Journal: EMBO Reports

Article Title: Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo

doi: 10.1038/s44319-024-00213-7

Figure Lengend Snippet: ( A ) Quantification of the tibial sciatic nerve area shows a significant decrease in Fam134b/c dKO compared with the other genotypes. ( B ) Representative immunofluorescence staining of ChAT (red) and NF200 (green) in tibial sciatic nerve sections from 4-weeks-old WT, Fam134b KO , Fam134c KO and Fam134b/c dKO mice. Scale bar, 50 µm. ( C , D ) Number of NF200 + ( C ) or ChAT + ( D ) axons per mm 2 showing no difference among genotypes. ( E , F ) Diameter distribution of NF200 + ( E ) or ChAT + ( F ) axons showing accumulation of smaller sized axons in Fam134b/c dKO compared with the other genotypes. ( G ) Representative immunofluorescence staining of AchR (red), NF200 (gray), and Syn1 (green) in EDL muscle from 4-weeks-old WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO mice. Scale bars, 100 µm and 25 µm, respectively, in the ×20 and ×63 magnification. ( H ) Fam134b/c dKO muscles show an increased percentage of denervated neuromuscular junctions (NMJs) compared with the other genotypes. ( I ) AchR mean positive area indicates no main changes among the different genotypes. ( J ) Fam134b/c dKO NMJs show a decreased number of branches compared with the other genotypes. ( K ) The ratio between the Syn1 and AChR positive area is reduced in Fam134b/c dKO NMJs compared to the other genotypes. Data information: n ≥ 4 animals/group in all experiments. Statistical significance was determined by one-way ANOVA ( A , C , D , H – K ) or two-way ANOVA ( E , F ), followed by Tukey’s multiple comparisons test. Data represent mean ± SEM. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical analysis and exact P values are included in the source data files.

Article Snippet: NF200 (rabbit) , SySy , 171102 , 1:200/1:1000 , IF/WB.

Techniques: Immunofluorescence, Staining, Muscles

Journal: EMBO Reports

Article Title: Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo

doi: 10.1038/s44319-024-00213-7

Figure Lengend Snippet: ( A ) Area quantification of tibial sciatic nerve showing a significant decrease in Fam134b/c dKO compared with the other genotypes. ( B ) Representative immunofluorescence staining of ChAT (red) and NF200 (green) in tibial sciatic nerve sections from WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO mice aged 15 weeks. Scale bar, 50 µm. ( C , D ) Number of NF200 + ( C ) or ChAT + ( D ) axons per mm 2 indicating a decreased density in Fam134b/c dKO compared with the other genotypes. ( E , F ) Diameter distribution of NF200 + ( E ) or ChAT + ( F ) axons showing accumulation of smaller sized axons in Fam134b/c dKO compared to the other genotypes. ( G ) Representative immunofluorescence staining of AchR (red), NF200 (gray), and Syn1 (green) in EDL muscle of WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO mice aged 15 weeks. Scale bars, 100 µm and 25 µm, respectively, in the ×20 and ×63 magnification. ( H ) Fam134b/c dKO muscles show an increased percentage of denervated neuromuscular junctions (NMJs) compared with the other genotypes. ( I ) AchR mean positive area showed there were no significant changes among the different genotypes. ( J ) Fam134b/c dKO NMJs show a decreased number of branches compared with the other genotypes. ( K ) The ratio between Syn1 and AChR positive area is reduced in Fam134b/c dKO NMJs compared with the other genotypes. Data information: n ≥ 4 animals/group in all experiments. Statistical significance was determined by one-way ANOVA ( A , C , D , H – K ) or two-way ANOVA ( E , F ) followed by Tukey’s multiple comparisons test. Data represent mean ± SEM. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical analysis and exact P values are included in the source data files. .

Article Snippet: NF200 (rabbit) , SySy , 171102 , 1:200/1:1000 , IF/WB.

Techniques: Immunofluorescence, Staining, Muscles

Journal: EMBO Reports

Article Title: Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo

doi: 10.1038/s44319-024-00213-7

Figure Lengend Snippet: ( A ) Western blot analysis showing protein expression of Fam134 proteins in brain, lumbar spinal cord, lumbar DRG, and sciatic nerve of 2-week-old mice with the indicated genotypes. Asterisks indicate a specific band. ( B ) Venn diagram showing significant differentially expressed proteins (DEPs) in sciatic nerves of Fam134b KO , Fam134c KO , and Fam134b/c dKO compared with WT. ( C ) Cellular components gene ontology enrichment in Fam134b/c dKO DEPs are represented as a percentage of total Fam134b/c dKO DEPs. ( D ) Heatmap based on significant Fam134b/c dKO DEPs annotated to ER and comparing WT, Fam134b KO , and Fam134c KO , and Fam134b/c dKO protein intensity values. The protein abundance scale is depicted on the top right. ( E ) Most relevant terms from Gene Ontology Biological Process (GO-BP) of Fam134b/c dKO DEPs annotated to ER are represented by dot plots indicating FDR, DEP count, and fold enrichment. n ≥ 3 animals/group. FDR < 0.05. ( F ) Western blot analysis showing Reep1 and Reep2 protein levels in the sciatic nerve of 2-week-old mice with the indicated genotypes. ( G ) Quantification of Reep1 and Reep2 protein levels normalized to vinculin. n ≥ 5 animals/group. ( H ) Western blot analysis showing total and hyperphospho- neurofilament heavy chain (NF200), beta-III-Tubulin, and beta-Actin protein levels in the sciatic nerve of 2-week-old mice with the indicated genotypes. ( I ) Protein quantification relative to ( H ) normalized to vinculin, or to total NF200 in the case of Hyperphospho-NF200. n ≥ 5 animals/group. Data information: Statistical significance was determined by one-way ANOVA ( G , I ) followed by Tukey’s multiple comparisons test. Data represent mean ± SEM. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical analysis and exact P values are included in the source data files. .

Article Snippet: NF200 (rabbit) , SySy , 171102 , 1:200/1:1000 , IF/WB.

Techniques: Western Blot, Expressing, Quantitative Proteomics

Journal: EMBO Reports

Article Title: Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo

doi: 10.1038/s44319-024-00213-7

Figure Lengend Snippet: ( A ) Western blot analysis showing protein expression of autophagy markers (Sqstm1/p62 and Lc3b), the ER marker Calnexin, and ER-phagy receptors (Atl3, Tex264, Ccpg1, and Rtn3) in sciatic nerve of 2-week-old mice with the indicated genotypes. ( B – D ) Protein level quantification of autophagy markers ( B ), Calnexin ( C ), and ER-phagy receptors ( D ). Normalized to vinculin. n ≥ 4 animals/group. ( E ) Representative immunofluorescence staining of ChAT (red) and NF200 (green) in tibial sciatic nerve sections of WT and Fam134b/c dKO mice aged 2 weeks. Scale bar, 50 µm. ( F ) Quantification of the tibial sciatic nerve area showing no difference between WT and Fam134b/c dKO mice. ( G – J ) Morphometric analysis of axons shows no alterations between WT and Fam134b/c dKO mice either in both the number of NF200 + ( G ) and ChAT + axons ( H ) or in their diameter distribution ( I , J ). n = 3 animals/group. Data information: Statistical significance was determined by one-way ANOVA ( B – D ) followed by Tukey’s multiple comparisons test or Student’s t- test ( F – H ). Data represent mean ± SEM. ns P > 0.05, * P < 0.05, ** P < 0.01. Statistical analysis and exact p-values are included in the source data files.

Article Snippet: NF200 (rabbit) , SySy , 171102 , 1:200/1:1000 , IF/WB.

Techniques: Western Blot, Expressing, Marker, Immunofluorescence, Staining

Journal: EMBO Reports

Article Title: Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo

doi: 10.1038/s44319-024-00213-7

Figure Lengend Snippet: Detailed information for antibodies and reagents.

Article Snippet: NF200 (rabbit) , SySy , 171102 , 1:200/1:1000 , IF/WB.

Techniques: Plasmid Preparation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Strong Twirling-Rotating Manual Acupuncture with 4 r/s Is Superior to 2 r/s in Relieving Pain by Activating C-Fibers in Rat Models of CFA-Induced Pain

doi: 10.1155/2021/5528780

Figure Lengend Snippet: The activation of primary sensory neurons in the DRG by twirling-rotating MA with different frequencies (n = 3). (a) The representative images of C‐FOS (+) co-labeled with CGRP (+) in DRG among each group; (b) comparison of the proportion of CGRP‐positive neurons excited by twirling-rotating MA with two forms of frequencies. (c) The representative images of C‐FOS(+) co‐labeled with NF200(+) in DRG among each group; (d) comparison of the proportion of NF200‐positive neurons excited by twirling‐rotating MA with two forms of frequencies. Red represents C‐FOS; green represents NF200 or CGRP. The white arrow indicates examples of co-labeled neurons. The bar = 50 um. ∗ P < 0.05; ∗∗ P < 0.01, compared with the control group; ★ P < 0.05; ★★ P < 0.01, compared with the 2 r/s MA group.

Article Snippet: The primary antibodies were C-FOS antibody (1:200, EL900233, EterLife, UK), NF200 antibody (1:200, EL900264, EterLife, UK), and CGRP antibody (1:200, EL901165, EterLife, UK).

Techniques: Activation Assay, Labeling, Comparison, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Strong Twirling-Rotating Manual Acupuncture with 4 r/s Is Superior to 2 r/s in Relieving Pain by Activating C-Fibers in Rat Models of CFA-Induced Pain

doi: 10.1155/2021/5528780

Figure Lengend Snippet: The activation of the DRG neurons and acupuncture analgesia of MA with different frequencies affected by RTX (n = 5). (a, b) The representative images of C‐FOS co-labeled with CGRP-positive neurons and C‐FOS co‐labeled with NF200‐positive neurons in the DRG among each group. The white arrows indicated examples of co-labeled neurons. Bar = 50 um. (c, d) Comparison of the proportion of CGRP-positive neurons and NF200‐positive neurons between the 4 r/s MA, 4 r/s MA+ RTX, 2 r/s MA, 2 r/s MA + RTX, and CFA groups. (e). The analgesic effects of 2 forms of MA affected by RTX. The white arrow indicates examples of co-labeled neurons. ⋆ P < 0.05; ⋆⋆ P < 0.01, compared with the control group; # P < 0.05; ## P < 0.01, compared with the CFA group. ▲ P < 0.05; ▲▲ P < 0.01, compared with the CFA + 4 r/s MA + RTX group. ★★ P < 0.01, compared with the CFA + 2 r/s MA + RTX group.

Article Snippet: The primary antibodies were C-FOS antibody (1:200, EL900233, EterLife, UK), NF200 antibody (1:200, EL900264, EterLife, UK), and CGRP antibody (1:200, EL901165, EterLife, UK).

Techniques: Activation Assay, Labeling, Comparison, Control

Journal: Bioactive Materials

Article Title: Micropatterns and peptide gradient on the inner surface of a guidance conduit synergistically promotes nerve regeneration in vivo

doi: 10.1016/j.bioactmat.2021.07.010

Figure Lengend Snippet: (a) Merged immunofluorescence staining images of S100β (green), NF200 (red), and DAPI (blue) for SCs, axons and cell nuclei in proximal parts of newborn nerves post surgery for 9 and 14 d, respectively. (b) Merged immunofluorescence staining images of CD163 (green), CD86 (red), and DAPI (blue) for M2, M1 and cell nuclei in the proximal segments of regenerated nerves post surgery for 9 and 14 d, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The immunofluorescence staining of NF200 (mouse anti-rat, 1:500; Peprotech Co., UK), S100β (rabbit anti-rat, 1:500; Peprotech Co., UK) and DAPI in the middle of regenerated nerves after 8 and 16 w implantation were observed by a fluorescence microscope and merged by the Image J software.

Techniques: Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Micropatterns and peptide gradient on the inner surface of a guidance conduit synergistically promotes nerve regeneration in vivo

doi: 10.1016/j.bioactmat.2021.07.010

Figure Lengend Snippet: Microstructures assay in vivo . Merged immunofluorescence staining images of green S100β (SCs), red NF200 (axons), and blue DAPI (cell nuclei) of newborn nerves guided by (a,a1) Flat, (b,b1) FG, (c,c1) MU, and (d,d1) MG conduits, and (e,e1) autografts at 16 w post-surgery, respectively. The white arrows point the axon fibers wrapped by myelin sheath in the regenerated nerves. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The immunofluorescence staining of NF200 (mouse anti-rat, 1:500; Peprotech Co., UK), S100β (rabbit anti-rat, 1:500; Peprotech Co., UK) and DAPI in the middle of regenerated nerves after 8 and 16 w implantation were observed by a fluorescence microscope and merged by the Image J software.

Techniques: In Vivo, Immunofluorescence, Staining

Journal: Tissue Engineering. Part A

Article Title: Long-Term Evaluation of Functional Outcomes Following Rat Volumetric Muscle Loss Injury and Repair

doi: 10.1089/ten.tea.2019.0126

Figure Lengend Snippet: Comparison of vasculature and neuronal innervation in the TA muscle of the TEMR max responders. (A) Cross-sectional schematic of the TA indicating where IF measurements were made in the contralateral control muscles (A[i]), and in the TEMR max responder muscles (A[ii]). Red shading indicates the region in the cortex where nominal de novo muscle regeneration was observed at the site of the original defect. Representative capillary and vessel staining in the (B[i]) representative TEMR max responder TA. Sections were stained for α-SMA (smooth muscle cells) and CD31 (endothelial cells) and the number of capillaries (white asterisks) and vessels (white arrows) were manually counted in the same region of interest in each sample, as described in the section “Materials and Methods.” Representative staining is shown in a higher magnification inset (B[ii]) that corresponds to the outlined area shown in (B[i]). (C) Capillaries and (D) vessels were quantified. There were no statistically significant differences between TEMR max responders and the contralateral control TA muscles (p > 0.05, Mann–Whitney test). Because the data were not normally distributed, the values are expressed as the median ± range; sample sizes are in parentheses. (E) Representative neuron staining in the rat TEMR max responder muscle. Punctate dots were counted as occurrences of positive staining. The inset shows a higher magnification of NF200 staining. (F) TEMR max responders had levels of innervation comparable to the native contralateral controls (by unpaired t-test, ns = not significant, p = 0.7). Values are expressed as the mean ± SEM; sample sizes are in parentheses. IF, immunofluorescence; SMA, smooth muscle actin. Color images are available online.

Article Snippet: Samples were incubated with antibodies against rabbit CD31 (1:250, NB100-2284; Novus Biologicals), mouse smooth muscle actin (SMA) conjugated to Alexa Fluor 488 (1:250, F3777; Sigma-Aldrich), mouse CD68 (1:100, MCA341R; Bio-Rad), rabbit CD163 (1:400, ab182422; Abcam), chicken neurofilament 200 (NF200, 1:1000; EnCor CPCA-NF-H), mouse myosin heavy chain slow (1:25, BA-F8; DSHB), mouse myosin heavy chain fast (1:25, A4.74; DSHB), and mouse myosin heavy chain type IIB (1:25, BF-F3; DSHB) overnight at 4°C.

Techniques: Staining, MANN-WHITNEY, Immunofluorescence