts were subjected to F/G‐actin fractionation. Overexpression of mDia2 enhanced actin polymerization, and silencing XPO6 did not affect F/G‐actin in the cells. mDia2+/si‐XPO6 cells showed increased nuclear F‐actin. Lower, ELISA confirmed that mDia2+/si‐XPO6 cells expressed increased nuclear F‐actin, decreased E‐cadherin, but increased N‐cadherin and vimentin compared to mDia2+/oligo cells. ** p < 0.01 versus oligo ( n = 6). B) mDia2+/si‐XPO6 cells expressed increased β‐catenin, SMAD2, and SMAD3 in the nuclei compared to mDia2+/oligo cells. C) Left, HEK293T, HaCaT, and MCF‐7 cells were co‐transfected with mDia2 and XPO6 siRNAs, and processed to chamber migration assays for indicated time points. mDia2+/si‐XPO6 cells showed enhanced cell migration compared to mDia2+/oligo cells. Right, The transfected cells were cultured in basal medium with 650 µ m H 2 O 2 for 24 h. mDia2+/si‐XPO6 cells showed enhanced cell survival compared to mDia2+/oligo cells. ** p < 0.01 versus oligo ( n = 6 ). D) HEK293T cells were transfected with the control vector or mDia2 with or without IPO9 siRNAs. ELISA showed that mDia2+/si‐IPO9 cells expressed decreased nuclear F‐actin, increased E‐cadherin, but decreased N‐cadherin and vimentin compared to the mDia2+/oligo treated cells. ** p < 0.01 versus oligo ( n = 6). E) mDia2+/si‐IPO9 cells displayed cuboidal epithelial shape compared to mDia2+/oligo cells. The ratios of cell length/width were quantified, which showed that silencing IPO9 decreased cell elongation ** p < 0.01 versus oligo ( n = 25). F) HEK293T cells were transfected with mDia2 siRNAs with or without XPO6 siRNAs and processed for nuclear extraction. The cells and nuclear extracts were subjected to F/G‐actin fractionation. ELISA showed that si‐mDia2/si‐XPO6 cells expressed increased nuclear G‐actin, increased E‐cadherin, but decreased N‐cadherin and vimentin compared to mDia2+/oligo cells. ** p < 0.01 versus oligo ( n = 6). G) ELISA showed that si‐mDia2/si‐IPO9 cells expressed decreased nuclear G‐actin, decreased E‐cadherin, but increased N‐cadherin and vimentin expression compared to mDia2+/oligo treated cells. ** p < 0.01 versus oligo ( n = 6).

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Binding Assay, Expressing, Transfection, Fractionation, Over Expression, Enzyme-linked Immunosorbent Assay, Migration, Cell Culture, Plasmid Preparation, Extraction

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Overexpression of the nuclear F/G‐actin with constructs modulates EMT. A) Upper, HEK293T cells were transfected with YFP‐NLS‐β‐actin (NLS‐β‐actin), YFP‐NLS‐β‐actin S14C (S14C), YFP‐NLS‐β‐actin G13R (G13R), pmCherry‐NLS‐β‐actin R62D (R62D), and the vector. Transfection of NLS‐β‐actin expressed both F‐actin and G‐actin (β‐actin) in the nuclei, transfection of S14C expressed F‐actin (β‐actin), while transfection of G13R or R62D expressed G‐actin (β‐actin). Lower, The nuclear extracts were subjected to Western blotting. Transfection with S14C enhanced β‐catenin, SMAD2, and SMAD3, while transfection with G13R or R62D enhanced MYBBP1A, NKRF, and MYPOP levels in the nuclei. B) Transfection with S14C repressed E‐cadherin, and enhanced N‐cadherin and vimentin expression, while expression of G13R or R62D enhanced E‐cadherin, and repressed N‐cadherin and vimentin. C) ELISA showed that transfection with S14C repressed E‐cadherin, and enhanced N‐cadherin and vimentin expression, while expression of G13R or R62D enhanced E‐cadherin, and repressed N‐cadherin and vimentin. ** p < 0.01 versus vector ( n = 6). D) HEK293T cells were transfected with NLS‐β‐actin, S14C, G13R, or R62D. Immunofluorescence showed that transfection with S14C enhanced nuclear F‐actin and cell N‐cadherin levels, but repressed E‐cadherin levels. Transfection with G13R or R62D enhanced nuclear G‐actin and cellular E‐cadherin levels, but repressed N‐cadherin levels. E) HEK293T cells were transfected with S14C and G13R. Immunofluorescence showed the co‐localization of MYBBP1A, NKRF, and MYPOP with the nuclear G‐actin in the G13R‐transfected cells. F) Upper, HEK293T, MDA‐MB‐231, MDA‐MB‐468, HaCaT, and MCF‐7 cells were transfected with the above constructs, and processed to chamber migration assays for indicated time points. Transfection with actin S14C enhanced cell migration, while transfection with G13R or R62D repressed cell migration. Lower, The cells were cultured in basal medium with 700 µ m H 2 O 2 for 24 h. Transfection with S14C enhanced cell survival, while transfection with G13R or R62D repressed cell survival. ** p < 0.01 versus vector ( n = 6).

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Over Expression, Construct, Transfection, Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Migration, Cell Culture

Journal: Advanced Science

Article Title: Nuclear Actin Polymerization Regulates Cell Epithelial‐Mesenchymal Transition

doi: 10.1002/advs.202300425

Figure Lengend Snippet: Association of EMT with wound repair. A) Absolute values of E‐cadherin, N‐cadherin, vimentin, and nuclear F‐actin/G‐actin protein levels were quantified by ELISA in 133 cell lines including, MDA‐231‐BoM‐1833, 786‐O, 4T1, 4T07, 66C14, 67NR, A431, A549, A2058, A2780, AC16, ACHN, ARH77, AU565, B16, BC3, BEAS‐2B, BTH‐1, BT‐20, BT‐474, BT‐549, BxPC‐3, C2C12, Caco‐2, Caki‐1, Caki‐2, CI3K, CO‐115, COLO‐201, COLO‐205, COLO‐775, CW‐9019, CRL‐1476, Cos‐1, Cos‐7, CV‐1, DLD‐1, DU145, EMT6, ES2, FHC, H460, HaCaT, HCC1393, HCT6, HCT8, HCT15, HCT116, HDL100, HEK293T, Hela, Hep3B, HepG2, HEY, HGF, HL‐1, HL‐60, HLE, HT‐1080, HT‐29, HTB‐123, HTB‐126, Ho, Hs5787, Huh6, Huh7, ICE6, ICE18, JHH‐1, Jurkat, JR75‐1, JR‐75‐30, K652, KTC‐1, LAPC‐4, LAPC‐9, Li7, LNCaP, MC3T3, MCF, MCF‐7, MCF‐10A, MDA‐MB‐157, MDA‐MB‐175, MDA‐MB‐231, MDA‐MB‐436, MDA‐MB‐468, MDA‐MB‐453, NIH3T3, NMuMG, PAN3, PANC‐1, PC3, PC12, PLC/PRF/5, OV‐2008, OVCAR‐3, Raji, Rat2, RD, RFL‐6, RH1, RH2, RH3, RH4, RH6, RH14, RH18, RH28, RH30, RIE‐1, SK‐NEP‐1, SNU‐16, SNU‐378, SNU‐449, Saos‐2, SW48, SW480, SW620, SW837, SW1116, SW1353, T3M‐4, T47V, T860, TOV‐112, SK‐BR‐3, UO‐31, U87, U118, U343, U937, and YPEN‐1. Pearson correlation analysis showed that E‐cadherin levels were negatively correlated with N‐cadherin in the cell lines. p < 0.0001, n = 133, R 2 = 0.6741. B) Pearson correlation analysis showed a positive correlation between the ratio of nuclear F‐actin/G‐actin proteins and N‐cadherin/E‐cadherin. p < 0.0001, n = 133, R 2 = 0.6818. C) A total of 52 wound healing samples were selected, which were collected from 6 day‐wounded mice. All samples contained normal skin and wound healing skin in the same section, which was confirmed by H&E staining. A typical image of wound healing sample is shown. D) Typical z‐stack images (xy, xz, and yz projection and orthogonal view) showing nuclear F‐actin (Phalloidin staining, red) and G‐actin (Deoxyribonuclease I staining, green) in wound healing and normal skin cells (epidermis). The images for wound healing skins were randomly selected from central wound healing areas, while the normal skin images were randomly selected from the normal epidermis areas far from the wound region. E) ImageJ analysis showed that the wound healing cells expressed higher levels of nuclear F‐actin and lower levels of nuclear G‐actin than the normal skin cells. The intensity of Phalloidin (F‐actin)/Deoxyribonuclease I staining (G‐actin) within the cell nucleus (DAPI staining) was analyzed by ImageJ. Phalloidin/Deoxyribonuclease I staining regions that overlapped with DAPI were defined as nuclear F/G‐actin stained. The average intensity value of five cells from each image represented F/G‐actin intensity of the sample image. Phalloidin/Deoxyribonuclease I stained areas around the edge of the nucleus were excluded, and only the regions stained away from the nuclear edge were counted as stained positive for nuclear F/G‐actin. ** p < 0.01 versus normal ( n = 6). F) A diagram showing that nuclear actin polymerization regulates cell epithelial‐mesenchymal transition process.

Article Snippet: Human E‐cadherin (10204), N‐cadherin (11039) and vimentin (10028) proteins, and polyclonal antibody against actin (101273) were purchased from Sino Biological.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific reports

Article Title: PAFAH1B3 is a KLF9 target gene that promotes proliferation and metastasis in pancreatic cancer.

doi: 10.1038/s41598-024-59427-3

Figure Lengend Snippet: Figure 6. PAFAH1B3 affects the expression of EMT-related proteins in pancreatic cancer cells (A) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing PAFAH1B3-Flag or NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting. (B) SW1990 and MIA Paca-2 cells were transduced with lentivirus containing sh-PAFAH1B3 or sh-NC, and the protein expression levels of PAFAH1B3, E-cadherin, N-cadherin, Vimentin, Snail1 and MMP2 were measured via Western blotting. β-Actin was used as an internal control. The data represent the average of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: rabbit anti-PAFAH1B3 polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA), mouse anti-β-actin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-PCNA polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA), rabbit anti-E-cadherin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-Ncadherin polyclonal antibody (1:1000; Boster Biological Technology, Ltd), rabbit anti-Snail1 polyclonal antibody 3 Vol.

Techniques: Expressing, Transduction, Western Blot, Control

Journal: BMC Genomics

Article Title: Benzo pyrene-induced DNA adducts and gene expression profiles in target and non-target organs for carcinogenesis in mice

doi: 10.1186/1471-2164-15-880

Figure Lengend Snippet: Gene expression changes validation with RT - PCR

Article Snippet: The assay codes were: GAPDH -4352339E, Trp53 -Mm00441964_g1, Pcna -Mm00448100_g1, Ccng1 -Mm00438084_m1, Ephx1 -Mm00468752_m1, Cyp1a1 -Mm00487218_m1, Cdh1 -Mm00486906_m1, Ctnnb1 -Mm01350394_m1, Apc -Mm00545877_m1, Stat3 -Mm00456961_m1, Erbb2 -Mm00658541_m1, Src -Mm00436783_m1, Hspa8 -Mm01731394_gH, Hspa5 -Mm00517691_m1, Fos -Mm00487425_m1, Myc -Mm00487804_m1, NFκB -Mm00476361_m1, Tnfα -Mm00443258_m1, Cav -Mm00483057_m1, Tubb5 -Mm00495804_m1, Cdkn2a -Mm00494449_m1, Ccnd2 -Mm00438071_m1.

Techniques: Gene Expression, Biomarker Discovery, Transformation Assay