Journal: The Journal of Clinical Investigation

Article Title: Lysyl hydroxylase 2 glucosylates collagen VI to drive lung cancer progression

doi: 10.1172/JCI189197

Figure Lengend Snippet: ( A ) Kaplan-Meier survival analysis of patients with LUAD based on Col6a3 mRNA levels above (high) or below (low) the median value using GENT2 ( http://gent2.appex.kr/gent2/ ) database. ( B ) Primary orthotopic lung tumors (left dot plot) and metastases to mediastinal nodes and contralateral lung (right dot plot) in syngeneic, immunocompetent mice injected with shRNA-transfected 344SQ cells. Control shRNA, shctrl. Col6a3 shRNA, shcol6a3. ( C ) Western blot (WB) confirmation that a CRISPR-mediated targeting approach activates endogenous Col6a3 expression in H358 cells. CRISPR-activated Col6a3 (CA-Col6a3) mass populations (numbers 1 and 2). Control transfectants (Ctl) were transfected with a vector in which guide RNAs were replaced with 20 noncoding nucleotides. ( D ) Flank tumor weights. Nu/nu mice injected with cells in C . ( E ) Numbers of orthotopic lung tumors (left dot plot) and metastases to mediastinal nodes and contralateral lung (right dot plot). Nu/nu mice injected with cells in C . ( F – K ) WST-1 proliferation assays ( F and G ) and Boyden chamber assays of migratory ( H and I ) and invasive ( J and K ) activities. For proliferation assays, mean values were calculated from replicate wells (dots). For Boyden chamber assays, mean values were calculated from multiple fields (dots) from replicate wells. P value was analyzed using Student’s 2-tailed t test ( D and E ) or 1-way ANOVA ( B and H – K ) or 2-way ANOVA ( F and G ).

Article Snippet: The human COL6A3 CRISPR activation plasmid (sc-402899-ACT) and Control CRISPR Activation Plasmid (sc-437275) were purchased from Santa Cruz.

Techniques: Injection, shRNA, Transfection, Control, Western Blot, CRISPR, Expressing, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: Lysyl hydroxylase 2 glucosylates collagen VI to drive lung cancer progression

doi: 10.1172/JCI189197

Figure Lengend Snippet: ( A ) IP/WB analysis of H157 cells transfected with Flag-tagged LH2. Col6a3 coimmunoprecipitated with HA-tagged LH2. The LH2 chaperone FKBP65 included as a positive control. Total cell lysates (input). ( B ) In vitro GGT activity assay on purified LH2 reacted with PGGHG-pretreated Col6 as substrate. Each dot represents a replicate reaction. ( C and D ) Boyden chamber migration assays. 344P cells were seeded on membranes coated with Col6, PGGHG-pretreated Col6 (deglu-Col6), or BSA. Migrated cells were imaged ( C ) and quantified ( D ). ( E ) Immunocytochemical detection of activated ITGβ1 (arrows) in 344SQ cells. Scale bar 50 μm. ( F ) Quantification of activated ITGβ1 per cell (dot). ( G ) WB analysis of 344SQ cells. Densitometric quantification of p-FAK normalized based on total FAK (values under gel). ( H ) Boyden chamber migration assay on 344SQ cells treated with ITGα2 inhibitor TC-I 15 in the presence of soluble Col6 or BSA. ( I ) Scratch wound closure rates for control (NC) and CA-Col6a3 H358 cells in the presence or absence of TC-I 15. ( J and K ) Boyden chamber migration assays on 344SQ cells treated with neutralizing antibodies against ITGβ1 ( J ) or ITGα2 ( K ) followed by soluble Col6 or BSA. ( L and M ) Boyden chamber migration assays on shITGβ1- ( L ) or siITGα2- ( M ) transfected 344SQ cells treated with soluble Col6 or BSA. ( N ) Quantification of activated ITGβ1 per cell (dot). 344SQ cells were seeded on surfaces coated with BSA, Col6, or PGGHG-treated Col6 (deglu-Col6) and immunostained with anti-activated ITGβ1 antibody. ( O ) Solid phase binding assays on purified ITGα2/β1 heterodimers incubated with PGGHG-treated (deglu-Col6) or untreated Col6. P values were determined using 1-way ANOVA ( B , D , F , and H – N ) or 2-way ANOVA ( O ).

Article Snippet: The human COL6A3 CRISPR activation plasmid (sc-402899-ACT) and Control CRISPR Activation Plasmid (sc-437275) were purchased from Santa Cruz.

Techniques: Transfection, Positive Control, In Vitro, GGT Activity Assay, Purification, Migration, Control, Binding Assay, Incubation

Journal: The Journal of Clinical Investigation

Article Title: Lysyl hydroxylase 2 glucosylates collagen VI to drive lung cancer progression

doi: 10.1172/JCI189197

Figure Lengend Snippet: ( A ) Abundance of glucosylated Col6a3 K2049 peptides in parental (P) and LH2-WA tumor samples (dots). Values normalized to total Col6a3. ( B ) Numbers of orthotopic lung tumors (left dot plot) and metastases to mediastinal nodes (middle dot plot) and contralateral lung (right dot plot). Syngeneic, immunocompetent mice were injected intrathoracically with parental or CRISPR/Cas-9-edited 344SQ cells that bear homozygous Col6a3 K2049R mutations (Col6-KR). ( C ) Flank tumor weights (left dot plot) and numbers of metastases (right dot plot) in syngeneic, immunocompetent mice injected with parental or Col6-KR 344SQ cells. ( D and E ) Boyden chamber migration ( D ) and invasion ( E ) assays on parental and Col6-KR 344SQ cells. ( F ) Boyden chamber migration assays on parental or Col6-KR 344SQ cells that have ectopic expression of LH2 or empty vector (vector). ( G and H ) Numbers of orthotopic lung tumors ( G ) and metastases to mediastinal nodes and contralateral lung ( H ) in syngeneic, immunocompetent mice. P values were determined using 1-way ANOVA. **** P < 0.0001.

Article Snippet: The human COL6A3 CRISPR activation plasmid (sc-402899-ACT) and Control CRISPR Activation Plasmid (sc-437275) were purchased from Santa Cruz.

Techniques: Injection, CRISPR, Migration, Expressing, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: Lysyl hydroxylase 2 glucosylates collagen VI to drive lung cancer progression

doi: 10.1172/JCI189197

Figure Lengend Snippet: ( A ) Areas of FAs (dots) in anti-p-paxillin–stained parental (P) and Col6-KR 344SQ cells (n ≥ 50 per group). ( B ) Representative TIRF micrograph of mCherry-paxillin-transfected 344SQ cells to detect FAs (arrows), Original magnification, ×60. ( C ) Disassembly T 1/2 of FAs (dots) in Col6-KR 344SQ cells determined based on 60 minute time-lapse sequences. ( D ) Areas of FAs (dots) in anti-p-paxillin–stained parental or LH2-WA 344SQ cells (n ≥ 50 per group). ( E ) Disassembly T 1/2 of FAs (dots) in parental and LH2-WA 344SQ cells determined based on 60 minute time-lapse sequences. ( F ) Quantification of activated ITGβ1 per cell (dot) by immunocytochemical analysis with antiactivated ITGβ1 antibody. ( G ) WB analysis of phospho- and total FAK in parental and Col6-KR 344SQ cells. Densitometric values normalized based on total FAK are under gel. ( H ) IP/WB analysis of Col6a3/ITGα2. WB analysis to detect Col6a3 was carried out on anti-ITGα2–immunoprecipitated proteins isolated from parental or Col6-KR 344SQ cells. Relative densitometric values are under gels. Total cell lysates (input). ( I ) Schematic illustration of findings. P values were determined using 1-way ANOVA ( A , D , and F ) or Student’s 2-tailed t test ( C and E ). **** P < 0.0001.

Article Snippet: The human COL6A3 CRISPR activation plasmid (sc-402899-ACT) and Control CRISPR Activation Plasmid (sc-437275) were purchased from Santa Cruz.

Techniques: Staining, Transfection, Immunoprecipitation, Isolation

Journal: Nature Communications

Article Title: Characterization of a pluripotent stem cell-derived matrix with powerful osteoregenerative capabilities

doi: 10.1038/s41467-020-16646-2

Figure Lengend Snippet: a Lysates of hMSCs harboring constructs expressing scrambled siRNA (scr), siRNA directed against collagen type VIα3 transcript (KD6), or collagen type XIIα1 transcript (KD12) were blotted and probed with antibodies against type VI collagen (Col VI) or type XII collagen (Col XII). Blots were performed independently three times. b Transcription of collagen VIα1, VIα3, and XIIαl in KD6 hMSCs measured by qRT-PCR. Results expressed as fold change compared to scr hMSCs. nd = signal not detected. c As panel b , but for KD12 hMSCs. d Growth curves of hMSCs over 15 days after an original seeding of 100 cells per cm 2 . e Single cell-derived colonies generated per 100 plated cells. f Average colony area. g Monolayer mineralization determined by ARS staining for calcium deposits after 7–21 days of exposure to basal osteogenic media supplemented with dexamethasone. Entire wells (left in each data set) or micrographs (right in each data set) are presented (bar = 100 μm). h Quantification of ARS determined by extraction and spectrophotometric quantification. i ALP activity (above) of intact monolayers after 8 days of exposure to basal osteogenic media. Rate of OPG secretion (below) after 4 days of exposure to basal osteogenic media. j KD6, KD12, or scrambled hMSCs were probed with anti-collagen VI (red) and anti-collagen XII (green) and stained for nuclei with DAPI (blue). Intercellular contacts harboring yellow punctae of co-localized collagen VI and XII (closed arrows) are detectable in scrambled cultures but not in the case of KD6 or KD12 even though contacts are evident (open arrows). In KD12 cells, punctae of collagen VI are randomly distributed throughout cell bodies. In the case of the scrambled micrograph, orthogonal images are provided. Representative imaging from three independent experiments. Bar = 20 μm. Statistics: Data are presented with means and SD (error bars) and analyzed using one-way ANOVA with Tukeyʼs or Dunnettʼs post-test. * p < 0.05, ** p < 0.01, *** p < 0.005. Panels b – f and h , n = 3, for panel i n = 6, or n = 12 for scr. Source data are provided as a Source Data file.

Article Snippet: Cells were transduced at a multiplicity of infection of 10 with lentiviral particles expressing shRNA directed against the collagen VIα3 transcript (sc-94560-V, Santa Cruz), the collagen XIIα1 transcript (sc-72958-V, Santa Cruz) or non-targeting (scrambled) control shRNA (sc-108080, Santa Cruz) for 24 h in the presence of 9 μg mL −1 polybrene (Sigma).

Techniques: Construct, Expressing, Quantitative RT-PCR, Derivative Assay, Generated, Staining, Extraction, Activity Assay, Imaging

Journal: Animal Models and Experimental Medicine

Article Title: Development, validation, and preliminary phenotypic characterization of a Col6a3 knockout mouse model targeting exon 3

doi: 10.1002/ame2.70063

Figure Lengend Snippet: Col6a3 d3/d3 mice design and validation. (A) Schematic presenting the targeting strategy. (B) Schematic of the reading frame analysis. The upper sequence represents DNA with capital letter representing coding sequences. Alternate red and black letters designate codons and the resulting amino acid sequence is illustrated at the bottom beginning with methionine. (C) Representative genotyping PCR to detect Col6a3 wild‐type (upper) and Col6a3 knockout (lower) of DNA extracted from ear from Col6a3 d3/d3 , Col6a3 d3/+ , Col6a3 +/+ mice. (D) Sanger sequencing of DNA extracted from Col6a3 −/− mouse skin using the Col6a3 knockout primers.

Article Snippet: Col6a3 d3/+ mouse founders were purchased from Cyagen LLC.

Techniques: Biomarker Discovery, Sequencing, Knock-Out

Journal: Animal Models and Experimental Medicine

Article Title: Development, validation, and preliminary phenotypic characterization of a Col6a3 knockout mouse model targeting exon 3

doi: 10.1002/ame2.70063

Figure Lengend Snippet: Impact of the Col6a3 exon 3 deletion on the resulting mRNA transcripts. (A) Relative gene expression using primers within (exon 3), upstream (exon 1) and downstream (exon 9) of the CRISPR‐deleted sequence in Col6a3 d3/d3 and Col6a3 +/+ mice. The data are expressed as mean ± the standard deviation, n = 4. Student's t‐test was used to measure statistical significance (** p ≤ 0.01). ns = non‐significant. (B) Sanger sequencing of cDNA from Col6a3 d3/d3 mouse skin using the Col6a3 knockout primers.

Article Snippet: Col6a3 d3/+ mouse founders were purchased from Cyagen LLC.

Techniques: Gene Expression, CRISPR, Sequencing, Standard Deviation, Knock-Out

Journal: Animal Models and Experimental Medicine

Article Title: Development, validation, and preliminary phenotypic characterization of a Col6a3 knockout mouse model targeting exon 3

doi: 10.1002/ame2.70063

Figure Lengend Snippet: Col6a3 d3/d3 mice display a body size decrease phenotype. (A and B) Gross representative picture of Col6a3 d3/d3 , Col6a3 d3/+ , Col6a3 +/+ male (A) and female (B) mice at 24 weeks. (C and D) Line chart of the body weight of Col6a3 d3/d3 , Col6a3 d3/+ , Col6a3 +/+ of male (C) and female (D) mice from 3 to 24 weeks. One‐way ANOVA (Kruskal‐Wallis) test, followed by Dunn's multiple comparison for the post hoc analysis, was used to compare the difference in weight between the groups. *Significant difference between Col6a3 d3/d3 and Col6a3 +/+ ( p < 0.05). +Significant difference between Col6a3 d3/d3 and Col6a3 d3/+ ( p < 0.05). (E and F) Bar graph of the food intake per day per animal across Col6a3 d3/d3 , Col6a3 d3/+ , Col6a3 +/+ mice genotype. One‐way ANOVA (Kruskal‐Wallis) test, followed by Dunn's multiple comparison for the post hoc analysis, was used to measure significance between the groups. *Significant difference ( p < 0.05) between female Col6a3 d3/+ and Col6a3 d3/d3 .

Article Snippet: Col6a3 d3/+ mouse founders were purchased from Cyagen LLC.

Techniques: Comparison

Journal: Animal Models and Experimental Medicine

Article Title: Development, validation, and preliminary phenotypic characterization of a Col6a3 knockout mouse model targeting exon 3

doi: 10.1002/ame2.70063

Figure Lengend Snippet: Analysis of the fat, muscle and bone percentage in Col6a3 d3/d3 mice. (A) Representative non‐segmented (left) and segmented images highlighting muscle (green), fat (blue) and bone (red) tissues. (B) Bar graph representing the muscle: Body weight ratio (left), fat: Body weight ratio (middle), and bone: Body weight ratio (right) of 6‐month‐old male Col6a3 d3/d3 ( n = 5), Col6a3 d3/+ ( n = 5), and Col6a3 +/+ ( n = 5) mice. One‐way ANOVA was used to measure statistical significance, but none was observed.

Article Snippet: Col6a3 d3/+ mouse founders were purchased from Cyagen LLC.

Techniques:

Journal: Animal Models and Experimental Medicine

Article Title: Development, validation, and preliminary phenotypic characterization of a Col6a3 knockout mouse model targeting exon 3

doi: 10.1002/ame2.70063

Figure Lengend Snippet: Histological analysis of quadriceps muscles of Col6a3 d3/d3 mice. (A) Representative H&E of quadriceps muscles from Col6a3 d3/d3 , Col6a3 d3/+ and Col6a3 +/+ . Arrows point to internally nucleated muscle cells. Scale bar = 100 μm. (B‐D) Bar graphs representing the average muscle fiber diameter regardless of the internalized or peripheral location of nuclei (B), percentage of the internally nucleated muscle cells (C) and average muscle fiber diameter of the internalized and peripheral nuclei in Col6a3 d3/d3 (D). The data are expressed as mean ± SD; n = 4. The data were analyzed with one way ANOVA (Kruskal‐Wallis) test, followed by Dunn's multiple comparison for the post hoc analysis. *** p ≤ 0.001, ns = non‐significant. Each dot represents the value from one biological replicate (one mouse).

Article Snippet: Col6a3 d3/+ mouse founders were purchased from Cyagen LLC.

Techniques: Muscles, Comparison

Journal: Animal Models and Experimental Medicine

Article Title: Development, validation, and preliminary phenotypic characterization of a Col6a3 knockout mouse model targeting exon 3

doi: 10.1002/ame2.70063

Figure Lengend Snippet: Histological analysis of peripheral nerves of Col6a3 d3/d3 mice. (A) Representative H&E of sciatic nerves from Col6a3 d3/d3 , Col6a3 d3/+ and Col6a3 +/+ . Scale bar = 100 μm. (B) Bar graph representing the sciatic nerve diameter across genotypes Col6a3 d3/d3 , Col6a3 d3/+ and Col6a3 +/+ . (C) Representative immunohistochemistry results using anti‐myelin associated glycoprotein (MAG) (upper) and anti‐myelin binding protein (MBP) (lower) in sciatic nerves from Col6a3 d3/d3 , Col6a3 d3/+ and Col6a3 +/+ . Scale bar = 250 μm. One‐way ANOVA was used to measure statistical significance, but none was observed.

Article Snippet: Col6a3 d3/+ mouse founders were purchased from Cyagen LLC.

Techniques: Immunohistochemistry, Binding Assay

Journal: Matrix Biology

Article Title: Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

doi: 10.1016/j.matbio.2011.12.003

Figure Lengend Snippet: Immunofluorescence analysis of collagen VI α3 and α 6 chains in normal muscle cultures. a: The collagen VI α3 chain (red fluorescence) is detected in the extracellular matrix of both proliferating and long-term (after ascorbic acid treatment for 7 days post-confluence) samples, however the arrangement of the collagen VI network appears better organized in long term cultures. Bar, 100 μm. Nuclei were counterstained with DAPI (blue). b: Collagen VI α6 chain (red fluorescence) deposits are detectable in the extracellular matrix of muscle cultures after ascorbic acid treatment for 7 days post-confluence. α6-containing structures (arrows, left upper panel) show a filamentous arrangement, as detected at higher magnification (right upper panel). Lower panels show double labeling with antibodies against the anti-α6 (red fluorescence) and α3 (green fluorescence) chains. Filamentous α6 chain-containing structures (red) co-localize with α3 chain-containing positive microfilaments, as revealed by the merged image (lower, right panel), however, an independent α3 chain-containing network (green fluorescence) can be also detected. Bar, 20 μm. Nuclei were counterstained with DAPI (blue). c: Real Time-PCR analysis of COL6A3 and COL6A6 transcripts. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts. The relative ratios of COL6A3/ Actin-GAPDH and COL6A6 /Actin-GAPDH mRNAs in proliferating cells were normalized to 1. Both COL6A3 and COL6A6 transcript levels were increased in long-term cells (1.7:1 for COL6A6 and 2.9:1 for COL6A3). d: Immunofluorescence analysis of collagen VI α3 and α6 chains in long-term normal muscle cell cultures in the absence of ascorbic acid treatment. Samples were double-labeled for desmin (upper panels, green fluorescence) and for α-smooth muscle actin (α-SMA; lower panels, green fluorescence). The collagen VI α3 chain (red fluorescence, left, upper panel) appears to be absent in desmin-positive cells and accumulates in desmin-negative cells, indicating that the fibroblasts are the main source of α3 chain. Similarly, the α6 chain (red, right, upper panel) is absent in desmin-positive, but different from the α3 chain, it is detected intracellularly only in a subset of desmin-negative cells. Cell producing collagen VI α6 chain (red) are positive for α-smooth muscle actin (green, right, lower panel), indicating that they could be myofibroblasts. Bar, 20 μm. Nuclei were counterstained with DAPI (blue).

Article Snippet: In order to quantify the steady state level of COL6A5 and COL6A6 transcripts, commercially available TaqMan expression assays (Applied Biosystems) were used for target genes ( COL6A3 : Hs00915102_m1 Ex 23–24; COL6A5 (COL29A1) : Hs00542046_m1 exons 35–36; COL6A6 : Hs01029204_m1 exons 12–13) and for β actin and GAPDH as housekeeping reference genes (ACTB Endogenous Control- GAPDH endogenous control).

Techniques: Immunofluorescence, Fluorescence, Labeling, Real-time Polymerase Chain Reaction

Journal: Matrix Biology

Article Title: Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

doi: 10.1016/j.matbio.2011.12.003

Figure Lengend Snippet: Immunofluorescence and Real Time-PCR analysis of α6 chain in normal muscle cultures after TGFβ1 treatment. a: After 10 days TGFβ1 treatment most of interstitial cells express α-SMA (left upper panel, green fluorescence) indicating trans-differentiation into a myofibroblasts-like phenotype. In the absence of ascorbic acid (right upper panel) most of α-SMA expressing cells accumulated the collagen VI α6 chain (red) intracellularly. After ascorbic acid treatment (left and right middle panels), the α6 chain (red) was abundantly secreted and arranged in a wide filamentous network in the extracellular matrix. Left and right lower panels show double labeling with antibodies against the α6 and α3 chains on samples treated with TGFβ1 for 10 days in presence of ascorbic acid. The α6 chain (red fluorescence) mainly co-localizes with the α3 chain (green) labeling. However, a distinct α3−containing network can also be detected (green fluorescence). Bar, 20 μm . Nuclei were counterstained with DAPI (blue). b : Real Time-PCR analysis. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts The relative ratios of COL6A3 /Actin-GAPDH and COL6A6 /Actin-GAPDH mRNAs in untreated cells were normalized to 1. COL6A3 and COL6A6 transcript levels are strongly reduced in cells after long-term (10 days) TGFβ1 treatment (0.48:1 for COL6A3 and 0.16:1 for COL6A6). Analysis of mRNA levels at different time points revealed constantly low transcriptional levels of COL6A6 mRNA starting from 24 hour of treatment. In contrast, an initial slight up-regulation of COL6A3 mRNA at 24–48 hours, was followed by a gradual decrease after prolonged TGFβ1 treatment.

Article Snippet: In order to quantify the steady state level of COL6A5 and COL6A6 transcripts, commercially available TaqMan expression assays (Applied Biosystems) were used for target genes ( COL6A3 : Hs00915102_m1 Ex 23–24; COL6A5 (COL29A1) : Hs00542046_m1 exons 35–36; COL6A6 : Hs01029204_m1 exons 12–13) and for β actin and GAPDH as housekeeping reference genes (ACTB Endogenous Control- GAPDH endogenous control).

Techniques: Immunofluorescence, Real-time Polymerase Chain Reaction, Fluorescence, Expressing, Labeling

Journal: Matrix Biology

Article Title: Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

doi: 10.1016/j.matbio.2011.12.003

Figure Lengend Snippet: Immunofluorescence analysis of α6 chain in DMD patients muscle biopsies. a: Immunofluorescence analysis of the collagen VI α6 chain (red) and the laminin γ1 chain (green) in muscle sections of three patients affected by Duchenne muscular dystrophy (DMD815, DMD1, DMD23). α6 chain labeling appears increased in the endomysium and perimysium, with its increase correlating with the degree of fibrosis. The α6 chain localization is strictly interstitial, as demonstrated by absence of co-localization with the laminin γ1 chain (right panels, green fluorescence),a marker for basal lamina. Bar, 100 μm. b: Immunofluorescence analysis of the collagen VI α3 chain in DMD815 muscle sections revealed a marked increase in fibrotic ares (red fluorescence, left panel). Double labeling with antibodies against –the laminin γ1 chain (green) reveals the presence of α3 chain at the basal lamina of muscle fibers (yellow fluorescence in merge image, right panel). c: High magnification of a muscle section from patient DMD815 double labeled with antibodies against collagen VI α6 (red fluorescence) and -α3 (green fluorescence) chains showing the presence of undulated/wavy bundles of fibrils of different size containing both the α6 and α3 chains. In the basement membrane of a muscle fiber, only the α3 chain is found (arrows). Bar, 20 μm. Nuclei were counterstained with DAPI (blue).

Article Snippet: In order to quantify the steady state level of COL6A5 and COL6A6 transcripts, commercially available TaqMan expression assays (Applied Biosystems) were used for target genes ( COL6A3 : Hs00915102_m1 Ex 23–24; COL6A5 (COL29A1) : Hs00542046_m1 exons 35–36; COL6A6 : Hs01029204_m1 exons 12–13) and for β actin and GAPDH as housekeeping reference genes (ACTB Endogenous Control- GAPDH endogenous control).

Techniques: Immunofluorescence, Labeling, Fluorescence, Marker, Membrane

Journal: Matrix Biology

Article Title: Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis

doi: 10.1016/j.matbio.2011.12.003

Figure Lengend Snippet: Western blot and RT-PCR analysis of collagen VI chains in muscle biopsies of DMD patients. a: Western blot analysis of skeletal muscles extracts from a healthy control (CTRL1,2) and from DMD815, DMD1112 and DMD1179 patients. Proteins were separated by SDS-PAGE under reducing conditions and detected with polyclonal antibodies specific for the collagen VI α6 and α3 and α1 chains; actin was used as loading control. b: Real Time-PCR analysis of COL6A6 transcripts in DMD skeletal muscle biopsies. Reported values are the mean of two different experiments utilizing ACTB and GAPDH as reference transcripts. The relative ratios of COL6A3 /Actin-GAPDH and COL6A6 /Actin-GAPDH mRNAs in control muscle were normalized to 1. The increased amount of collagen VI α3 and α6 chains in DMD muscles matched with an increase of the corresponding mRNAs. The COL6A6 transcript level showed a fold change that ranged from 10 to 14 in the three analyzed samples whereas the variation in the COL6A3 mRNA level was lower (2–4 fold change).

Article Snippet: In order to quantify the steady state level of COL6A5 and COL6A6 transcripts, commercially available TaqMan expression assays (Applied Biosystems) were used for target genes ( COL6A3 : Hs00915102_m1 Ex 23–24; COL6A5 (COL29A1) : Hs00542046_m1 exons 35–36; COL6A6 : Hs01029204_m1 exons 12–13) and for β actin and GAPDH as housekeeping reference genes (ACTB Endogenous Control- GAPDH endogenous control).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Muscles, Control, SDS Page, Real-time Polymerase Chain Reaction