Fig 2: Chronic elevation of endogenous insular glutamate decreased intraepidermal nerve fiber density (IENFD). Immediately following animal dispatch, the most distal papillae from glabrous skin of both hindpaws was removed with a razor blade and placed in 2% Zamboni's fixative for 4–6 hours. Tissue was rinsed in 30% sucrose in 1X PBS overnight and embedded in OCT in 4565 Tissue-Tek molds the next day. Thirty micron cross-sections from the largest area of the papillae were sectioned and stained with protein gene product 9.5 (PGP9.5) at 1/2500, visualized with Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488; Thermofisher; Pittsburgh; PA; Cat #: A-11034) preadsorbed (ab150065), and mounted on slides with prolong gold with 4',6-diamidino-2-phenylindole (DAPI). A technician blinded to experimental group, using a Nikon microphot FXA upright microscope with the X-Cite 120Q light source and 40x Plan Apo lens, counted images manually. Fibers that cross over the basement membrane and into the epidermis were counted as positive fibers (A; denoted by white dots on each image). Distance was measured in mm and final data is presented as fibers per mm. Hind paw IENFD was compared between experimental groups (B): Naïve (n=7; blue circles; no surgery/treatment; mean ± SEM: 14.36 ± 3.42), Ringer's (n=6; black squares; insula-administered Ringer's vehicle solution; 13.03 ± 2.65), and PDC1 (n=6; at least one microinjector in the insula delivering PDC; 11.22 ± 2.29) or PDC2 (n=4; both microinjectors in the insula delivering PDC; 9.23 ± 1.33). In the PDC1 column, the yellow triangles indicate rats that received unilateral injections and the red triangles represent rats that received bilateral injections from the PDC2 group. For each rat, IENFD was not different between left and right hind paws. A nonsignificant reduction in IENFD was observed in PDC1 rats compared to the Naïve and Ringer's groups (H(3) = 3.25, p = 0.201). In a secondary analysis restricted to PDC2 rats, IENFD was significantly reduced following PDC administration (H(3) = 6.33, p = 0.034), signifying that bilateral insular administration of PDC is necessary to significantly reduce hindpaw IENFD. Post hoc analysis revealed a decrease in IENFD in the PDC2 group compared to the Naïve group (p = 0.047). No other post hoc comparisons were significant.

Fig 3: Morphological equivalents of mast cell CPA3 secretory pathways. Primary antibodies used: Rabbit polyclonal to CPA3 antibody (ab251696) (a–u) (AbCam), and Mouse monoclonal [NK1/C3] to CD63 antibody (ab1318) (f) (AbCam). Secondary antibodies used: (a–c,g–l,n–q,s,t) AmpliStain anti-Rabbit 1-Step HRP (#AS-R1-HRP), SDT GmbH, Baesweiler, Germany, label-HRP. (d–f,m,r,u) Goat anti-rabbit IgG Ab (#A-11034) Invitrogen, label-Cy3. (f) Goat anti-mouse IgG Ab (#A-11029), Invitrogen, label Alexa Fluor 488. (a,b) Tonsil. Compound CPA3 exocytosis with probable involvement of piecemeal degranulation. Selective accumulation of CPA3 in targets of a specific tissue microenvironment (indicated by an arrow) (a), accumulation of protease in the pericellular space of the extracellular matrix (indicated by an arrow) (b). (c) Tonsil. Formation of MC loci with the most intense CPA3 secretion (indicated by an arrow). (d,e) Skin. Secretion of CPA3 into the extracellular matrix by the mechanism of compound exocytosis and piecemeal degranulation, with the accumulation of protease in the pericellular space of neighboring cells (indicated by an arrow) (d). Formation of a separate MC cytoplasmic locus for CPA3 secretion (indicated by an arrow) (e). (f) Melanoma of the skin. High content of exosomes in CPA3+ mast cell. (g,r) Morphological variants of targeted degranulation of CPA3 by the mechanism of exocytosis to cellular targets in the specific tissue microenvironment of the tonsilla (g–j) and skin (k–m) (indicated by an arrow). (n–p) Tonsil. The initial stages of mast cell degranulation by the mechanism of exocytosis (n,o) (indicated by an arrow), leading to the distribution of CPA3 + secretory granules in the extracellular matrix (p) (indicated by an arrow). (q–r) Skin. Active exocytosis of mature secretory MC granules into the extracellular matrix with the formation of loci of the tissue microenvironment with a high content of CPA3+ granules (indicated by an arrow). (s–u) skin process of formation of CPA3+ macrovesicles of small (s) and large sizes (t,u) (indicated by an arrow). Scale bar: 5 µm.

Fig 4: Cytotopography and morphological features of CPA3 MC biogenesis. Primary antibodies used: Rabbit polyclonal to CPA3 antibody (ab251696) (AbCam, Cambridge, UK). Secondary antibodies used: (a,b,f–i,k) AmpliStain anti-Rabbit 1-Step HRP (#AS-R1-HRP), SDT GmbH, Baesweiler, Germany, label-HRP. (c–e,j,l–p) Goat anti-rabbit IgG Ab (#A-11034) Invitrogen Darmstadt, Germany, label-Cy3. (a) Tonsilla. Initial stages of intragranular packing of CPA3 in MC (indicated by an arrow). (b) Stomach. Formation of several CPA3 + granules in MC, freely located in the cytoplasm (indicated by an arrow). (c) Melanoma of the skin. Filling the cytoplasm with MC CPA3 + granules. (d) Melanoma of the skin. Perinuclear localization of CPA3+ granules of varying degrees of maturity, the formation of type III granules (indicated by an arrow). (e–h) Melanoma of the skin. Different variants of the accumulation of mature CPA3+ granules in the cytoplasm of MC. (i–k) Melanoma of the skin. Denucleation of MC with the formation of a nuclear-free CPA3-positive fragment of the cytoplasm (k). (l) Skin. Predominant peripheral localization of secretory granules in the cytoplasm of the MC. (m) Skin. An elongated mast cell filled with granules along the periphery of the cytoplasm. (n) Skin. Peripheral localization of CPA3+ granules in the transversely cut elongated part of the mast cell. (o) Melanoma of the skin. General view of a mast cell filled with mature CPA3-positive granules of approximately equal size. The protease is located at the periphery of the granule (indicated by the arrow). A granule fusion process occurs, leading to an increase in the size of the larger granule (double arrow) (p) Melanoma of the skin. CPA3+ MC granules of various sizes, the protease is located at the periphery of the granule (indicated by the arrow). There are areas of cytoplasm free of granules (double arrow). Scale bar: 1 µm (p’), 5 µm (the rest).

Fig 5: DSB repair and non-canonical DNA structures. (A) Canonical B-form duplex DNA structure. (B) Different conformations of G-quadruplexes. Inset: G-quadruplex might be intramolecular (generated on one strand of the DNA) or intermolecular (generated by several strands of DNA). In each case, they can be parallel or anti-parallel depending on the orientation of the strands. (C) R-loops structures are formed by the base-paired annealing of an RNA molecule with a DNA strand and the consequent displacement its complementary one. (D) G-loops structures arise from the formation of a G4 in the displaced ssDNA strand of an R-loop. (E) Hybrid G4s are chimeric structures in which the G-quadruplex is formed by the interaction of G at both a ssDNA and RNA molecules of an R-loop, displacing the other strand of the DNA (F) RNase H overexpression rescues the resection defect observed after PIF1 depletion. DNA resection proficiency measured as the percentage of RPA-foci-positive U2OS cells in cells expressing FLAG-RNase H or a FLAG empty vector and with either an siRNA against PIF1 (Dharmacon, CAU?AUC?UGC?UAA?AGC?GAA?U) or control siNT. Briefly, cells were seeded and grown for 24 h on coverslips. The day of transfection, medium was replaced by fresh DMEM without antibiotics and cells were incubated with a mix of siRNA and Lipofectamine diluted for 6 h in Opti-MEM before transfection with the plasmids with FuGENE six Transfection Reagent (Promega). 48 h after siRNA transfection, cells were irradiated (10 Gy) and incubated at 37°C for 1 h. Coverslips were then washed once with PBS followed by treatment with pre-extraction buffer (25 mMTris-HCl, pH 7.5, 50 mMNaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose and 0.2% Triton X-100) for 5 min on ice. Cells were fixed with 4% paraformaldehyde (w/v) in PBS for 15 min. Following two washes with PBS, cells were blocked for 1 h with 5% FBS in PBS, co-stained with anti-RPA (Abcam ab2175) and anti-?H2AX (Cell Signaling 2,577) antibodies in blocking solution overnight at 4°C, washed again with PBS and then co-immunostained with the appropriate secondary antibodies (Alexa Fluor 594 goat anti-mouse (Invitrogen A-11032), Alexa Fluor 488 goat anti-rabbit (Invitrogen A-11034) in blocking buffer. After washing with PBS and dried with ethanol 70 and 100% washes, coverslips were mounted into glass slides using Vectashield mounting medium with DAPI (Vector Laboratories). RPA foci immunofluorescences were analyzed using a Leica Fluorescence microscope with a HCX PL APO 63x/1.4 OIL objective. In all cases, at least 200 cells were analysed per condition and the experiments were replicated independently at least three times. Significance was determined by Student’s t test comparing each condition to siNT cells. *p < 0.05.