Fig 2: Thalidomide increases oxidative stress in the hind paw, sciatic nerve, and spinal cord. a H2O2 levels in the hind paw, sciatic nerve, and lumbar (L4–L6) spinal cord at day 7 following intraperitoneal (i.p.) injection of thalidomide (Thal, 50 mg/kg) or Veh and 60 min after the administration of phenyl-α-tert-butyl nitrone (PBN, 100 mg/kg, i.p.) or Veh. b, c Representative images and mean fluorescence intensity of 4-hydroxynonenal (4-HNE) staining in the hind paw, sciatic nerve, and lumbar (L4–L6) spinal cord and TRPA1 and TRPV4 staining in the spinal cord, at day 7 following Thal (50 mg/kg, i.p.) and 60 min after the administration of PBN (100 mg/kg, i.p.) or Veh. Data are mean ± SEM (n = 6 mice). *P < 0.05 vs. Veh Thal/Veh PBN; §P < 0.05 vs. Thal/Veh PBN. One-way ANOVA followed by Bonferroni’s post hoc test

Fig 3: Oxidative stress targets TRPA1 and TRPV4. a Typical traces and pooled data of patch-clamp inward currents elicited by thalidomide (Thal), pomalidomide (Poma) and lenalidomide (Lena) (all, 100 µM), allyl isothiocyanate (AITC, 100 µM), 4a-phorbol 12,13-didecanoate (4a-PDD, 100 µM), capsaicin (CPS, 1 µM), or Veh in mouse dorsal root ganglion (DRG) neurons. Data are mean ± SEM, n = 4–5 cells. *P < 0.05 vs. Veh. One-way ANOVA followed by Bonferroni’s post hoc test. b, c Mechanical and cold allodynia at day 7 following the intraperitoneal (i.p.) injection of thalidomide (Thal, 50 mg/kg) or Veh, and after the administration of phenyl-a-tert-butyl nitrone (PBN, 100 mg/kg, i.p.) or Veh. Data are mean ± SEM, n = 6 mice. *P < 0.05 vs. Veh Thal/Veh PBN; §P < 0.05 vs. Thal/Veh PBN. Two-way ANOVA followed by Bonferroni’s post hoc test. d Pooled data of Ca2+ response to AITC (10 µM), 4a-PDD (1 µM), or Veh hTRPA1-HEK293 and hTRPV4-HEK293 cells in the presence of PBN (30 µM). e Concentration response (Ca2+ mobilization)-curve to H2O2 in cultured hTRPA1- and mTRPA1-HEK293 and hTRPV4- and mTRPV4-HEK293 transfected cells. f Pooled data of Ca2+ response to H2O2 (500 µM and 5 mM) or Veh in untransfected HEK293 or hTRPA1-HEK293, hTRPV4-HEK293 cells in the presence of HC-030031 (HC03, 30 µM) or HC-067047 (HC06, 10 µM). g Pooled data of the Ca2+ response to H2O2 (5 mM) in hTRPV4-HEK293 cells in the presence of dithiothreitol (DTT, 10 mM) or 2-mercaptoethanol (ß-ME, 50 mM). h Pooled data of the Ca2+ response to H2O2 (500 µM) in DRG neurons from Trpa1+/+ and Trpa1-/- mice and H2O2 (5 and 10 mM) in DRG neurons from Trpa1-/- mice in the presence of HC06 (10 µM). Data are mean ± SEM, n = 20–25 neurons or 80–100 cells. *P < 0.05 vs. Veh; §P < 0.05 vs. H2O2 (500 µM, 5 mM, or 10 mM). One-way ANOVA followed by Bonferroni’s post hoc test

Fig 4: TRPV4-Rac1 signaling axis plays a crucial role in the augmentation of intracellular stiffness and regulation of cytoskeletal remodeling in BMDMs. A, schematic diagram of atomic force microscopy (AFM) setup to determine the stiffness (Young’s modulus) of BMDMs. A detector records deflection of a laser beam by deformation of the cantilever attached to a circular symmetric quartz probe with a radius of 30 nm. Force curves generated by this process are fitted to the Hertz model to achieve Young’s modulus value (kPa). B, representative high-resolution AFM micrographs show distribution of lamellipodia/filopodial areas of indicated cell groups. Scale bars: 2 µm; n = 10 cells/group; 2 scanned areas/cell. Quantification of data from experiment shown in B: Histograms show percent area of filopodia (C), the number of filopodia (D), and size of filopodia (E). Student’s t test; $$p < 0.01 (KO, UT versus KO+Ad-TRPV4), **p < 0.01 (WT, UT versus WT+IL-4+GM-CSF), ***p < 0.001, and ###p < 0.001 (KO+Ad-TRPV4 versus KO+Ad-TRPV4+Rac1-I). F, quantification of Young’s modulus (kPa) of the dataset, and G, upper quartile data points acquired from the experiment shown in B. n = 70 data points/group; One-way ANOVA followed by Bonferroni test; ***p < 0.001. BMDM, bone marrow–derived macrophage; GM-CSF, granulocyte macrophage–colony stimulating factor; IL-4, interleukin-4; TRPV4, transient receptor potential vanilloid 4.

Fig 5: Recordings of single-channel activity (opening is downward) and corresponding amplitude histogram of single-channel currents through TRPV4, activated by GSK101, in the presence of PTX alone (A) and in the presence of PTX and NCS1 (B) in inside-out patches from CTR MDA-MB231. (C) Comparison of the mean Po in 12 different patches in the conditions described in (A) and (B), as indicated. * denotes significant increase (P = 0.0387). (D) Comparison of the mean inward and outward conductances in 10 different patches in the conditions described in (A) and (B), as indicated; conductances were calculated from linear regressions of the corresponding I-V relationships. * denotes significant increase (inward P = 0.0111; outward P = 0.0065). pA: picoamperes. pS: picosiemens.