interleukin 34 (il34)  (PeproTech)

Journal: Theranostics

Article Title: Inactivating IL34 promotes regenerating muscle stem cell expansion and attenuates Duchenne muscular dystrophy in mouse models

doi: 10.7150/thno.83817

Figure Lengend Snippet: Inactivation of IL34 hindered long-term skeletal muscle regeneration. (A) WT SCs were cultured in growth medium for 4 days and then switched to differentiation medium and cultured for 12 h, 24 h or 48 h. Immunoblot analysis of IL34 and MyHC expression in cultured WT SCs at different time points after induction of differentiation. (B) Quantitative analysis of IL34 protein levels at various time points in regenerating skeletal muscle (N=3 per group for each time point). *p<0.05. (C) Representative images of 3-day injured and 10-day injured TA muscle of WT mice costained for Pax7 and IL34, or MyoD and IL34. The nuclei were labeled using DAPI. Scale bar: 30 μm. (D) H&E staining analysis of transverse sections of uninjured and day 14 and 21 postinjury TA muscle from WT and IL34 KO mice. Scale bar: 50 μm. (E) Quantification of the average CSA of uninjured TA muscle from WT and IL34 KO mice. (F) Quantification of the average CSA of regenerating fibers (14 d.p.i.) TA muscle from WT and IL34 KO mice. N=4 mice in each group. ***p<0.001. (G) Quantification of the ratio of regenerating myofibers containing two or more centralized nuclei at day 14 post injury. ***p<0.001. (H) Quantification of the average CSA of regenerating fibers (21 d.p.i.) from WT and IL34 KO mice. N=4 mice in each group. ***p<0.001. (I) H&E staining analysis of transverse sections from day 14 postinjury of TA muscle from Ctrl and IL34 CKO mice. Scale bar: 50 μm. (J) Quantification of the fiber size of damaged TA muscle from Ctrl and IL34 CKO mice. N=4 mice in each group. ***p<0.001. (K) Quantification of the ratio of regenerating myofibers containing two or more centralized nuclei at day 14 post injury. **p<0.01.

Article Snippet: After one day in differentiation medium, the medium was supplemented with 100 ng/ml recombinant IL34 (Origene); the same volume of 0.1% BSA was used as a control.

Techniques: Cell Culture, Western Blot, Expressing, Labeling, Staining

Journal: Theranostics

Article Title: Inactivating IL34 promotes regenerating muscle stem cell expansion and attenuates Duchenne muscular dystrophy in mouse models

doi: 10.7150/thno.83817

Figure Lengend Snippet: Inactivating IL34 promotes the expansion and self-renewal of Pax7 + cells. (A) Representative images of WT and IL34-KO SC-induced differentiation after 1 day, with staining for Pax7, MyoG and DAPI. Scale bar: 30 μm. (B) Quantification of the percentages of Pax7 + MyoG - , Pax7 + MyoG + and Pax7 - MyoG + cell populations in WT and IL34-KO cultures. **p<0.01. (C) Representative overlapping images of 72 h cultured myofibers from the EDL muscle of WT and IL34-KO mice costained for Pax7, MyoG and DAPI. Scale bar: 50 μm. (D) Percentage of Pax7 + MyoG - and Pax7 - MyoG + cell populations in WT and IL34-KO myofiber-associated SCs. **p<0.01. (E) WT and IL34-KO SCs were cultured in differentiation medium for 2 days, and representative merged images of cultures were costained with MyHC, Ki67 and DAPI. Scale bar: 30 μm. (F) Measurement of the fusion index was calculated by the frequency of MyHC + cells with 2 or more nuclei relative to total MyHC + cells. **p<0.01. (G) One day differentiated WT SCs were treated with BSA or IL34 recombinants, and representative merged images of cultures were costained with MyHC and DAPI. Scale bar: 30 μm. (H) Measurement of the differentiation index was calculated by the frequency of nuclei in MyHC + cells relative to total nuclei. *p<0.05. (I) IL34-KO myoblasts induced to differentiation by supplementing with supernatant medium collected from two day differentiated WT or IL34-KO SCs, respectively. Representative merged images of cultures were costained with MyHC, Ki67 and DAPI. Scale bar: 50 μm. (J) Measurement of the differentiation index was calculated by the frequency of nuclei in MyHC + cells relative to total nuclei. **p<0.01. (K) FACS-isolated WT and IL34-KO SCs cultured in differentiation medium for 1 day. The cells were then fixed and labeled with MyoD, Ki67 and DAPI. Representative merged photomicrographs of WT and IL34-KO cultures after labeling with MyoD, Ki67 and DAPI. Scale bars: 30 μm. (L) Quantitative analysis of the frequency of undifferentiated MyoD - Ki67 + to MyoD + Ki67 + cells in WT and IL34-KO cultures. *p<0.05. (M) Representative merged images of transverse sections on day 14 postinjury TA muscle from WT and IL34-KO mice stained for Pax7 and laminin. Scale bar: 50 μm. (N) Quantification of the number of Pax7 + cells per area in WT and IL34-KO mice. *p<0.05. (O) After experiencing a period of 14 days of recovery, the damaged muscle was subjected to a second round of injury via injection of 1.2% BaCl 2 . Representative merged photomicrographs of transverse sections of TA muscle 5 days after a second round injury immunostained for eMyHC, laminin and DAPI. Scale bar, 50 μm. (P) Quantification of the average CSA of eMyHC + fibers from WT and IL34 KO mice suffering from a second round of damage. N=3 mice in each group. *p<0.05. (Q) Single myofibers isolated from 14 day injured EDL muscle of Ctrl and IL34 CKO mice and immediately fixed to stain with DAPI. Scale bar, 50 μm. (U) Measurement of myonuclear domain between Ctrl and IL34 CKO fibers. *p<0.05.

Article Snippet: After one day in differentiation medium, the medium was supplemented with 100 ng/ml recombinant IL34 (Origene); the same volume of 0.1% BSA was used as a control.

Techniques: Staining, Cell Culture, Isolation, Labeling, Injection

Journal: Theranostics

Article Title: Inactivating IL34 promotes regenerating muscle stem cell expansion and attenuates Duchenne muscular dystrophy in mouse models

doi: 10.7150/thno.83817

Figure Lengend Snippet: IL34 represses the transcription of Igfbp5 during muscle repair. (A) Heatmaps of genes with up- and downregulated expression in differentiated SCs after inactivation of IL34. (B) Relative Igfbp5 mRNA levels in day 1 differentiated WT and IL34-KO cultures detected using RT-qPCR. *p<0.05. (C) Western blot analysis of Igfbp5 and unrelated β-tubulin in day 1 differentiated WT and IL34-KO cultures. (D) Relative Igfbp5 protein levels in day 1 differentiated WT and IL34-KO cultures. *p<0.05. (E) Experimental procedure outlining the use of stable shIgfbp5 and shScr lentivirus-infected IL34-KO SCs for cell fate determination analysis. (F) Representative merged images of stably infected IL34-KO SCs stained for Pax7, MyoG and DAPI. Scale bar: 30 μm. (G and H) Quantitative analysis of the frequency of MyoG + (G) and Pax7 + (H) cells in shScr- and shIgfbp5-infected IL34-KO cultures. **p<0.01. (I) Representative immunofluorescence analysis of MyoD, Ki67 and DAPI costaining in shScr- and shIgfbp5-infected IL34-KO cultures. Scale bar: 30 μm. (J) Quantification of the percentage of differentiated MyoD + Ki67 - cells from shScr- and shIgfbp lentivirus-treated IL34-lacking SCs. *p<0.05. (K) Representative merged photomicrographs of stably infected IL34-KO SCs stained with EdU and DAPI. Scale bar: 50 μm. (L) Proportion of EdU + cells in shIgfbp5- and shScr-treated IL34-KO SCs. *p<0.05. (M) Primary myoblasts were established from the hind limbs of WT mice and then expanded to induce differentiation for 1 day in medium containing BSA or Igfbp5 recombinant when cells were 70% confluent. Representative immunofluorescence analysis of MyoG and DAPI costaining in BSA- and recombinant Igfbp5-treated WT cultures. Scale bar: 30 μm. (N) Quantification of the percentage of differentiated MyoG cells from BSA- and recombinant Igfbp5-treated WT cultures. **p<0.01. (O) Primary WT myoblasts were cultured in differentiation medium containing BSA or Igfbp5 recombinant for 1 day. Myogenic differentiation was determined by immunostaining for MyHC. Scale bar: 30 μm. (P) Measurement of the differentiation index was determined by the frequency of nuclei in MyHC + cells. *p<0.05.

Article Snippet: After one day in differentiation medium, the medium was supplemented with 100 ng/ml recombinant IL34 (Origene); the same volume of 0.1% BSA was used as a control.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Stable Transfection, Staining, Immunofluorescence, Recombinant, Cell Culture, Immunostaining

Journal: Theranostics

Article Title: Inactivating IL34 promotes regenerating muscle stem cell expansion and attenuates Duchenne muscular dystrophy in mouse models

doi: 10.7150/thno.83817

Figure Lengend Snippet: IL34-Igfbp5 axis-mediated SC function through upregulation of PI3K-AKT activity. (A) Left panel, immunoblot analysis of relative protein levels of p-AKT, total AKT and unrelated β-tubulin in 1-day differentiated WT and IL34-KO cultures. Right panel, relative p-Akt protein levels in 1-day differentiated WT and IL34-KO cultures. ***p<0.001. (B) Left panel, western blot analysis of p-AKT, total AKT, and unrelated β-tubulin protein levels in 1-day differentiated IL34-depleted SCs stably infected with shScr or shIgfbp5 lentivirus. Right panel, relative p-Akt protein levels in 1-day differentiated IL34-depleted SCs stably infected with shScr or shIgfbp5 lentivirus. **p<0.01. (C) Left panel, western blot analysis of p-AKT, total AKT, and unrelated β-tubulin protein levels in 1-day differentiated WT SCs treated with BSA or recombinant Igfbp5. Right panel, relative p-Akt protein levels in 1-day differentiated WT SCs treated with BSA or recombinant Igfbp5. *p<0.05. (D) Left panel, western blot analysis of p-AKT, AKT and unrelated β-tubulin protein levels in proliferating myoblasts and 1-day differentiated myoblasts from WT mice. Right panel, relative p-Akt protein levels in proliferating myoblasts and 1-day differentiated myoblasts from WT mice. ***p<0.001. (E) Left panel, immunoblot analysis of the relative protein levels of p-AKT, total AKT and unrelated β-tubulin in 1-day differentiated WT SCs treated with DMSO or LY294002 for 24 h. Right panel, relative p-Akt protein levels in proliferating myoblasts and 1-day differentiated WT SCs treated with DMSO or LY294002 for 24 h. **p<0.01. (F) FACS-isolated SCs were first plated in growth medium to expand and then switched to differentiation medium plus DMSO or LY294002 when cells were 70% confluent. Representative immunofluorescence analysis of MyoG and DAPI costaining in WT SC cultures treated with DMSO or LY294002. Scale bar: 30 μm. (G) Quantification of the percentage of differentiated MyoG cells from differentiated WT SCs treated with DMSO or LY294002. **p<0.01. (H) Representative overlapping images of DMSO- or LY294002-treated differentiated WT SCs stained against MyoD, Ki67 and DAPI. Scale bar: 30 μm. (I) Quantification of the frequency of MyoD + Ki67 - cells from WT SCs in differentiation medium supplemented with DMSO or LY294002. **p<0.01. (J) Representative merged photomicrographs of Pax7-stained WT SCs cultured in differentiation medium containing DMSO or LY294002. Nuclei were labeled with DAPI. Scale bar: 30 μm. (K) Frequency of undifferentiated Pax7 + cells in WT SCs cultured in differentiation medium containing DMSO or LY294002. *p<0.05. (L) Representative immunofluorescence analysis of WT SCs cultured in differentiation medium containing DMSO or LY294002. MyHC and Ki67 were stained to visualize the differentiation index, and DNA was stained with DAPI. Scale bar: 30 μm. (M) Measurement of the differentiation index detected by calculating the percentage of nuclei in MyHC-positive cells. ***p<0.001. (N) LY294002 hindered AKT activity in skeletal muscle regeneration. The beginning of LY294002 treatment was on the 5 th day after injury via intramuscular injection, the later injection was performed every 2 days via intraperitoneal injection, and samples were collected 1 day after the fifth LY294002 treatment. (O) Representative H&E-stained transverse sections of TA muscle from WT mice treated with vector or LY294002 at 14 days postinjury. Scale bar: 50 μm. (P) Quantification of the average CSA of myofibers with centralized nuclei. ***p<0.001.

Article Snippet: After one day in differentiation medium, the medium was supplemented with 100 ng/ml recombinant IL34 (Origene); the same volume of 0.1% BSA was used as a control.

Techniques: Activity Assay, Western Blot, Stable Transfection, Infection, Recombinant, Isolation, Immunofluorescence, Staining, Cell Culture, Labeling, Injection, Plasmid Preparation

Journal: Theranostics

Article Title: Inactivating IL34 promotes regenerating muscle stem cell expansion and attenuates Duchenne muscular dystrophy in mouse models

doi: 10.7150/thno.83817

Figure Lengend Snippet: IL34 inactivation resulting in overactivated NFKB1 activity upregulates Igfbp5 transcription. (A) Schematic diagram presenting 2 potential NFKB1 binding sites in the Igfbp5 promoter. (B) Western blot analysis of NFKB1, Igfbp5 and unrelated GAPDH protein levels in 1-day differentiated WT and IL34-KO cultures. (C) Relative NFKB1 protein levels in 1-day differentiated WT and IL34-KO cultures. **p<0.01. (D) Representative immunofluorescence analysis of WT and IL34-KO primary myoblasts cultured in differentiation medium for 1 day. NFKB1 and MyoD were stained to visualize the nuclear localization signal of NFKB1, and DNA was stained with DAPI. Scale bar: 20 μm. (E) Left panel, immunoblot analysis of the protein levels of Igfbp5, p-AKT, total AKT and unrelated β-tubulin and GAPDH in 1-day differentiated IL34-KO cultures treated with DMSO or (-)-DHMEQ. Right panel, relative p-Akt protein levels in 1-day differentiated IL34-KO cultures treated with DMSO or (-)-DHMEQ. **p<0.01. (F) Representative overlapping images of 1-day differentiated IL34-KO SCs treated with DMSO or (-)-DHMEQ after labeling with MyoG and DAPI. Scale bar: 30 μm. (G) Quantification estimation of the ratio of MyoG + cells in 1-day differentiated IL34-KO SCs treated with DMSO or (-)-DHMEQ. **p<0.01. (H) Representative merged photomicrographs of 1-day differentiated IL34-KO SCs treated with DMSO or (-)-DHMEQ after staining with Pax7 and DAPI. Scale bar: 30 μm. (I) Percentage of Pax7 + cells in differentiated IL34-KO cultures treated with DMSO or (-)-DHMEQ. *p<0.05. (J) Relative luciferase activity in C2C12 cells electrotransfected with phRL-TK plasmid and pGL3-basic empty vector (pGL3 basic), pGL3-control vector (pGL3 control), wild-type Igfbp5 promoter (Igfbp5-WT), or mutant Igfbp5 promoter (Igfbp5-mut) with mutation of NFKB1 binding sites under normal conditions. ***p<0.001. (K) Isolated WT myoblasts from the adult mice were initially cultured in growth medium then induced to differentiation by switching growth medium into differentiation medium with or without Stattic. Western blot analysis of the levels of NFKB1, p-STAT3, STAT3 and an unrelated protein (GAPDH) in WT myoblasts grown in differentiation medium after the addition of 2 μM Stattic. (L) Isolated WT myoblasts from the adult mice were initially undergone expansion in growth medium and then induced to differentiation in medium with or without OSM. Western blot analysis of the levels of NFKB1, p-STAT3, STAT3 and an unrelated protein (GAPDH) in SCs grown in differentiation medium containing 50 ng/ml OSM.

Article Snippet: After one day in differentiation medium, the medium was supplemented with 100 ng/ml recombinant IL34 (Origene); the same volume of 0.1% BSA was used as a control.

Techniques: Activity Assay, Binding Assay, Western Blot, Immunofluorescence, Cell Culture, Staining, Labeling, Luciferase, Plasmid Preparation, Mutagenesis, Isolation

Journal: Theranostics

Article Title: Inactivating IL34 promotes regenerating muscle stem cell expansion and attenuates Duchenne muscular dystrophy in mouse models

doi: 10.7150/thno.83817

Figure Lengend Snippet: IL34 inactivation attenuates Duchenne muscular dystrophy in mouse models. (A) Representative overlapping images of eMyHC + EdU + cells on transverse sections of the Gas muscle from mdx and mdx::IL34 -/- mice at 8 weeks of age. Scale bar: 30 μm. (B) Quantification number of eMyHC + EdU + cells per unit field. *p<0.05. (C) Representative merged images of Pax7 and laminin coimmunostained TA sections from mdx and mdx::IL34 -/- mice. Nuclei was labeled with DAPI. Scale bar: 30 μm. (D) Number of Pax7 + cells per field. **p<0.01. (E) Left, H&E-stained TA sections from mdx and mdx::IL34 -/- mice at 8 weeks of age; the marked area presents regenerating muscle fibers. Scale bar: 30 μm. Right, representative merged images of eMyHC and laminin coimmunostained serial TA muscle sections. The same marked area presents eMyHC-positive regenerating muscle fibers. Scale bar: 50 μm. (F) H&E staining analysis of transverse sections of Gas muscle from mdx and mdx::IL34 -/- mice. Scale bar: 50 μm. (G) Quantification of regenerating fiber size (frequency) distribution of mdx and mdx::IL34 -/- mice. N=4 mice in each group. *p<0.05 and **p<0.01. (H) Quantification of necrotic area in Gas muscle *p<0.05. (I) Masson's staining analysis of fibrosis accumulation in transverse sections of Gas muscle from mdx and mdx::IL34 -/- mice. Scale bar: 50 μm. (J) Measurement of the fibrotic area. ***p<0.001. (K) Running distance to exhaustion on the downhill treadmill test. *p<0.05.

Article Snippet: After one day in differentiation medium, the medium was supplemented with 100 ng/ml recombinant IL34 (Origene); the same volume of 0.1% BSA was used as a control.

Techniques: Labeling, Staining