Fig 2: CAP2 inactivation increased G-actin levels in MEFs. (A) Immunoblots showing CAP2 expression in two CTR MEF lines and CAP2 inactivation in two KO MEF lines. GAPDH was used as loading control. (B) Representative micrographs showing localization of GFP-tagged CAP2 (green) in CTR MEFs (left panel). Merge micrograph (middle panel) includes counterstaining with DNA dye Hoechst (blue). Box indicates area shown at higher magnification. Representative micrograph of GFP-transfected CTR MEF (right panel). (C) Fluorescence recovery curve after photobleaching in GFP-actin transfected CTR (red) and KO MEFs (green) as well as in CTR MEFs treated with 200 nM jasplakinolide (JASP; black). (D) Half-recovery time of actin turnover and (E) stable actin fraction in CTR and KO MEFs as well as in JASP-treated CTR MFEs. (F) Representative immunoblots showing actin in soluble (G) and insoluble (F) protein fractions. (G) Immunoblot showing total actin levels in CTR and KO MEFs (left). Quantification of relative protein levels from 5 biological replicates (see Fig. S2). Signal intensity of actin was first normalized to tubulin, and then ratio of KO vs CTR was calculated (right). (H) Area quantification of CTR and KO MEFs. (I) Solidity index of CTR and KO MEFs. Scale bar in (B): 10 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant.

Fig 3: HA fragments decomposed by Exo-PH20 induced DC maturation. (a) Characterization of HA fragments generated by Exo-PH20. Top: Size determination of HA (> 10 kDa) by agarose gel electrophoresis. Bottom: FACE analysis of HA fragment from the centrifugation filtrate (< 10 kDa). Oligo-HA of defined sizes (4–12 disaccharides) were used as standards. (b) Flow cytometric analysis of the phenotypic maturation of DCs in vitro. BMDCs incubated with HMW-HA or Exo-PH20-decomposed HA fragments (< 10 kDa) for 24 h were stained with specific monoclonal antibodies against CD11c, CD40, CD86 and CCR7 and analysed by flow cytometry. The data represents the percentage (%) of CD40, CD86, CCR7 expression on BMDC. Each value represents the mean ± SD (*p < 0.05, ** p < 0.01, ***p < 0.001 versus the BMDC-only group as assessed by one-way ANOVA with Tukey’s post test). (c) Western blot analysis of the TLR-4 mediated-DC maturation pathway. BMDCs were incubated with HMW-HA or Exo-PH20-decomposed HA fragments (< 10 kDa) for 6 h. The contents of phospho-p38, phospho-p44/42, total p38, total p44/42 and GAPDH were detected in cell lysates and the NF-?B content was detected in nuclear extracts.

Fig 4: Simultaneous knockdown of multiple genes in primary human islets following electroporation. (A) Graph shows RT-qPCR results of islets electroporated with non-targeting control siRNA or siRNA targeting RB and p130 (L-003299). (B) Graph shows RT-qPCR results of islets electroporated with non-targeted control siRNA or siRNAs targeting the indicated gene products. The housekeeping gene GAPDH was used as the control. Representative experiment shown from 3 separate experiments. Error bars indicate standard deviation. (C) Western blot for RB protein in human islet lysates after electroporation of control or targeting siRNA. GAPDH was used as a loading control. RB appears as a characteristic doublet representing the hypo and hyperphosphorylated protein. Representative experiment shown from 3 different experiments.

Fig 5: Protein expression of FNDC5. The presence of a 25 kDa band was detected in the gastrocnemius with anti-FNDC5 antibodies after 4 and 8 weeks of dietary intervention and training; GAPDH was used as a loading control. HFD-Ex: high-fat diet and exercise; HFD-Sed: sedentary high-fat diet; CD-Ex: chow diet and exercise; CD-Sed: sedentary chow diet. The bars represent the significant differences between the indicated groups. The results are presented as means ± SEM (p < 0.05).