Journal: Journal of Functional Biomaterials

Article Title: A Borophosphate Glass Doped with Cobalt Oxide Improves Skeletal Muscle Structure and Function in Myopathic Mice

doi: 10.3390/jfb17030155

Figure Lengend Snippet: CoO-TRIM increased myofibers with centrally nucleated myofibers (CLN) and modulated proteolytic activity. ( A ) Representative images of TA muscle cross-sections at 14 dpt. Laminin (white): basal laminae; DAPI (blue): nuclei. Yellow arrows depicting position of central nuclei. ( B ) Summary values ( n = 4–5/group) for total myofibers with CLN normalized to TA muscle cross-section area (mm 2 ) at 14 dpt. ( C ) Summary values ( n = 4–5/group) for total myofibers (CLN + + CLN − ) normalized to TA muscle cross-section (mm 2 ) at 14 dpt. ( D ) Representative immunoblot for αII-Spectrin. The 145 kDa cleavage byproduct was normalized to total protein per lane, represented by the 40 kDa band from the total protein stain, and analyzed as a ratio of the 250 kDa band of αII-Spectrin. Mean densitometric data revealed a significant reduction in cleaved αII-Spectrin abundance in TA muscles of D2. mdx TRIM mice at 14 dpt. ( E ) Representative immunoblot of LC3B II and mean densitometric data at 14 dpt revealed reduced autophagosome number following CoO-TRIM administration in D2. mdx mice. ( n = 5/group); Summary values are means ± SEM. Comparisons made vs. WT and vehicle controls by 2-Way ANOVA, p < 0.05 = significant. Scale bars = 100 µm.

Article Snippet: Sections were washed 3× in TBS, incubated with secondary antibodies in blocking buffer for 60 min at RT, washed 3× in TBS, and mounted in InvitrogenTM ProLongTM Gold antifade reagent with DAPI (Cat.# P36941 , Fisher Scientific, Hampton, NJ, USA).

Techniques: Activity Assay, Western Blot, Staining, Muscles

Journal: Journal of Functional Biomaterials

Article Title: A Borophosphate Glass Doped with Cobalt Oxide Improves Skeletal Muscle Structure and Function in Myopathic Mice

doi: 10.3390/jfb17030155

Figure Lengend Snippet: Comparison of CLN in myofibers of WT and D2. mdx treated with Saline and TRIM mice. ( A , E ) Representative images of TA muscle cross-sections at 70 dpt and 140 dpt. Laminin (white): basal laminae; DAPI (blue): nuclei. Yellow arrows identify position of central nuclei. ( B , F ) Summary values for total myofibers with CLN (Top) and total myofibers (CLN + + CLN − ) (Bottom) normalized to TA muscle cross-section area (mm 2 ). Summary values presented for ( B ) 70 dpt and ( F ) 140 dpt. ( n = 7–8/group); scale bars = 100 µm. Proteolytic activity is altered following CoO-TRIM treatment. ( C , G ) Representative immunoblots for αII-Spectrin. The 145 kDa cleavage byproduct was normalized to total protein per lane, represented by the 40 kDa band from the total protein stain, and analyzed as a ratio of the 250 kDa band of αII-Spectrin. Mean densitometric data revealed a significant reduction in cleaved αII-Spectrin abundance in TRIM mice at 70 dpt. There was no difference at 140 dpt. ( D , H ) Representative immunoblots of LC3B II and mean densitometric data at ( D ) 70 dpt, revealed that TRIM reduced autophagosome number. No differences were observed at ( H ) 140 dpt. ( n = 7–8/group); summary values are means ± SEM. Comparisons made vs. vehicle controls by two-tailed Student’s t -test; p < 0.05 = significant.

Article Snippet: Sections were washed 3× in TBS, incubated with secondary antibodies in blocking buffer for 60 min at RT, washed 3× in TBS, and mounted in InvitrogenTM ProLongTM Gold antifade reagent with DAPI (Cat.# P36941 , Fisher Scientific, Hampton, NJ, USA).

Techniques: Comparison, Saline, Activity Assay, Western Blot, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A , B ) Representative micrographs of cilia ( A ) and quantification of the cilia length ( B ), stained with antibody to acetylated tubulin (Ac-tub), in the WT and BBS4 KO/KO RPE1 cells. Scale bar, 5 µm. Medians with interquartile range from three independent experiment ( n = 96 cilia). ( C ) Expansion microscopy of the cilia axoneme of the WT and BBS4 KO/KO RPE1 cells visualized via staining with antibody to acetylated tubulin. Enlarged insets and white arrows point to bulges at cilia tips observed in BBS4 KO/KO cells. Scale bar, 2 µm. ( D ) Representative micrographs of cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) and staining of F-actin (Phalloidin) in WT, BBS1 KO/KO , and BBS4 KO/KO RPE1 cells treated with the ROCK1 inhibitor Y27632 or CDC42 inhibitor ML141 for 2 h. Cilia base at bottom. Scale bar, 5 µm. ( E ) Quantification of the cilia length based on Ac-tub signal in the WT, BBS1 KO/KO , and BBS4 KO/KO RPE1 cells non-treated (NT) or treated with Y27632 for 2 h. Medians with interquartile range from three independent experiments ( n = 160–290 cilia). ( F ) Quantification of the cilia length based on Ac-tub signal in the WT, BBS1 KO/KO , BBS4 KO/KO , and BBS9 KO/KO RPE1 cells treated with vehicle or ML141 for 2 h. Medians with interquartile range from three independent experiments ( n = 145–270 cilia). ( G ) Merged micrographs (left) show representative cilia stained with antibodies against ARL13B (green) and Ac-tubulin (red), along with the corresponding intensity line scans (dashed line next to the cilium) from the cilia base (B) to the cilia tip (T) in WT RPE1 cells. The arrow indicates the foci at the tip of the top cilium, defined as the ARL13B signal extending (~0.5 μm) beyond the Ac-tubulin signal, as illustrated in the intensity plot on the right. The bottom cilium does not exhibit foci based on this criterion. Scale bar, 5 µm. ( H ) Quantification of the frequency of ciliary tip foci observed in ( G ). Mean and SD of three independent experiments ( n = 100–200 cilia). ( I , J ) Representative micrographs ( I ) and quantification of the cilia length ( J ), in WT and Bbs4 KO/KO MEFs treated with vehicle or ML141 and stained with antibodies to acetylated tubulin (Ac-tub) and ARL13B. Cilia base at bottom. Scale bar, 5 µm. Medians with interquartile range from three independent experiments ( n = 300–400 cilia). ( K ) Representative Western blots from total cell lysates (TCL, left blots) and EVs purifications (right blots) prepared from GPR161-mCherry WT and Bbs4 KO/KO MEFs treated with SAG and vehicle or ML141 for 2.5 h probed with antibodies to IFT88 (red star—middle band), mCherry, TSG101, CD9 and Calnexin. ( L , M ) Quantification of the GPR161-mCherry ( L ) and IFT88 ( M ) present in the ciliary EV fraction purified by ultracentrifugation of cell supernatants of GPR161-mCherry WT and Bbs4 KO/KO MEFs treated with SAG and vehicle or ML141 for 2.5 h. Mean and SD of four independent experiments. Data information: Statistical significance was calculated using the two-tailed Mann–Whitney test ( B , E , F , J ), two-tailed paired t-test ( H ), and one-tailed paired t-test ( L , M ) and the obtained p -values are indicated. Merged micrographs show nuclei staining by DAPI—blue ( D , G , I ). .

Article Snippet: ProLongTM Gold Antifade Mountant with DNA Stain DAPI , Invitrogen , P36941.

Techniques: Staining, Microscopy, Western Blot, Purification, Two Tailed Test, MANN-WHITNEY, One-tailed Test

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Expansion microscopy of the cilia axoneme of the BBS1 KO/KO and BBS7 KO/KO RPE1 cells visualized by staining with antibody to acetylated tubulin. White arrows point to bulges at the cilia tips. Scale bar, 2 µm. ( B ) The graph shows the frequency of bulges at the cilia tips visualized by expansion microscopy of cilia in WT and BBS4 KO/KO RPE1 cells. The number of observed bulges out of the total count of imaged cilia is shown. Statistical analysis was done using the contingency table and one-sided Chi-square test. ( C ) Representative micrographs of the cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) and staining of actin cytoskeleton with Phalloidin-TexasRed in the WT RPE1 cells treated with inhibitors of ROCK1/RHOA (Y27632), CDC42 (ML141), RAC1 ( CAS 1177865-17-6 (CAS)), ARP2/3 (CK666), and actin polymerization (Cytochalasin D) for 2 h. Scale bar, 5 µm. ( D ) Quantification of the cilia length based on acetylated tubulin signal in the WT and BBS4 KO/KO RPE1 cells treated with DMSO or CK666 or Cytochalasin D (CytoD) for 2 h as in ( C ). Medians with interquartile range from three independent experiments ( n = 220–320 cilia). ( E ) Quantification of the cilia length based on acetylated tubulin signal in the WT and BBS4 KO/KO RPE1 cells treated with vehicle or CAS for 2 h as in ( C ). Medians with interquartile range from three independent experiments ( n = 250–280 cilia). ( F ) Representative micrographs of the cilia visualized by staining with antibody to acetylated tubulin (Ac-tub) and ARL13B in the WT and BBS4 KO/KO RPE1 cells treated with DMSO or ML141 for 2 h. Scale bar, 5 µm. ( G ) Quantification of the cilia length in WT, BBS4 KO/KO , and BBS4 KO/KO RPE1 cells expressing YFP-BBS4 treated with DMSO or ML141 for 2 h and stained with antibodies to acetylated tubulin (Ac-tub) and ARL13B. Medians with interquartile range from three independent experiments ( n = 200–250 cilia). ( H ) Quantification of the frequency of ARL13B foci at the cilia tips in WT, BBS4 KO/KO , and BBS4 KO/KO RPE1 cells expressing YFP-BBS4 treated with DMSO or ML141 for 2 h in ( G ). Mean and SD of three independent experiments ( n = 200–250 cilia). ( I ) Intensity plot profiles of the Ac-tub and ARL13B signal measured from the cilia base (B) to the cilia tip (T) in Bbs4 KO MEFs treated with ML141 in Fig. . The ARL13B signal extends (~0.5 μm) beyond the Ac-tub signal. ( J ) Representative chromatogram analysis of DNA sequencing data for WT ST2 and Ift88 KO ST2 cell clones reveals a homozygous mutation characterized by the insertion of a T nucleotide between bases 248 and 249 of the Ift88 gene (NM_009376.3, arrow). The position of the sgRNA and PAM sequence used for targeting is indicated. ( K ) Representative Western blot from total cell lysates prepared from WT MEFs, SAG/DMSO treated GPR161-mCherry WT MEFs (experiments #1–4 in Fig. ), WT and Ift88 KO ST2 cells and Ift88 KO ST2 cells expressing the IFT88-mNeonGreen and probed with antibodies to IFT88 and β-actin. The arrow highlights the IFT88 band (red star) corresponding to the expected size of 100 kDa, which is absent in the Ift88 KO ST2 cells. Data information: Statistical significance was calculated using the one-sided Chi-square test ( B ), two-tailed Mann–Whitney test ( D , E , G ) and two-tailed paired t-test ( H ) and the obtained p -values are indicated. Merged micrographs show nuclei staining with DAPI—blue ( F ).

Article Snippet: ProLongTM Gold Antifade Mountant with DNA Stain DAPI , Invitrogen , P36941.

Techniques: Microscopy, Staining, Expressing, DNA Sequencing, Clone Assay, Mutagenesis, Sequencing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: ( A ) Representative micrographs depict cilia visualized via staining with antibodies to acetylated tubulin (Ac-tub) and GPR161 in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs. Cilia base at bottom. Scale bar, 5 µm. ( B , C ) Quantification of the frequency of GPR161 positive cilia ( B ) and of the frequency of GPR161 foci at the cilia tip ( C ) in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs in ( A ). Mean and SD of three independent experiments ( n = 160–210 cilia). ( D ) Representative micrographs depict cilia visualized via staining with antibodies to acetylated tubulin (Ac-tub) and GPR161 in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs concomitantly treated with ML141. Cilia base at bottom. Scale bar, 5 µm. ( E ) Quantification of the cilia length, in non-treated (nt) and SAG induced WT and Bbs4 KO/KO MEFs concomitantly treated with DMSO or ML141 and stained with antibodies to acetylated tubulin (Ac-tub) and GPR161. Medians with interquartile range from three independent experiments ( n = 170–190 cilia). ( F ) Representative micrographs depict cilia visualized via staining with antibodies to acetylated tubulin (Ac-tub) and GPR161 (Merge and insets), in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing the cilia targeted GFP-CDC42 WT or DN variant. Cilia base at bottom. Scale bar, 5 µm. ( G ) Quantification of the cilia length, in non-treated (nt) and SAG induced—2 h, WT, and Bbs4 KO/KO MEFs expressing cilia targeted GFP-CDC42 WT or DN variant stained with antibodies to acetylated tubulin (Ac-tub) and GPR161. Medians with interquartile range from three independent experiments ( n = 160–220 cilia). ( H ) Quantification of the frequency of GPR161 positive cilia in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing cilia targeted GFP-CDC42 WT or DN variant. Mean and SD of three independent experiments ( n = 160–190 cilia). ( I ) Quantification of the frequency of GPR161 tip foci in non-treated (nt) and SAG induced—2 h, WT and Bbs4 KO/KO MEFs expressing cilia targeted GFP-CDC42 WT or DN variant in ( H ). Mean and SD of three independent experiments ( n = 160–190 cilia). Data information: Statistical significance was calculated using two-tailed paired t-test ( B , C , H , I ), and two-tailed Mann–Whitney test ( E , G ) and the obtained p -values are indicated. Merged micrographs show nuclei staining with DAPI—blue ( A , D , F ). .

Article Snippet: ProLongTM Gold Antifade Mountant with DNA Stain DAPI , Invitrogen , P36941.

Techniques: Staining, Expressing, Variant Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Reports

Article Title: BBSome-deficient cells activate intraciliary CDC42 to trigger actin-dependent ciliary ectocytosis

doi: 10.1038/s44319-024-00326-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: ProLongTM Gold Antifade Mountant with DNA Stain DAPI , Invitrogen , P36941.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Staining, Modification, Protease Inhibitor, Software, Imaging, Bicinchoninic Acid Protein Assay, Microscopy