fibulin 2 ko mice  (Jackson Laboratory)

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. Representative images of SGC cultures stained for the SGCs markers FABP7, and GS, the Schwann cell marker S100B, the neuron marker TUJ1 and the fibroblast marker PDGFRα. Scale bar 100μm. B. RT-qPCR analysis of SGC cultures for known SGCs markers ( Fabp7, Glul, GjaI, Ednrb, Gfap ), Schwann cells ( Scn7a, Mpz, Ncam ), macrophages ( Aif, Itgam, Cx3cr1 ), neurons ( Nfeh, Prph ), and fibroblasts ( Fgf13, Fgf9 ). N=2. C. Transcriptional similarity between cultured cells and primary tissue cell types. Heatmap showing Spearman correlation coefficients between bulk RNA-seq profiles from cultured samples (columns) and pseudobulk expression profiles from major cell classes in the single-cell atlas (rows). Correlation was computed using 5,000 highly variable genes following joint TMM normalization. Values within cells indicate correlation coefficients; color scale ranges from blue (low correlation) to red (high correlation). Hierarchical clustering was performed using Euclidean distance. D. Bar plot showing mRNA expression levels (counts per million, CPM) for a panel of cell-type-specific marker genes in bulk RNA-seq from primary SGC cultures. Bars are colored by cell type: Glial/SGC (blue), Schwann (green), Neuronal (black), Fibroblast (purple), Endothelial (orange), Mural (red), and Immune (yellow). Individual sample values are shown as points; circles indicate serum-free (24h) samples, triangles indicate serum-containing samples. Error bars represent standard deviation. E. Representative western blot (from 3 independent experiments) of DRG and SGC lysate from WT and Fibulin-2 KO mice, probed for Fibulin-2. GAPDH and Ponceau are used as loading controls. F. Representative western blot of SGC-CM from WT and Fibulin-2 KO mice, probed for Fibulin- 2. Ponceau staining is used as a loading control. G. Ponceau staining of western blot showed in for protein loading control.

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Staining, Marker, Quantitative RT-PCR, Cell Culture, RNA Sequencing, Expressing, Single Cell, Standard Deviation, Western Blot, Control

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. GO pathway analysis (cellular component) of the SGC-secreted proteins showing enrichment for extracellular proteins. B. SGC-secreted proteins were analyzed using the matrisome analyzer. C. Representative images of cultured SGCs immunostained for Fibulin-2, along with the indicated cellular markers. Lamp1-Lysosome, CD63-multivesicular bodies, VAMP1/2/3 (note that the antibody recgonizes VAMP1, VAMP2 and VAMP3) and VAMP5-secretory vesicles, and GM130-Golgi apparatus. 10-15 fields per group were imaged using confocal microscopy. D. Quantification of the Pearson correlation coefficient for the co-localization of Fibulin2 with the different cellular compartment markers. Each data point in the bar graph represents the colocalization coefficient from an individual cell (LAMP1 n=25 cells, CD63 n=25 cells, VAMP5 n=15cells, VAMP1/2/3 n=35 cells, GM130 n=23 cells). E. SGC-CM obtained from SGC cultures treated with Brefeldin-A was analyzed by western blot for Fibulin-2. CD9, a marker of EV, is used as a control. Ponceau staining is shown for loading control. n= 3 independent experiments. F. SGC cell pellet obtained from SGC cultures treated with Brefeldin-A was analyzed by western blot for Fibulin-2. GAPDH is used as a loading control. Ponceau staining is shown as another loading control. n= 3 independent experiments. G. Quantification of Fibulin-2 level in SGC-CM shown in E. n=3 T-test; *** P < 0.001. H. Quantification of Fibulin-2 level in SGC cell pellet shown in F. n=3, T-test; * P < 0.05. I. EVs were isolated from SGC-CM prepared from cultures from WT or Fibulin2-KO mice and analyzed by western blot alongside the SGC lysate for Fibulin2, CD9 (EVs marker) and GM130 (Golgi-associated vesicles, negative control for EV). GAPDH is used as a loading control. n= 3 independent experiments.

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Cell Culture, Confocal Microscopy, Western Blot, Marker, Control, Staining, Isolation, Negative Control

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A. Representative images of DRG sections from WT and Fibulin-2 KO mice immunostained for Fibulin-2, FABP7, TUJ1, and DAPI. Scale bar 50μm. Two DRG sections from n=3 mice were imaged for each group. B. Super-resolution imaging of DRG tissue section highlighting the subcellular localization of Fibulin-2 in SGCs near the perinuclear region. Fibulin-2 (Red), FABP7 (White), TUJ1 (Green), and DAPI (Blue). Scale bar 2μm C. Electron micrograph of DRG sections showing the portion of an SGC covering the neuron soma. The SGC cytoplasm contains multivesicular bodies (white arrow), vesicle fusion/budding at the plasma membrane (red arrow), and a Golgi apparatus (asterisk). Scale bar 1μm D. Immunofluorescence of DRG section showing localization of Fibulin-2 in SGCs (labeled for Fabp7 or PDPN) surrounding neurons labeled with NeuN, NF200, TRPV1, TH, TRKA, and IB4. Scale bar 50μm. E. Quantification of the size distribution of the indicated DRG neurons subtypes. The size of neurons surrounded by Fibulin-2 positive SGC is indicated by a red dotted line. A total of 8956 neurons were analyzed from n=7 mice, with three DRG sections per mice. F. Quantification of the percentage of neuronal subtype surrounded by Fibulin-2-positive SGCs. Each data point in the bar graph represents an independent biological replicate. NF200 (n=5 mice), TRPV1 (n=5 mice), TRKA (n=3 mice), IB4 (n=4 mice), TH (n=4). Two sections from each biological replicate were imaged and used for quantification. Error bars represent SEM.

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Imaging, Clinical Proteomics, Membrane, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: Satellite Glial Cells Control Sensory Neuron Excitability via the Release of Fibulin-2

doi: 10.64898/2026.02.13.705760

Figure Lengend Snippet: A . Voltage protocols for measurement of different types of K + currents: total ( I Total ), K-type ( I K ) and A-type ( I A ) K + currents. B . Sample traces of voltage-dependent K + currents I total (left), I K (middle) and I A (right) evoked by the protocols in ( A ) from Control (upper panel) and rFibulin-2 treated DRG cells (lower panel). C . rFibulin-2 increases voltage-dependent K + currents I Total (left), I K (middle) and I A (right) in DRG cells. Insert bar graphs are K + currents at membrane potential of -10 mV (around voltage threshold level), indicating that rFibulin-2 decreases excitability mainly mediated by enhancement of I A conductance, which reduces input resistance. Number of cells tested from 3 independent experiments: control n = 10; rFibulin-2: n = 8. D . Phrixotoxin-1 (PaTx1) was used to isolate Kv4 current evoked by voltage ramp (-100 to +20 mV, 100 mV/s). Sample traces of ramp-evoked K + currents before (a) and during (b) application of PaTx1, and the PaTx1-sensitive current (c, c = a - b). Currents were normalized to membrane capacitance for better comparison. E . I-V curves were constructed from the ramp-evoked Kv4 current (mean current value over 0.1 mV intervals from averages of five trials for each cell to approximate quasi-steady-state current). Note PaTx1 significantly increases the Kv4 current when the membrane potentials are depolarized to positive values greater than -25 mV. Number of cells tested from 3 independent experiments: control n = 6; rFibulin-2: n = 6; T-test; * P < 0.05; ** P < 0.01. F . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.2. GAPDH is used as a loading control. G . Quantification of Kv4.2 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; ** P < 0.01. H . Representative western blot of control and Fibulin-2 KO DRG lysate analyzed for Fibulin-2 and Kv4.3. GAPDH is used as a loading control. I . Quantification of Kv4.3 expression in control and Fibulin-2 KO mice. n=3 WT and n=3 Fibulin-2 KO mice. T-test; *** P < 0.001 J . Fibulin-2 KO mice show hypersensitivity to mechanical stimuli compared to controls, measured by the Von Frey Test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. K . Fibulin-2 KO mice exhibit hypersensitivity to heat stimuli compared to controls, measured by the Hot-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. L . Fibulin-2 KO mice exhibit hypersensitivity to cold stimuli compared to controls, measured by the Cold-Plate test. 12 WT and 8 Fibulin-2 KO mice were used. Two-Way Anova. ∗p < 0.05, ∗∗p < 0.01, ***p<0.001. M . Representative immunofluorescence images of the hindpaw of control and Fibulin-2 KO mice immunostained for PGP9.5 (white) and DAPI (blue). Three sections from n=3 mouse per group were used. N . Quantification of intraepidermal nerve fiber density (IENFD). n=3 mice per genotype. T-test, ns- non-significant

Article Snippet: Fibulin-2 KO mice (B6.129S1-Fbln2tm1Chu/J) were obtained from The Jackson Laboratory.

Techniques: Control, Membrane, Comparison, Construct, Western Blot, Expressing, Hot Plate Test, Immunofluorescence