Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 1. Biological activity of hLMP2 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#2 (T type) clone and flat revertants of the SKN-LMP2#122 (F type) clone (magnification 100). Changes in human uterine LMS cell line, SKN-transfectants, SKN-CEM9 (T type) clone, and SKN-LMP2wt (F type) clone xenograft volumes in mice (n = 8). Representative photographs of xenografts in mice (Left). Tumor growth of SKN-LMP2 was markedly reduced in comparison with that of the control transfectant SKN-CEM9 (T type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-CEM9 (T type) clone and SKN-LMP2 (F type) clone (Right). (B) RT-PCR experiments revealed hLMP2, hLMP7, Calponin h1, SRF, cyclin B and b-actin mRNA expression in tumors. Precursor LMP2 or LMP7 (pre-LMP2, pre- LMP7) and mature LMP2 or LMP7 (LMP2, LMP7) are shown. (C) Western blotting revealed LMP2, LMP7, calponin h1, SRF, cyclin B, and b-actin in SKN-transfectant clones. (D) The luciferase reporter vectors containing the hCalponin h1 promoter with wild type SRF binding sites (Calponin-wt-Luc.), mutant SRF binding sites (Calponin-mut-Luc.), or empty luciferase reporter vector (Basic-Luc.) [23] were transiently co-transfected with pSV-b-galactosidase in SKN-transfectants, SKN-CEM9#2, SKN-LMP2#121, or SKN- LMP2#122 clones for the final 48 h, and then luciferase activities were measured. Values were normalized to those obtained with the co-transfected pSV-b-galactosidase expression vector. Each assay was performed at least three times and in triplicate. Luciferase reporter assays showed that LMP2 expression markedly induced calponin h1 promoter activation. Data are presented as the mean from three independent experiments (⁄S.D.). The experiments were performed four times with similar results. SKN transformantsa, CEM9 SKN-CEM9#2; LMP2, SKN-LMP2wt#121, SKN-LMP2wt#122. Detail is shown in SFig. 2, SFig. 3 and STable 3. RT-PCRb, total RNA samples were isolated from the individual xenografted-tumors, which were removed at 5 weeks after xenografting. W.B.c, W.B. are performed with the total cell lysates from SKN transformants.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, Comparison, Control, Transfection, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Clone Assay, Luciferase, Binding Assay, Mutagenesis, Plasmid Preparation, Activation Assay, Isolation

Journal: FEBS letters

Article Title: Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: morphological significance of calponin h1.

doi: 10.1016/j.febslet.2012.05.029

Figure Lengend Snippet: Fig. 2. Biological activity of calponin h1 in uterine leiomyosarcoma (LMS). (A) Phase-contrast micrographs of the parental transformed SKN-CEM9#1Scr.shRNA (T type) clone, SKN-CEM9#2 calponin h1shRNA (T type) clone, SKN-CEM9#2 (T type) clone, SKN-LMP2#1Scr.shRNA (F type) clone, and SKN-LMP2#2Calponin h1shRNA (T type) clone of the SKN-LMP2 (F type) clone (magnification 60). The growth rates of the SKN-transfectant clones were measured as population doubling time (PDT). (B) Western blotting and RT-PCR experiments revealed calponin h1, precursor LMP2 (pre-LMP2), mature LMP2 (LMP2), and b-actin in SKN-transfectant clones. SKN transformantsa, CEM9#3 Scr.shRNA, CEM9#4 Calponin h1shRNA, LMP2#1 Scr.shRNA, LMP2#2 Calponin h1shRNA, Detail is shown in Table 1 and SFig. 5 and STable 3. (C) Changes in the human uterine LMS cell line, SKN-transfectant, SKN-CEM9#2 (T type) clone, SKN-LMP2wt#2/Calponin h1shRNA (T type) clone, and SKN-LMP2wt#1/ Scr.shRNA (F type) clone xenograft volumes in mice (n = 3). Representative photographs of xenografts in mice (Left). Tumor growth of the SKN-LMP2wt#2/Calponin h1shRNA (T type) clone is mildly increased in comparison with that of the SKN-LMP2wt#1/Scr.shRNA (F type) clone. Tumor growth kinetics after subcutaneous injection of the SKN-transfectant clones (Right). RT-PCR experiments revealed hCalponin h1, hLMP2 and b-actin mRNA expression in tumors (Bottom). Experiments were performed three times with similar results. SKN-CEM9c, SKN- CEM9#2; LMP2wt+Calponin h1shRNAd, SKN-LMP2wt#2/ CalponinshRNA; LMP2wt/Scr.shRNAe, SKN-LMP2wt#1/Scr.shRNA. Details of SKN transfectants are shown in Table 1, SFig. 5 and STable 3. RT-PCRf, total RNA samples were isolated from the individual xenografted-tumors, which were removed from BALB/c nu/numice at 5 weeks after xenografting. Xenograftsg, BALB/c nu/nu mice were inoculated with SKN-CEM9#2, SKN-LMP2wt#2/CalponinshRNA or SKN-LMP2wt#1/Scr.shRNA.

Article Snippet: LMP2 expression vector was co-transfected into SKN cells with shRNA vector. c shRNA, Calponin h1 shRNA or Scramble shRNA were co-transfected into SKN cells with LMP2wt expression vector using manufacturer’s recomendations (Santa Cruz Biotechnology, Inc., CA, USA). d Morphology.

Techniques: Activity Assay, Transformation Assay, shRNA, Transfection, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Comparison, Injection, Expressing, Isolation

Journal: Nature Communications

Article Title: Identification of evolutionarily conserved regulators of muscle mitochondrial network organization

doi: 10.1038/s41467-022-34445-9

Figure Lengend Snippet: a , b , c Fibrillar flight muscles (IFMs) stained for F - actin and mitochondria expressing mito-gfp driven by DMef2-Gal4 showing parallel aligned mitochondria that are large, tube-like, and packed between myofibrils (Scale Bars: 5 μm). d , e Representative 3D rendering of electron microscopic images of mitochondrial arrangement (yellow) and ER (magenta) in flight muscles. f , g , h Tubular jump muscles show mitochondria that are thin and elongated arranged in parallel mitochondrial networks (Scale Bars: 5 μm). i , j Representative 3D rendering of mitochondrial networks (yellow) and ER (magenta) in jump muscles. k In walking (leg) muscles, DMef2-Gal4 driven mito-gfp shows both parallel and grid-like mitochondrial networks (marked with dashed line, scale bar: 20 μm). l , m Tubular leg muscle Fiber I showing primarily parallel mitochondrial networks. n , o Tubular leg muscle Fiber II showing a grid-like mitochondrial network. p , q Tubular leg muscle Fiber III showing grid-like mitochondria. (Scale Bars: 5 μm for all). r , s Representative 3D rendering of mitochondrial network organization (yellow) and ER (magenta) in leg muscles. t Mitochondrial volume as a percent of total muscle volume (Flight muscles (IFM) ( UAS-mito-gfp;Dmef2-Gal4 ), n = 5 animals; Jump muscles (TDT) ( UAS-mito-gfp;Dmef2-Gal4), n = 5 animals; Leg Fiber I, n = 7 animals; Leg Fiber II, n = 9 animals; Leg Fiber III, n = 5 animals). u Quantification of mitochondrial network orientation. Dotted line represents parallel equal to perpendicular (IFM, n = 11 animals; Jump muscles (TDT), n = 12 animals; Leg Fiber I, n = 10 animals; Leg Fiber II, n = 9 animals; Leg Fiber III, n = 6 animals). v Endoplasmic reticulum (ER) volume as a percent of total muscle volume (IFM, n = 5 animals; Jump muscles (TDT), n = 5 animals; Leg Fiber I, n = 3 animals; Leg Fiber II, n = 4 animals; Leg Fiber III, n = 5 animals). Each point represents value for each animal dataset. Bars represent mean ± SD. Significance determined as p < 0.05 from one way ANOVA with Tukey’s (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, non-significant).

Article Snippet: Three-dimensional rendering of mitochondria networks was performed using IMARIS 9.7.0 ( http://www.bitplane.com/imaris/imaris ).

Techniques: Muscles, Staining, Expressing

Journal: Nature Communications

Article Title: Identification of evolutionarily conserved regulators of muscle mitochondrial network organization

doi: 10.1038/s41467-022-34445-9

Figure Lengend Snippet: a Adult wild-type flight muscles (fibrillar) stained for F-actin (phTRITC). b salm KD muscle fiber stained for muscles showing tubular muscle type (Scale Bars: 100 μm). c , d , e Wildtype flight muscles show elongated, parallel mitochondria (mito-gfp) between myofibers (phTRITC). f , g , h The knockdown of salm (UAS-salm RNAi;UAS-mito-gfp;mef2) in flight muscles results in fiber conversion to tubular muscle type and mitochondria to a grid-like network. (i) Wild type leg muscles show tubular muscle type in the coxa. Inset shows a cross-section of leg muscles displaying well-aligned fibers. j , k Wildtype leg muscles show parallel mitochondria (mito-mcherry) aligned next to myofibrils (phTRITC). l salm OE converts muscle fibers to fibrillar in nature. Inset, well-defined fibrillar fibers in cross-section of leg muscles. m , n salm OE leg muscles have parallel mitochondrial networks (mito-mcherry) along the myofibrils (phTRITC) (Scale Bars: 5 μm for all). o Quantification of mitochondrial network orientation. Dotted line represents parallel equal to perpendicular. mito-gfp;mito-mcherry;mef2-Gal4 used as Wildtype, WT. (WT-IFM, n = 9 animals; salm -KD IFM, n = 7 animals; WT-Leg Fiber I, n = 6 animals; salm -OE Leg Fiber I, n = 8 animals). Each point represents value for each animal dataset. Bars represent mean ± SD. Significance determined as p < 0.05 from one way ANOVA with Tukey’s (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, non-significant).

Article Snippet: Three-dimensional rendering of mitochondria networks was performed using IMARIS 9.7.0 ( http://www.bitplane.com/imaris/imaris ).

Techniques: Muscles, Staining, Knockdown

Journal: Nature Communications

Article Title: Identification of evolutionarily conserved regulators of muscle mitochondrial network organization

doi: 10.1038/s41467-022-34445-9

Figure Lengend Snippet: a , b , c Fibrillar flight muscles (IFMs) stained for F-actin (phTRITC) and mitochondria (mito-GFP) showing parallel aligned mitochondria between myofibrils. d H15 KD shows fibrillar muscles switched to tubular muscle type. e , f H15 KD muscles show parallel mitochondria along muscle fibers. g , h , i Tubular jump muscles show parallel mitochondria (thin and elongated). j H15 KD in jump muscles shows tubular fibers (phTRITC). k , l Upon H15 KD, tubular jump muscles show a change in mitochondrial networks to more grid-like (Scale Bars: 5 μm for all). m Wildtype coxa leg muscle Fiber I showing parallel mitochondrial networks. n Wildtype leg muscle Fiber II and o Fiber III showing grid-like mitochondrial networks. p H15 KD leg muscle Fiber I showing conversion to a grid-like mitochondrial network. q , r H15 KD leg muscle fibers II and III show grid-like structures similar to their wild type counterparts (Scale Bars: 5 μm for all). s Quantification of mitochondrial network orientation. Dotted line represents parallel equal to perpendicular. mito-gfp;mito-mcherry;mef2-Gal4 used as wildtype, WT; indirect flight muscles, IFM; Jump muscles, TDT. (WT-IFM, n = 9 animals; H15 KD-IFM, n = 6 animals; WT-TDT, n = 8 animals; H15 KD-TDT, n = 8 animals). t Quantification of mitochondrial network orientation in leg (walking) Fibers. (WT-leg Fibers I, n = 5 animals; H15 KD-IFM, n = 7 animals; WT-leg Fiber II, n = 9 animals; H15 KD-Leg Fiber II, n = 5 animals; WT-Leg Fiber III, n = 6 animals, H15 KD-Leg Fiber III, n = 5 animals). Each point represents value for each dataset. Bars represent mean ± SD. Significance determined as p < 0.05 from one way ANOVA with Tukey’s (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, non-significant).

Article Snippet: Three-dimensional rendering of mitochondria networks was performed using IMARIS 9.7.0 ( http://www.bitplane.com/imaris/imaris ).

Techniques: Muscles, Staining

Journal: Nature Communications

Article Title: Identification of evolutionarily conserved regulators of muscle mitochondrial network organization

doi: 10.1038/s41467-022-34445-9

Figure Lengend Snippet: a , b , c Fibrillar flight muscles (IFMs) stained for F-actin (phTRITC) and mitochondria (MitoTracker) showing parallel aligned mitochondria between myofibrils. d , e , f salm KD ( UAS-salm RNAi;UAS-mito-gfp;mef2) shows fibrillar muscles switched to tubular muscle type and mitochondria (mito-gfp) converted to grid-like networks. g , h , i salm KD; cut KD shows fibrillar fiber type and parallel mitochondrial networks (MitoTracker) in IFMs similar to wildtype. j , k , l cut -OE shows tubular fiber type and grid-like mitochondrial networks (MitoTracker) in IFMs similar to salm KD ( UAS-salm RNAi;UAS-mito-gfp;mef2) (Scale Bars: 5 μm for all). m Quantification of mitochondrial network orientation. Dotted line represents parallel equal to perpendicular (WT IFM, n = 3 animals; salm KD( UAS-salm RNAi;UAS-mito-gfp;mef2) IFM, n = 3 animals; salm KD; cut KD IFM, n = 3 animals ; cut-OE, n = 4 animals). n Wildtype fibrillar IFM stained for nuclei (DAPI), and salm antibody showing salm expression in the nuclei. o salm KD IFM showing decreased salm expression. p cut KD IFM and q salm KD; cut KD showing restored salm expression in the nuclei. r cut -OE showing absence of salm expression in the nuclei (Scale Bars: 5 μm for all). s Quantification of salm transcript levels (WT IFM, n = 5; salm KD ( UAS-salm RNAi;UAS-mito-gfp;mef2) IFM, n = 5; cut KD, n = 3; salm KD; cut KD IFM, n = 3 ). t Quantification(qPCR) of cut , salm, and H15 transcript levels. Each point represents value for each dataset. Bars represent mean ± SD. Significance determined as p < 0.05 from one way ANOVA with Tukey’s (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, non-significant).

Article Snippet: Three-dimensional rendering of mitochondria networks was performed using IMARIS 9.7.0 ( http://www.bitplane.com/imaris/imaris ).

Techniques: Muscles, Staining, Expressing

Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Expression levels of cardiac markers and cardiac transcription factors in LAP2a CRISPR clones after 7 days in differentiation medium . ( A ) Whole cell protein extracts were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (Prolif.) or incubated for 7 days in differentiation medium (Diff.) and analysed by western blot. The latter were revealed either with mouse anti Actin alpha 1 cardiac muscle Ab and successively with mouse anti cardiac troponin T monoclonal Ab, or with mouse anti Myosin-2 Ab. Red Ponceau staining prior to incubation with antibodies is also shown. ( B ) The graphs depict the relative protein amount (mean ± s.e.m normalized to Red Ponceau) for a1 cardiac Actin, cardiac troponin T2 (TNNT2) and Myosin-2 in cells grown in proliferation (P) or differentiation (D) medium. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). The graphs present the individual values and means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 or 5 independent differentiation experiments for the LAP2a -/- clone). * p<0.05, ** p<0.01 (Mann Whitney test). ( C ) The graphs depict relative mRNA levels normalized to Hmbs for Actc1 , Tnnt2 and Myh7 in cells grown in proliferation (P) or differentiation (D) medium, as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 or 5 independent differentiation experiments for the LAP2a -/- clone). ** p<0.01, *** p<0.001 (Mann Whitney test). ( D ) The graphs depict relative mRNA levels normalized to Hmbs for Gata4 , Mef2c and P300 in cells grown in proliferation (P) or differentiation (D) medium, as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 3 independent differentiation experiments for each WT +/+ clone, N = 1 or 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 independent differentiation experiments for the LAP2a -/- clone). * p<0.05, ** p<0.01, *** p<0.001 (Mann Whitney test).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Expressing, CRISPR, Clone Assay, Incubation, Western Blot, Staining, MANN-WHITNEY, Quantitative RT-PCR

Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Changes in LAP2a and LAP2b expression levels in vitro upon cardiac differentiation. ( A ) mRNAs were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (P) or incubated for 7 days in differentiation medium (D). Their relative amount was evaluated by RT-qPCR using specific primers (see Materials and Methods). The graphs depict relative mRNA amount (normalized to Hmbs ) for Lap2a and Lap2b. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). For each analysis, samples of LAP2a +/+ in proliferation (green circle) were used to arbitrarily define a reference value equal to 1.00. Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 3 or 4 independent differentiation experiments for the LAP2a -/- clone). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 (Mann Whitney test). ( B ) Whole cell protein extracts were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (Prolif., P) or incubated for 7 days in differentiation medium (Diff., D) and analysed by western blot. The latter were revealed with the rabbit anti TMPO Ab to detect LAP2a (alpha), LAP2b (beta) and a short LAP2b-like isoform (bsh). ( C-E ) The graphs depict the relative protein amount (i.e. mean ECL signal intensity (a.u) ± s.e.m normalized to Red ponceau) for LAP2a, LAP2 b, LAP2a:LAP2b, and LAP2bsh as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). For each analysis, samples of LAP2a +/+ in proliferation were used to arbitrarily define a reference value equal to 1.00 (green circle). The graphs present the individual values and means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 3 to 5 independent differentiation experiments for the LAP2a -/- clone). * p<0.05; ** p<0.01; *** p<0.001 (Mann Whitney test).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Expressing, In Vitro, CRISPR, Clone Assay, Incubation, Quantitative RT-PCR, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 1. IgLON expressions in murine C2C12 myoblasts and primary MSCs during myogenic differentiation, and IgLON4 protein expression in regenerating mouse muscle: (A) mRNA and protein expressions of IgLONs (IgLON1 to 5) in C2C12 myoblasts on differentiation day 2 (DD2) as determined by real-time RT-PCR, and protein expressions of IgLON4 and 5 on DD2 as determined by Western blot. Locations of IgLON4 protein were determined by immunocytochemistry. (B) mRNA expressions of IgLONs in mouse primary MSCs on DD2 as determined by real-time RT-PCR, and protein expressions of IgLON4 and 5 as determined by Western blot analysis. (C) Expressions of IgLON4 in control and CTX-injected mouse gastrocnemius muscles during regeneration (differentia- tion days 3 and 7), as determined by immunofluorescence; muscle was collected and embedded with paraffin and then stained with IgLON4 and laminin (Green: IgLON4, Red: Laminin, Blue: Nucleus). (D) Expressions of IgLON4 in control and CTX-injected mouse gastrocnemius muscles during regen- eration (differentiation day 7), as determined by H&E staining and immunohistochemistry. Protein expressions of PAX7 and IgLON4 were determined by Western blot analysis. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SD (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Control, Injection, Muscles, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 2. Inhibitory effect of IgLON4 gene and protein expression on C2C12 myoblast differentiation: (A) mRNA and protein expressions of IgLON4 as determined by real-time RT-PCR and Western blot analysis in Wt and IgLON4kd C2C12 myoblasts on differentiation days (DDs) 2, 4, and 6. mRNA levels on DDs 2, 4 and 6 were normalized versus Wt levels. (B) Expression of MYH by immunocytochemistry in Wt and IgLON4kd C2C12 myoblasts on DD4, and IgLON4kd fusion indices. (C) mRNA and protein levels of MYOD, IgLON5, MYOG, and MYH as determined by real-time RT-PCR and Western blot analysis in Wt and IgLON4kd cells on DDs 2, 4, and 6. mRNA and protein levels were compared at each time point. (D) Morphologies of cells treated with or without IgLON4 antibody on DD2 or 4. mRNA and protein expressions of IgLON4, IgLON5, MYOD, MYOG, and MYH in cells treated with or without IgLON4 antibody on DDs 2 or 4. mRNA and protein levels at each time-point were compared. Wt indicates transfection with scrambled vector. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SDs (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunocytochemistry, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 3. Differentiation, adhesion, and proliferation of Wt, IgLON4kd, IgLON5kd, and Dbkd C2C12 myoblasts: (A) The mRNA and protein expressions of IgLON4 and IgLON5 in Wt and Dbkd C2C12 myoblasts on differentiation day 2 (DD2) as determined by real-time RT-PCR and Western blot. (B) mRNA and protein expressions of MYOD, MYOG, and MYH in Wt and Dbkd cells on DDs 2, 4, or 6 as determined by real-time RT-PCR and Western blot analysis. mRNA and protein levels were compared at each time point. (C) Wt and Dbkd C2C12 myoblast morphologies, MYH expressions (as determined by immunocytochemistry), and fusion indices of Dbkd cells on DD4. (D,E) MTS assays

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Quantitative RT-PCR, Western Blot, Immunocytochemistry

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 4. IgLON4 protein expression and myotube orientation in Wt and IgLON4kd C2C12 myoblasts during differentiation: (A) IgLON4 protein locations were determined by immunocytochemistry on differentiation days (DDs) 2 and 4. Phalloidin and IgLON4 were fluorescently labeled green and red, respectively. Phalloidin was used for counterstaining cytoskeletal actin filaments. (B) Myotube for- mation was observed by MYH immunocytochemistry in Wt and IgLON4kd cells on DD4. Directional analysis of myotube formation by Wt and IgLON4kd cells was performed using ImageJ on DD4. Wt indicates transfection with a scrambled vector.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Expressing, Immunocytochemistry, Labeling, Transfection, Plasmid Preparation

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 5. Locations of IgLON4 protein and lipid rafts on membranes, and the inhibitory effects of IgLON4 mRNA and protein on lipid raft during myoblast differentiation: (A) Locations of IgLON4 protein and lipid rafts as determined by immunocytochemistry on DD2. (B) Locations of IgLON5, NCAM, and CDH15 proteins and lipid rafts as determined by immunocytochemistry on DD2. (C) mRNA and protein expressions of NCAM, CDH15, WASP, CAV1, CAV2, CAV3, and FLOT1 in Wt and IgLON4kd C2C12 myoblasts on DD2 as determined by real-time RT-PCR and Western blot analysis. (D) Extraction of lipid rafts from Wt and IgLON4kd C2C12 myoblasts on DD2, and the protein expressions of IgLON4, IgLON5, CDH15, and FLOT1 by Western blot analysis. FLOT1 and β-actin were used as markers of lipid rafts and total cell lysates, respectively. (E) Locations of IgLON4 protein and lipid rafts in C2C12 myoblasts treated with or without IgLON4 antibody as determined by immunocytochemistry on DD2. Wt indicates transfection with a scrambled vector. Lipid rafts were labeled green using cholera toxin and IgLON4, IgLON5, NCAM, and CDH15 were labeled red using Alexa Fluor 594. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SDs (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Immunocytochemistry, Quantitative RT-PCR, Western Blot, Extraction, Transfection, Plasmid Preparation, Labeling, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 6. Effect of using striped culture plates on C2C12 myoblasts differentiation: (A) The formation of myotubes in normal and striped plate cultures was observed by MYH immunocytochemistry. (B) mRNA and protein expressions of myogenic markers, muscle-specific adhesion proteins, and IgLON family members in C2C12 myoblasts cultured in normal and striped plates on DD2 and 4 as determined by real-time RT-PCR and Western blot analysis. (C) Location of IgLON4 protein as determined by immunocytochemistry on DD2 in C2C12 myoblasts cultured on striped plates. Labeling was performed using fluorescently labeled phalloidin and IgLON4 (green and red, respec- tively). Phalloidin was used to counterstain cytoskeletal actin filaments. (D) Locations of lipid rafts and IgLON4 protein as determined by immunocytochemistry on DD2. Lipid rafts were stained using labeled cholera toxin (green fluorescence) and IgLON4 with Alexa Fluor 594 (red fluorescence). (E) Morphologies of C2C12 myoblasts treated with or without IgLON4 antibody on DD2. Real-time PCR results were normalized to each control (con, before differentiation) and analyzed by t-test. Means ± SD (n ≥3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Immunocytochemistry, Cell Culture, Quantitative RT-PCR, Western Blot, Labeling, Staining, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: IgLON4 Regulates Myogenesis via Promoting Cell Adhesion and Maintaining Myotube Orientation.

doi: 10.3390/cells11203265

Figure Lengend Snippet: Figure 7. The proposed role of IgLON4 during myoblast differentiation. (A) Schematic structure of IgLON4, its membrane location, and its association with lipid raft accumulation during my- oblast differentiation (B) Illustrations of the hypothetical structure of IgLON4-blocked muscle and normal muscle.

Article Snippet: Sections were then incubated with IgLON4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and laminin antibodies (1:50) at 4 ◦C overnight and then with secondary antibody (1:100) from the Alexa Fluor 594 goat mouse antibody SFX kit.

Techniques: Membrane