Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a GSEA of HKCs profiles with PRMT1 silencing by siRNA versus up- or down- regulated signatures from HKCs with ULK3 silencing, P < 0.001, permutation-based p. The Venn diagram of genes modulated by ULK3 silencing and bound by ULK3 in ChIP-seq in SCC13 cells is in Supplementary Fig. . The list of the genes modulated by siPRMT1 and shULK3 in HKCs is in Supplementary Fig. . b Proximity ligation assays (PLA) of two SCC cell lines with anti -ULK3 and -PRMT1 or -PRMT5 antibodies, with anti-ULK3 alone or IgG as controls. Representative images and quantification of the magenta puncta from the juxtaposition of ULK3 and PRMT1 or PRMT5 antibodies, n (cells/condition) = 75, mean ± SEM, P < 0.01, two-tailed unpaired t-test. Scale bar 5 µm. c Immunoprecipitations (IP) with anti -ULK3 (ULK3) or IgG antibodies from SCC13 cells followed by PRMT1 and PRMT5 immunoblotting (left), and IPs with anti -PRMT1, -PRMT5, or IgG antibodies followed by ULK3 immunoblotting (right). Inputs were run on the same gel. d ULK3 protein map with indicated the kinase and the microtubule-interacting (MIT1/2) domains, the positions of inactivating mutations (K139R and K44H) , , C-terminus truncation (∆C) and epitope tags (FLAG or V5). Analysis of HEK293T cells transfected with the indicated expression vectors followed by IP with anti-epitope antibodies (IP TAG) and immunoblotting with the indicated antibodies. e Analysis of HeLa cells mock-transfected or transfected with the indicated vectors followed by IP with FLAG antibodies (IP FLAG) and immunoblotting with the indicated antibodies. f PLA with anti -PRMT1 or -PRMT5 and anti -phosphorylated Ser/Thr (P-S/T) antibodies in HeLa cells infected with an ULK3 -silencing (shULK3#1) versus control (shCTRL) lentivirus. IgGs and anti-PRMT antibodies alone served as controls. Representative images and quantification of the magenta puncta from the juxtaposition of the antibodies. n (fields/condition) = 6, mean ± SEM, P < 0.01, two-tailed unpaired t -test. Scale bar 10 µm. g HeLa cells were either mock transfected (M) or transfected with expression vectors for FLAG-PRMT1 or FLAG-PRMT5 alone or in combination with wild-type ULK3 (ULK3 + PRMT1 and ULK3 + PRMT5). Total extracts (Inputs) and the proteins eluted (fractions E1–E3) from a phosphoprotein-binding resin (Talon), were analyzed by FLAG immunoblotting and quantified.

Article Snippet: PLA analysis of PRMT1 and PRMT5 phosphorylation in HeLa cells plus/minus ULK3 silencing was similarly carried out by using anti-phospho Ser/Thr antibodies (Abcam #17464, 1:100 dilution) in combination with anti -PRMT1 Santa Cruz (# 166963, 1:100 dilution), -PRMT5 (Santa Cruz # 424245, 1:100 dilution), or nonimmune control antibodies (1:100 dilution), with the addition of phosphatase inhibitors (Thermo Fisher) in all the buffers.

Techniques: ChIP-sequencing, Ligation, Two Tailed Test, Western Blot, Transfection, Expressing, Infection, Control, Binding Assay

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a Immunoblot analysis of recombinant FLAG-tagged -PRMT1 (200 ng) or -PRMT5 (200 ng) admixed with His-tagged ULK3 (200 ng) and pulled-down with a Ni-NTA resin (NTA). The same amounts of proteins were incubated with the resin singularly as controls. Sequential FLAG and ULK3 immunoblots. b Recombinant PRMT1 and PRMT5 were incubated with ULK3 as above before PRMT pull-down with anti-FLAG antibodies. Immunoblots were probed as indicated. c Recombinant PRMT1 and PRMT5 plus/minus heating for 20 min at 90 °C ( h ) before incubation with ULK3, NTA pull-down, and sequential immunoblot analysis as in ( a ). d Recombinant PRMT1 and PRMT5 were incubated with recombinant ULK3 for 30 minutes at 37°C in kinase buffer containing 200 µM ATP. Proteins were separated on a Phos-TAG gel, which delays the migration of phosphorylated proteins , followed by immunoblot analysis with the indicated antibodies. Parallel kinase reactions contained 1 and 10 µM ULK3 inhibitor SU6668 . The immunoblot analysis of the same samples in Fig. 7d separated by SDS-PAGE is in Supplementary Fig. . A kinase assay with PRMT1 plus/minus heat treatment prior to ULK3 kinase assay is in Supplementary Fig. . e In vitro ULK3 kinase assay using recombinant GFP as substrate. Proteins were admixed as in (d) before western blot analysis using the indicated antibodies. f , g Impact of ULK3 on PRTM1 and PRMT5 activity. Increasing amounts of PRMT1 or PRMT5 (0, 50, 100, 200, 400 ng) were incubated with (red bars) or without ULK3 (white bars) as in ( d ), followed by the methylation reaction (with 50µM S-adenosyl-methionine) of recombinant histone 4 (H4) (0.5 µg). Quantification of H4 arginine 3 asymmetric-, for PRMT1 activity ( f ), or symmetric-, for PRMT5 activity ( g ), dimethylation by dot blots analysis with antibodies specific for these epigenetic modifications. Mean ± SD, n (dot blot/condition) = 3, P < 0.001, two-tailed unpaired t -test. Supplementary Fig. shows symmetric and asymmetric dimethylated arginine immunoblottings of methylation assays using ULK3 as a substrate. Supplementary Fig. shows an immunoblot with the same antibodies of ULK3 and truncated ULK3 IP from transfected HEK293 cells.

Article Snippet: PLA analysis of PRMT1 and PRMT5 phosphorylation in HeLa cells plus/minus ULK3 silencing was similarly carried out by using anti-phospho Ser/Thr antibodies (Abcam #17464, 1:100 dilution) in combination with anti -PRMT1 Santa Cruz (# 166963, 1:100 dilution), -PRMT5 (Santa Cruz # 424245, 1:100 dilution), or nonimmune control antibodies (1:100 dilution), with the addition of phosphatase inhibitors (Thermo Fisher) in all the buffers.

Techniques: Western Blot, Recombinant, Incubation, Migration, SDS Page, Kinase Assay, In Vitro, Activity Assay, Methylation, Dot Blot, Two Tailed Test, Transfection

Journal: Nature Communications

Article Title: The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

doi: 10.1038/s41467-023-36410-6

Figure Lengend Snippet: a Immunoblots against the indicated proteins of SCC13 cells with ULK3 silencing by two shRNAs (shULK3 #1, #2) for 1 week or with two siRNAs (siULK3#1, 2) for 48 h, with corresponding controls. b Immunoblots of nuclear (N) and cytosolic (C) fractions from SCC13 cells with ULK3 silencing by two shRNA-specific versus control lentiviruses. The blots were sequentially probed with the indicated antibodies. c PRMT1, PRMT5 and ULK3 immunostaining (green) of SCC13 cells plus/minus ULK3 silencing with two lentiviruses versus control virus. Phalloidin labeled actin cytoskeleton (magenta). Scale bar 5 µm. d ChIP with anti -PRMT1 and -PRMT5 antibodies from SCC13 cells plus/minus ULK3 silencing followed by qPCR amplification of four regions of the PKM and LDHA genes. The position of amplified regions (magenta lines) and TSS (arrow) are shown. Results are expressed as enrichment fold relative to IgGs as in . e ChIPs with anti-histone 3 (H3) antibodies followed by PRMT1, PRMT5 and H3 immunoblotting from SCC13 cells plus/minus ULK3 silencing. For H3, the immunoblots of inputs and immunoprecipitations are shown at different exposure times. f Asymmetrically (H4R3DA) and symmetrically (H4R3DS) dimethylated arginine 3 of histone 4 immunoblotting in SCC cells plus/minus ULK3 silencing for one week, with anti-histone H4 as a loading control. An additional H4R3DS and H4R3DA immunoblot upon ULK3 silencing is in Supplementary Fig. . g H4R3DA (green) immunofluorescence of two SCC cell lines plus/minus ULK3 silencing, with DAPI (blue) as nuclear staining. Representative images and quantification of individual cells fluorescence signal, Cal27: n (cell/condition) = 35; SCC13: n (cells/condition) = 30, mean ± SD, P < 0.0001, one-way ANOVA. Scale bar 5 µm. An additional experiment is in Supplementary Fig. . Supplementary Fig. show immunoblot and immunostaining with anti -H4R3DS, -H4R3DA, and H4 antibodies of HeLa cells plus/minus ULK3 silencing. h H4R3DA, H4R3DS, total H4 and ULK3 immunoblotting of SCC13 cells with CRISPR/Cas9-mediated ULK3 gene deletion with three guide RNA (gRNA) versus control (SCR), as in Fig. . A similar analysis of HEK293T cells plus/minus CRISPR/Cas9-mediated ULK3 gene deletion is in Supplementary Fig. .

Article Snippet: PLA analysis of PRMT1 and PRMT5 phosphorylation in HeLa cells plus/minus ULK3 silencing was similarly carried out by using anti-phospho Ser/Thr antibodies (Abcam #17464, 1:100 dilution) in combination with anti -PRMT1 Santa Cruz (# 166963, 1:100 dilution), -PRMT5 (Santa Cruz # 424245, 1:100 dilution), or nonimmune control antibodies (1:100 dilution), with the addition of phosphatase inhibitors (Thermo Fisher) in all the buffers.

Techniques: Western Blot, shRNA, Control, Immunostaining, Virus, Labeling, Amplification, Immunofluorescence, Staining, Fluorescence, CRISPR

Journal: Sports Medicine and Health Science

Article Title: Exercise preconditioning prevents immobilization-induced skeletal muscle atrophy by activating Prmt1-p38/ATF2-Sesn1 signaling axis in C57BL/6J mice

doi: 10.1016/j.smhs.2025.04.001

Figure Lengend Snippet: Figure 4. Protein arginine methyltransferase 1 (Prmt1)-Sestrin1 (Sesn1) 757

Article Snippet: ChIP grade antibodies to Prmt1 (1:100, Proteintech, 256 USA) were used to perform immunoprecipitation.

Techniques: