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<t>MAP4</t> phosphorylation is induced at the wound edge and promotes wound repair. (A) Immunofluorescence staining of p-MAP4 in normal unwounded skin (day 0), day 5, day 9, and day 15 wound sections obtained from wild-type (WT) C57BL/6J mice. Wounds were close to reepithelialization on day 9 and fully re-epithelialized on day 15 (n = 10). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis (day 5 and day 9). Epi, epidermis; Derm, dermis. (B) Western blotting was performed to detect the phosphorylation of MAP4 at S737, S760 and S667 in mouse epidermis as well as the activation of p38/MAPK. β-Actin was used as a loading control (n = 10). (C) Images of skin wound sites taken 0, 3, 5, 7, and 9 days post wounding. Full-thickness excisional wounds (5 mm in diameter) were made on dorsal skin of KI mice and their corresponding WT littermates (n = 10). (D) Graphs showing the rate of wound closure. Areas around the wounds were measured with ImageJ software. The results are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (E) Wound healing was monitored by histological staining of skin sections (day 5 and day 9 post injury) at the wound edge. Scale bar = 200 μm. Dotted lines indicate dermal-epidermal boundaries. Arrows denote the leading edges of the epidermis (n = 10). (F) Graph shows the average wound gap quantified at the indicated time after wounding. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group.

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1) Product Images from "Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation"

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.35440

MAP4 phosphorylation is induced at the wound edge and promotes wound repair. (A) Immunofluorescence staining of p-MAP4 in normal unwounded skin (day 0), day 5, day 9, and day 15 wound sections obtained from wild-type (WT) C57BL/6J mice. Wounds were close to reepithelialization on day 9 and fully re-epithelialized on day 15 (n = 10). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis (day 5 and day 9). Epi, epidermis; Derm, dermis. (B) Western blotting was performed to detect the phosphorylation of MAP4 at S737, S760 and S667 in mouse epidermis as well as the activation of p38/MAPK. β-Actin was used as a loading control (n = 10). (C) Images of skin wound sites taken 0, 3, 5, 7, and 9 days post wounding. Full-thickness excisional wounds (5 mm in diameter) were made on dorsal skin of KI mice and their corresponding WT littermates (n = 10). (D) Graphs showing the rate of wound closure. Areas around the wounds were measured with ImageJ software. The results are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (E) Wound healing was monitored by histological staining of skin sections (day 5 and day 9 post injury) at the wound edge. Scale bar = 200 μm. Dotted lines indicate dermal-epidermal boundaries. Arrows denote the leading edges of the epidermis (n = 10). (F) Graph shows the average wound gap quantified at the indicated time after wounding. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group.

Figure Legend Snippet: MAP4 phosphorylation is induced at the wound edge and promotes wound repair. (A) Immunofluorescence staining of p-MAP4 in normal unwounded skin (day 0), day 5, day 9, and day 15 wound sections obtained from wild-type (WT) C57BL/6J mice. Wounds were close to reepithelialization on day 9 and fully re-epithelialized on day 15 (n = 10). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis (day 5 and day 9). Epi, epidermis; Derm, dermis. (B) Western blotting was performed to detect the phosphorylation of MAP4 at S737, S760 and S667 in mouse epidermis as well as the activation of p38/MAPK. β-Actin was used as a loading control (n = 10). (C) Images of skin wound sites taken 0, 3, 5, 7, and 9 days post wounding. Full-thickness excisional wounds (5 mm in diameter) were made on dorsal skin of KI mice and their corresponding WT littermates (n = 10). (D) Graphs showing the rate of wound closure. Areas around the wounds were measured with ImageJ software. The results are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (E) Wound healing was monitored by histological staining of skin sections (day 5 and day 9 post injury) at the wound edge. Scale bar = 200 μm. Dotted lines indicate dermal-epidermal boundaries. Arrows denote the leading edges of the epidermis (n = 10). (F) Graph shows the average wound gap quantified at the indicated time after wounding. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group.

Techniques Used: Immunofluorescence, Staining, Western Blot, Activation Assay, Software

MAP4 phosphorylation promotes the proliferation of epidermal keratinocytes. (A) Representative pictures of confocal images of EdU staining (green) and pankeratin (red) of WT and KI mice wounds on day 5 and day 9 after injury (n = 10). Nuclei were stained with DAPI (blue). Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis. Scale bar = 50 μm. (B) Quantification of the EdU-positive keratinocytes at times indicated after wounding (A). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (C) Quantification of the average epidermal thickness at times indicated after wounding (A). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (D) Representative pictures of EdU staining (green) of MKs isolated from the epidermis of KI and WT mice (n = 5). Nuclei were stained with DAPI (blue). (E) Graph shows quantification data of the EdU-positive keratinocytes shown in (D). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (F) Keratinocyte proliferation was evaluated using a CCK-8 assay according to manufacturer's instructions. The results are shown as means ± SEM (n = 5). * P < 0.05 versus the WT group. (G) Western blotting was performed to analyze the expression of PCNA and Ki67 in cultured keratinocytes isolated from the epidermis of KI and WT mice (n = 5). Representative bands of two samples in each group are shown. β-Actin was used as a loading control.

Figure Legend Snippet: MAP4 phosphorylation promotes the proliferation of epidermal keratinocytes. (A) Representative pictures of confocal images of EdU staining (green) and pankeratin (red) of WT and KI mice wounds on day 5 and day 9 after injury (n = 10). Nuclei were stained with DAPI (blue). Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis. Scale bar = 50 μm. (B) Quantification of the EdU-positive keratinocytes at times indicated after wounding (A). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (C) Quantification of the average epidermal thickness at times indicated after wounding (A). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (D) Representative pictures of EdU staining (green) of MKs isolated from the epidermis of KI and WT mice (n = 5). Nuclei were stained with DAPI (blue). (E) Graph shows quantification data of the EdU-positive keratinocytes shown in (D). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (F) Keratinocyte proliferation was evaluated using a CCK-8 assay according to manufacturer's instructions. The results are shown as means ± SEM (n = 5). * P < 0.05 versus the WT group. (G) Western blotting was performed to analyze the expression of PCNA and Ki67 in cultured keratinocytes isolated from the epidermis of KI and WT mice (n = 5). Representative bands of two samples in each group are shown. β-Actin was used as a loading control.

Techniques Used: Staining, Isolation, CCK-8 Assay, Western Blot, Expressing, Cell Culture

MAP4 phosphorylation promotes epidermal keratinocyte migration and regulates MT rearrangement. Scratch wound healing assays (A, B) and single-cell motility assays (C, D) were performed using MKs isolated from the epidermis of KI and WT mice (n = 5). Representative pictures of wound healing and the trajectories of keratinocytes are shown. Scale bar = 100 μm. Quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 vs. WT group. (E) Representative images of skin explant culture (day 4). Dotted lines denote the boundary of skin explant (left) or leading edge of epidermal outgrowth from the explant (right) (n = 10). Scale bar = 100 μm. (F) Quantification of the outgrowth of epidermal explants. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (G) Staining of MTs in the indicated keratinocytes (n = 5). The boxed areas show image at higher magnification to illustrate details. Scale bar = 25 μm. All experiments were repeated 3 times.

Figure Legend Snippet: MAP4 phosphorylation promotes epidermal keratinocyte migration and regulates MT rearrangement. Scratch wound healing assays (A, B) and single-cell motility assays (C, D) were performed using MKs isolated from the epidermis of KI and WT mice (n = 5). Representative pictures of wound healing and the trajectories of keratinocytes are shown. Scale bar = 100 μm. Quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 vs. WT group. (E) Representative images of skin explant culture (day 4). Dotted lines denote the boundary of skin explant (left) or leading edge of epidermal outgrowth from the explant (right) (n = 10). Scale bar = 100 μm. (F) Quantification of the outgrowth of epidermal explants. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (G) Staining of MTs in the indicated keratinocytes (n = 5). The boxed areas show image at higher magnification to illustrate details. Scale bar = 25 μm. All experiments were repeated 3 times.

Techniques Used: Migration, Isolation, Staining

MAP4 phosphorylation regulates hypoxia-induced epidermal keratinocyte proliferation and migration through MT rearrangement. (A) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of MAP4 phosphorylation by Western blotting. β-Actin was used as a loading control (n = 5). (B) Confirmation of adenovirus transfection at comparable levels in MKs. Total cell extracts from MKs after transfecting MAP4(Ala) or CMV-null adenovirus were analyzed by Western blotting (n = 5). (C) Graphs indicate the relative intensities as determined by Quantity one software. Results are shown as the means ± SEM. */# P < 0.05 vs. the corresponding CMV-null (CMV) group. (D) MKs isolated from the epidermis of KI or WT mice were subjected to hypoxia (2% O 2 , 24 h) after being transfected with CMV-null or MAP4 (Ala) for 48 h. The Western blot shows activation of p38/MAPK (n = 5); β-Actin was used as the loading control. Then, cell migration and motility were assessed by scratch wound healing assays (E) and single-cell motility assays (F) . The quantitative results are shown as the means ± SEM. * P < 0.05 vs. Hypo + WT + CMV group, # P < 0.05 vs. Hypo + KI + CMV group (n = 5). (G) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of the expression of PCNA and Ki67 using Western blotting (n = 5). Representative bands are shown. β-Actin was used as a loading control. (H) MKs isolated from the epidermis of KI and WT mice were subjected to hypoxia (2% O 2 , 24 h) after being transfected with CMV-null or MAP4(Ala) for 48 h. Representative blots show the expression of PCNA and Ki67 (n = 5); β-Actin was used as the loading control. (I) Representative pictures of EdU staining (green) of the indicated keratinocytes. Nuclei were stained with DAPI (blue). Scale bar = 50 μm (n = 5). ( J ) Quantification of the positive rate of EdU in indicated keratinocytes. The results are shown as the means ± SEM. * P < 0.05 vs. Hypo + WT + CMV group, # P < 0.05 vs. Hypo + KI + CMV group. (K) MTs stained in the indicated keratinocytes. The boxed areas show higher magnification to illustrate details (n = 5). Scale bar = 25 μm. All experiments were repeated 3 times. Hypo, hypoxia.

Figure Legend Snippet: MAP4 phosphorylation regulates hypoxia-induced epidermal keratinocyte proliferation and migration through MT rearrangement. (A) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of MAP4 phosphorylation by Western blotting. β-Actin was used as a loading control (n = 5). (B) Confirmation of adenovirus transfection at comparable levels in MKs. Total cell extracts from MKs after transfecting MAP4(Ala) or CMV-null adenovirus were analyzed by Western blotting (n = 5). (C) Graphs indicate the relative intensities as determined by Quantity one software. Results are shown as the means ± SEM. */# P < 0.05 vs. the corresponding CMV-null (CMV) group. (D) MKs isolated from the epidermis of KI or WT mice were subjected to hypoxia (2% O 2 , 24 h) after being transfected with CMV-null or MAP4 (Ala) for 48 h. The Western blot shows activation of p38/MAPK (n = 5); β-Actin was used as the loading control. Then, cell migration and motility were assessed by scratch wound healing assays (E) and single-cell motility assays (F) . The quantitative results are shown as the means ± SEM. * P < 0.05 vs. Hypo + WT + CMV group, # P < 0.05 vs. Hypo + KI + CMV group (n = 5). (G) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of the expression of PCNA and Ki67 using Western blotting (n = 5). Representative bands are shown. β-Actin was used as a loading control. (H) MKs isolated from the epidermis of KI and WT mice were subjected to hypoxia (2% O 2 , 24 h) after being transfected with CMV-null or MAP4(Ala) for 48 h. Representative blots show the expression of PCNA and Ki67 (n = 5); β-Actin was used as the loading control. (I) Representative pictures of EdU staining (green) of the indicated keratinocytes. Nuclei were stained with DAPI (blue). Scale bar = 50 μm (n = 5). ( J ) Quantification of the positive rate of EdU in indicated keratinocytes. The results are shown as the means ± SEM. * P < 0.05 vs. Hypo + WT + CMV group, # P < 0.05 vs. Hypo + KI + CMV group. (K) MTs stained in the indicated keratinocytes. The boxed areas show higher magnification to illustrate details (n = 5). Scale bar = 25 μm. All experiments were repeated 3 times. Hypo, hypoxia.

Techniques Used: Migration, Incubation, Western Blot, Transfection, Software, Isolation, Activation Assay, Expressing, Staining

P38/MAPK is involved in MAP4 phosphorylation-induced epidermal keratinocyte migration and proliferation under hypoxia. (A) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and cell proteins were harvested for detection of activated of p38/MAPK using Western blotting. β-Actin was used as a loading control (n = 5). (B) Confirmation of adenovirus transfection at comparable levels in MKs. Cell extracts from MKs after transfection with MKK6(Glu) adenovirus were analyzed by Western blotting (n = 5). (C) MKs were transfected with MKK6(Glu) adenovirus under normoxia or subjected to a specific p38/MAPK inhibitor SB203580 (SB, 5 μM) before hypoxia exposure (2% O 2 , 24 h). Western blot analysis showed the activities of p38/MAPK and MAP4 phosphorylation of MKs with the indicated treatments (n = 5). β-Actin was used as the loading control. (D) MTs stained in the indicated keratinocytes (n = 5). The boxed areas show higher magnification to illustrate details. Scale bar = 25 μm. (E) Western blotting was performed to analyze the expression of PCNA and Ki67 in the indicated keratinocytes (n = 5). (F) Representative pictures of EdU staining (green) of the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue), scale bar = 50 μm. (G) Graphs indicate the positive rate of EdU in the indicated MKs. Results are shown as the means ± SEM. * P < 0.05 vs. Norm + Con group, # P < 0.05 vs. Hypo + Con group. Then, scratch wound healing assays (H) and single-cell motility assays (I) were performed to determine the migration of indicated keratinocytes, and the quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 versus the Norm + Con group, # P < 0.05 versus the Hypo + Con group. (J) Western blotting was performed to analyze the activities of p38/MAPK and the expression of MAP4 in MKs transiently transfected with MAP4(Ala), MKK6(Glu) or both (n = 5). β-Actin was used as the loading control. Then, scratch wound healing assays (K) and single-cell motility assays (L) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the mean ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (M) Western blotting was performed to analyze the expression of PCNA and Ki67 in the indicated keratinocytes (n = 5). (N) Representative pictures of EdU staining (green) in the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (O) Graphs show the positive rate of EdU in the indicated MKs. The results are shown as the means ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (P) MTs stained in the indicated keratinocytes (n = 5). The boxed areas show higher magnification to illustrate details. Scale bar = 25 μm. All experiments were repeated 3 times. Con, control.

Figure Legend Snippet: P38/MAPK is involved in MAP4 phosphorylation-induced epidermal keratinocyte migration and proliferation under hypoxia. (A) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and cell proteins were harvested for detection of activated of p38/MAPK using Western blotting. β-Actin was used as a loading control (n = 5). (B) Confirmation of adenovirus transfection at comparable levels in MKs. Cell extracts from MKs after transfection with MKK6(Glu) adenovirus were analyzed by Western blotting (n = 5). (C) MKs were transfected with MKK6(Glu) adenovirus under normoxia or subjected to a specific p38/MAPK inhibitor SB203580 (SB, 5 μM) before hypoxia exposure (2% O 2 , 24 h). Western blot analysis showed the activities of p38/MAPK and MAP4 phosphorylation of MKs with the indicated treatments (n = 5). β-Actin was used as the loading control. (D) MTs stained in the indicated keratinocytes (n = 5). The boxed areas show higher magnification to illustrate details. Scale bar = 25 μm. (E) Western blotting was performed to analyze the expression of PCNA and Ki67 in the indicated keratinocytes (n = 5). (F) Representative pictures of EdU staining (green) of the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue), scale bar = 50 μm. (G) Graphs indicate the positive rate of EdU in the indicated MKs. Results are shown as the means ± SEM. * P < 0.05 vs. Norm + Con group, # P < 0.05 vs. Hypo + Con group. Then, scratch wound healing assays (H) and single-cell motility assays (I) were performed to determine the migration of indicated keratinocytes, and the quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 versus the Norm + Con group, # P < 0.05 versus the Hypo + Con group. (J) Western blotting was performed to analyze the activities of p38/MAPK and the expression of MAP4 in MKs transiently transfected with MAP4(Ala), MKK6(Glu) or both (n = 5). β-Actin was used as the loading control. Then, scratch wound healing assays (K) and single-cell motility assays (L) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the mean ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (M) Western blotting was performed to analyze the expression of PCNA and Ki67 in the indicated keratinocytes (n = 5). (N) Representative pictures of EdU staining (green) in the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (O) Graphs show the positive rate of EdU in the indicated MKs. The results are shown as the means ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (P) MTs stained in the indicated keratinocytes (n = 5). The boxed areas show higher magnification to illustrate details. Scale bar = 25 μm. All experiments were repeated 3 times. Con, control.

Techniques Used: Migration, Incubation, Western Blot, Transfection, Staining, Expressing

MAP4 phosphorylation induced by p38/MAPK promotes proliferation and migration in human keratinocytes under hypoxia. (A) HaCaT cells were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of MAP4 phosphorylation and the activities of p38/MAPK using Western blotting (n = 5). β-Actin was used as a loading control. (B) Confirmation of adenovirus transfection at comparable levels in HaCaT cells. Cell extracts from HaCaT cells after transfection by MKK6(Glu) adenovirus were analyzed by Western blotting (n = 5). (C) HaCaT cells were transfected with MKK6(Glu) adenovirus under normoxia or subjected to SB (5 μM) before hypoxia exposure (2% O 2 , 24 h). The Western blot showed the activities of p38/MAPK and MAP4 phosphorylation with the indicated treatments (n = 5). β-Actin was used as the loading control. (D) The expression levels of PCNA and Ki67 in the indicated HaCaT cells were analyzed using Western blotting (n = 5). (E) Representative pictures of EdU staining (green) of the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (F) Graphs show the positive rate of EdU in the indicated MKs (n = 5). The results are shown as the means ± SEM. * P < 0.05 vs. Norm + Con group, # P < 0.05 vs. Hypo + Con group. Then, scratch wound healing assays (G) and single-cell motility assays (H) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 versus the Norm + Con group, # P < 0.05 versus the Hypo + Con group. (I) Confirmation of adenovirus transfection at comparable levels in HaCaT cells. Cell extracts from HaCaT cells after transfection by MAP4 (Ala) adenovirus were analyzed by Western blotting (n = 5). (J) Western blotting was performed to analyze the activities of p38/MAPK and the expression of MAP4 in HaCaT cells transiently transfected with MAP4(Ala), MKK6(Glu) or both (n = 5). β-Actin was used as the loading control. Then, scratch wound healing assays (K) and single-cell motility assays (L) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (M) The expression of PCNA and Ki67 in the indicated HaCaT cells was detected using Western blotting (n = 5). (N) Representative pictures of EdU staining (green) of the indicated keratinocytes. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (O) Graphs show the positive rate of EdU in the indicated MKs (n = 5). The results were shown as the means ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. All experiments were repeated 3 times.

Figure Legend Snippet: MAP4 phosphorylation induced by p38/MAPK promotes proliferation and migration in human keratinocytes under hypoxia. (A) HaCaT cells were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of MAP4 phosphorylation and the activities of p38/MAPK using Western blotting (n = 5). β-Actin was used as a loading control. (B) Confirmation of adenovirus transfection at comparable levels in HaCaT cells. Cell extracts from HaCaT cells after transfection by MKK6(Glu) adenovirus were analyzed by Western blotting (n = 5). (C) HaCaT cells were transfected with MKK6(Glu) adenovirus under normoxia or subjected to SB (5 μM) before hypoxia exposure (2% O 2 , 24 h). The Western blot showed the activities of p38/MAPK and MAP4 phosphorylation with the indicated treatments (n = 5). β-Actin was used as the loading control. (D) The expression levels of PCNA and Ki67 in the indicated HaCaT cells were analyzed using Western blotting (n = 5). (E) Representative pictures of EdU staining (green) of the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (F) Graphs show the positive rate of EdU in the indicated MKs (n = 5). The results are shown as the means ± SEM. * P < 0.05 vs. Norm + Con group, # P < 0.05 vs. Hypo + Con group. Then, scratch wound healing assays (G) and single-cell motility assays (H) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 versus the Norm + Con group, # P < 0.05 versus the Hypo + Con group. (I) Confirmation of adenovirus transfection at comparable levels in HaCaT cells. Cell extracts from HaCaT cells after transfection by MAP4 (Ala) adenovirus were analyzed by Western blotting (n = 5). (J) Western blotting was performed to analyze the activities of p38/MAPK and the expression of MAP4 in HaCaT cells transiently transfected with MAP4(Ala), MKK6(Glu) or both (n = 5). β-Actin was used as the loading control. Then, scratch wound healing assays (K) and single-cell motility assays (L) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (M) The expression of PCNA and Ki67 in the indicated HaCaT cells was detected using Western blotting (n = 5). (N) Representative pictures of EdU staining (green) of the indicated keratinocytes. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (O) Graphs show the positive rate of EdU in the indicated MKs (n = 5). The results were shown as the means ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. All experiments were repeated 3 times.

Techniques Used: Migration, Incubation, Western Blot, Transfection, Expressing, Staining

Schematic illustrating that MAP4 phosphorylation is involved in keratinocyte migration and proliferation. Wound-induced hypoxia in the wound edge stimulates the activation of p38/MAPK in keratinocytes, i.e., increases in p38 phosphorylation. The activated p38/MAPK promotes the phosphorylation of MAP4 and, sequentially, the depolymerization of MTs, essential components of the cytoskeleton in the control of cell migration and proliferation.

Figure Legend Snippet: Schematic illustrating that MAP4 phosphorylation is involved in keratinocyte migration and proliferation. Wound-induced hypoxia in the wound edge stimulates the activation of p38/MAPK in keratinocytes, i.e., increases in p38 phosphorylation. The activated p38/MAPK promotes the phosphorylation of MAP4 and, sequentially, the depolymerization of MTs, essential components of the cytoskeleton in the control of cell migration and proliferation.

Techniques Used: Migration, Activation Assay

Image Search Results

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: MAP4 phosphorylation is induced at the wound edge and promotes wound repair. (A) Immunofluorescence staining of p-MAP4 in normal unwounded skin (day 0), day 5, day 9, and day 15 wound sections obtained from wild-type (WT) C57BL/6J mice. Wounds were close to reepithelialization on day 9 and fully re-epithelialized on day 15 (n = 10). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis (day 5 and day 9). Epi, epidermis; Derm, dermis. (B) Western blotting was performed to detect the phosphorylation of MAP4 at S737, S760 and S667 in mouse epidermis as well as the activation of p38/MAPK. β-Actin was used as a loading control (n = 10). (C) Images of skin wound sites taken 0, 3, 5, 7, and 9 days post wounding. Full-thickness excisional wounds (5 mm in diameter) were made on dorsal skin of KI mice and their corresponding WT littermates (n = 10). (D) Graphs showing the rate of wound closure. Areas around the wounds were measured with ImageJ software. The results are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (E) Wound healing was monitored by histological staining of skin sections (day 5 and day 9 post injury) at the wound edge. Scale bar = 200 μm. Dotted lines indicate dermal-epidermal boundaries. Arrows denote the leading edges of the epidermis (n = 10). (F) Graph shows the average wound gap quantified at the indicated time after wounding. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group.

Article Snippet: The primary antibodies used for immunoblotting were as follows: p-p38 (1:1000, Cell Signaling Technology), p38 (1:1000, Cell Signaling Technology), MAP4 (1:1000, Bethyl, USA), p-MAP4(S768) (Biolegend, 1:1000), p-MAP4(S696) (1:1000, GL Biochem), p-MAP4(S787) (1:1000, GL Biochem), p-MAP4(S737) (1:1000, GL Biochem), β-Actin (1:1000, Cell Signaling Technology), PCNA (1:1000, Abcam), and Ki67 (1:1000, Abcam).

Techniques: Immunofluorescence, Staining, Western Blot, Activation Assay, Software

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: MAP4 phosphorylation promotes the proliferation of epidermal keratinocytes. (A) Representative pictures of confocal images of EdU staining (green) and pankeratin (red) of WT and KI mice wounds on day 5 and day 9 after injury (n = 10). Nuclei were stained with DAPI (blue). Narrow dotted line: interface between epidermis and dermis or leading edge of migrating epidermis. Scale bar = 50 μm. (B) Quantification of the EdU-positive keratinocytes at times indicated after wounding (A). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (C) Quantification of the average epidermal thickness at times indicated after wounding (A). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (D) Representative pictures of EdU staining (green) of MKs isolated from the epidermis of KI and WT mice (n = 5). Nuclei were stained with DAPI (blue). (E) Graph shows quantification data of the EdU-positive keratinocytes shown in (D). The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (F) Keratinocyte proliferation was evaluated using a CCK-8 assay according to manufacturer's instructions. The results are shown as means ± SEM (n = 5). * P < 0.05 versus the WT group. (G) Western blotting was performed to analyze the expression of PCNA and Ki67 in cultured keratinocytes isolated from the epidermis of KI and WT mice (n = 5). Representative bands of two samples in each group are shown. β-Actin was used as a loading control.

Techniques: Staining, Isolation, CCK-8 Assay, Western Blot, Expressing, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: MAP4 phosphorylation promotes epidermal keratinocyte migration and regulates MT rearrangement. Scratch wound healing assays (A, B) and single-cell motility assays (C, D) were performed using MKs isolated from the epidermis of KI and WT mice (n = 5). Representative pictures of wound healing and the trajectories of keratinocytes are shown. Scale bar = 100 μm. Quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 vs. WT group. (E) Representative images of skin explant culture (day 4). Dotted lines denote the boundary of skin explant (left) or leading edge of epidermal outgrowth from the explant (right) (n = 10). Scale bar = 100 μm. (F) Quantification of the outgrowth of epidermal explants. The data are shown as the means ± SEM. * P < 0.05 vs. the corresponding WT group. (G) Staining of MTs in the indicated keratinocytes (n = 5). The boxed areas show image at higher magnification to illustrate details. Scale bar = 25 μm. All experiments were repeated 3 times.

Techniques: Migration, Isolation, Staining

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: MAP4 phosphorylation regulates hypoxia-induced epidermal keratinocyte proliferation and migration through MT rearrangement. (A) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of MAP4 phosphorylation by Western blotting. β-Actin was used as a loading control (n = 5). (B) Confirmation of adenovirus transfection at comparable levels in MKs. Total cell extracts from MKs after transfecting MAP4(Ala) or CMV-null adenovirus were analyzed by Western blotting (n = 5). (C) Graphs indicate the relative intensities as determined by Quantity one software. Results are shown as the means ± SEM. */# P < 0.05 vs. the corresponding CMV-null (CMV) group. (D) MKs isolated from the epidermis of KI or WT mice were subjected to hypoxia (2% O 2 , 24 h) after being transfected with CMV-null or MAP4 (Ala) for 48 h. The Western blot shows activation of p38/MAPK (n = 5); β-Actin was used as the loading control. Then, cell migration and motility were assessed by scratch wound healing assays (E) and single-cell motility assays (F) . The quantitative results are shown as the means ± SEM. * P < 0.05 vs. Hypo + WT + CMV group, # P < 0.05 vs. Hypo + KI + CMV group (n = 5). (G) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of the expression of PCNA and Ki67 using Western blotting (n = 5). Representative bands are shown. β-Actin was used as a loading control. (H) MKs isolated from the epidermis of KI and WT mice were subjected to hypoxia (2% O 2 , 24 h) after being transfected with CMV-null or MAP4(Ala) for 48 h. Representative blots show the expression of PCNA and Ki67 (n = 5); β-Actin was used as the loading control. (I) Representative pictures of EdU staining (green) of the indicated keratinocytes. Nuclei were stained with DAPI (blue). Scale bar = 50 μm (n = 5). ( J ) Quantification of the positive rate of EdU in indicated keratinocytes. The results are shown as the means ± SEM. * P < 0.05 vs. Hypo + WT + CMV group, # P < 0.05 vs. Hypo + KI + CMV group. (K) MTs stained in the indicated keratinocytes. The boxed areas show higher magnification to illustrate details (n = 5). Scale bar = 25 μm. All experiments were repeated 3 times. Hypo, hypoxia.

Techniques: Migration, Incubation, Western Blot, Transfection, Software, Isolation, Activation Assay, Expressing, Staining

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: P38/MAPK is involved in MAP4 phosphorylation-induced epidermal keratinocyte migration and proliferation under hypoxia. (A) MKs were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and cell proteins were harvested for detection of activated of p38/MAPK using Western blotting. β-Actin was used as a loading control (n = 5). (B) Confirmation of adenovirus transfection at comparable levels in MKs. Cell extracts from MKs after transfection with MKK6(Glu) adenovirus were analyzed by Western blotting (n = 5). (C) MKs were transfected with MKK6(Glu) adenovirus under normoxia or subjected to a specific p38/MAPK inhibitor SB203580 (SB, 5 μM) before hypoxia exposure (2% O 2 , 24 h). Western blot analysis showed the activities of p38/MAPK and MAP4 phosphorylation of MKs with the indicated treatments (n = 5). β-Actin was used as the loading control. (D) MTs stained in the indicated keratinocytes (n = 5). The boxed areas show higher magnification to illustrate details. Scale bar = 25 μm. (E) Western blotting was performed to analyze the expression of PCNA and Ki67 in the indicated keratinocytes (n = 5). (F) Representative pictures of EdU staining (green) of the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue), scale bar = 50 μm. (G) Graphs indicate the positive rate of EdU in the indicated MKs. Results are shown as the means ± SEM. * P < 0.05 vs. Norm + Con group, # P < 0.05 vs. Hypo + Con group. Then, scratch wound healing assays (H) and single-cell motility assays (I) were performed to determine the migration of indicated keratinocytes, and the quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 versus the Norm + Con group, # P < 0.05 versus the Hypo + Con group. (J) Western blotting was performed to analyze the activities of p38/MAPK and the expression of MAP4 in MKs transiently transfected with MAP4(Ala), MKK6(Glu) or both (n = 5). β-Actin was used as the loading control. Then, scratch wound healing assays (K) and single-cell motility assays (L) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the mean ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (M) Western blotting was performed to analyze the expression of PCNA and Ki67 in the indicated keratinocytes (n = 5). (N) Representative pictures of EdU staining (green) in the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (O) Graphs show the positive rate of EdU in the indicated MKs. The results are shown as the means ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (P) MTs stained in the indicated keratinocytes (n = 5). The boxed areas show higher magnification to illustrate details. Scale bar = 25 μm. All experiments were repeated 3 times. Con, control.

Techniques: Migration, Incubation, Western Blot, Transfection, Staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: MAP4 phosphorylation induced by p38/MAPK promotes proliferation and migration in human keratinocytes under hypoxia. (A) HaCaT cells were exposed to hypoxia (2% O 2 ) and incubated for the indicated times, and total proteins were harvested for detection of MAP4 phosphorylation and the activities of p38/MAPK using Western blotting (n = 5). β-Actin was used as a loading control. (B) Confirmation of adenovirus transfection at comparable levels in HaCaT cells. Cell extracts from HaCaT cells after transfection by MKK6(Glu) adenovirus were analyzed by Western blotting (n = 5). (C) HaCaT cells were transfected with MKK6(Glu) adenovirus under normoxia or subjected to SB (5 μM) before hypoxia exposure (2% O 2 , 24 h). The Western blot showed the activities of p38/MAPK and MAP4 phosphorylation with the indicated treatments (n = 5). β-Actin was used as the loading control. (D) The expression levels of PCNA and Ki67 in the indicated HaCaT cells were analyzed using Western blotting (n = 5). (E) Representative pictures of EdU staining (green) of the indicated keratinocytes (n = 5). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (F) Graphs show the positive rate of EdU in the indicated MKs (n = 5). The results are shown as the means ± SEM. * P < 0.05 vs. Norm + Con group, # P < 0.05 vs. Hypo + Con group. Then, scratch wound healing assays (G) and single-cell motility assays (H) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 versus the Norm + Con group, # P < 0.05 versus the Hypo + Con group. (I) Confirmation of adenovirus transfection at comparable levels in HaCaT cells. Cell extracts from HaCaT cells after transfection by MAP4 (Ala) adenovirus were analyzed by Western blotting (n = 5). (J) Western blotting was performed to analyze the activities of p38/MAPK and the expression of MAP4 in HaCaT cells transiently transfected with MAP4(Ala), MKK6(Glu) or both (n = 5). β-Actin was used as the loading control. Then, scratch wound healing assays (K) and single-cell motility assays (L) were performed to determine the migration of the indicated keratinocytes. The quantitative results are shown as the means ± SEM (n = 5). * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. (M) The expression of PCNA and Ki67 in the indicated HaCaT cells was detected using Western blotting (n = 5). (N) Representative pictures of EdU staining (green) of the indicated keratinocytes. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (O) Graphs show the positive rate of EdU in the indicated MKs (n = 5). The results were shown as the means ± SEM. * P < 0.05 vs. CMV group, # P < 0.05 vs. MKK6 + CMV group. All experiments were repeated 3 times.

Techniques: Migration, Incubation, Western Blot, Transfection, Expressing, Staining

Journal: International Journal of Biological Sciences

Article Title: Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

doi: 10.7150/ijbs.35440

Figure Lengend Snippet: Schematic illustrating that MAP4 phosphorylation is involved in keratinocyte migration and proliferation. Wound-induced hypoxia in the wound edge stimulates the activation of p38/MAPK in keratinocytes, i.e., increases in p38 phosphorylation. The activated p38/MAPK promotes the phosphorylation of MAP4 and, sequentially, the depolymerization of MTs, essential components of the cytoskeleton in the control of cell migration and proliferation.

Techniques: Migration, Activation Assay

Cardiac MAP4 hyperphosphorylation in hypoxic and pressure overload cardiomyopathy. (a, b) Representative WB (a) and quantitative analysis (b) depicting p-M (S696, S768 and S787) in TOF patients with hypoxemia. Data showed median (interquartile range). n = 8. (c, d) MAP4 phosphorylation was examined following MI at 0 min, 5 min, 60 min, 180 min and 360 min. n = 5. (e, f) WB analysis exhibited p-M (S667, S737 and S760) in mice with TAC for three weeks. n = 6. p-M, p-MAP4. The graph showed mean ± SEM (d and f). *P < 0.05, ***P < 0.001, ns, not statistically significant. P values were derived from non-parametric Mann-Whitney test (b), two-tailed Student's t -test (f) and one-way ANOVA with Bonferroni's post-test (d).

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: Cardiac MAP4 hyperphosphorylation in hypoxic and pressure overload cardiomyopathy. (a, b) Representative WB (a) and quantitative analysis (b) depicting p-M (S696, S768 and S787) in TOF patients with hypoxemia. Data showed median (interquartile range). n = 8. (c, d) MAP4 phosphorylation was examined following MI at 0 min, 5 min, 60 min, 180 min and 360 min. n = 5. (e, f) WB analysis exhibited p-M (S667, S737 and S760) in mice with TAC for three weeks. n = 6. p-M, p-MAP4. The graph showed mean ± SEM (d and f). *P < 0.05, ***P < 0.001, ns, not statistically significant. P values were derived from non-parametric Mann-Whitney test (b), two-tailed Student's t -test (f) and one-way ANOVA with Bonferroni's post-test (d).

Article Snippet: The following antibodies were used in this experiment: p38 MAPK (Cell Signaling Technology Cat# 9212, RRID:AB_330713, 1:1000), Phospho-p38 MAPK (p-P38) (Cell Signaling Technology Cat# 9211, RRID:AB_331641, 1:1000), VDAC (Cell Signaling Technology Cat# 4661, RRID:AB_10557420, 1:3000), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID:AB_2341188, 1:1000), Caspase-3 (Cell Signaling Technology Cat# 9662, RRID:AB_331439, 1:2000), GAPDH (Proteintech Group Cat# 60004-1-Ig, RRID:AB_2107436, 1:5000), p-MAP4 (S768) (Biolegend Cat# 621102, 1:5000), and rabbit polyclonal antibody against p-MAP4 (S696) (GL Biochem, 1:1000), p-MAP4 (S787) (GL Biochem, 1:1000) or polyclonal antibody against p-MAP4 (S737) (GL Biochem, 1:1000), α-tubulin (Proteintech Group Cat# 11224-1-AP, RRID:AB_2210206, 1:2000), cytochrome c (Santa Cruz Biotechnology Cat# sc-13156, RRID:AB_627385, 1:5000), ANP (Santa Cruz Biotechnology Cat# sc-515701, 1:1000), BNP (Santa Cruz Biotechnology Cat# sc-271185, RRID:AB_10609757, 1:1000), MYH7 (Santa Cruz Biotechnology Cat# sc-53089, RRID:AB_2147281, 1:1000), COL1A1 (Santa Cruz Biotechnology Cat# sc-293182, 1:1000), COL1A2 (Santa Cruz Biotechnology Cat# sc-393537, 1:1000), COL3A1 (Santa Cruz Biotechnology Cat# sc-271249, RRID:AB_10613985, 1:1000), RIP3 (Santa Cruz Biotechnology Cat# sc-374639, RRID:AB_10992232, 1:2000), p-MLKL (Abcam Cat# ab196436, RRID:AB_2687465, 1:1000), MLKL (Abcam Cat# ab194699, 1:2000), FN (Abcam Cat# ab2413, RRID:AB_2262874, 1:5000), α-SMA (Abcam Cat# ab5649, 1:4000), MAP4 (Bethyl Cat# A301-489A, RRID:AB_999616, 1:1000), E-cadherin (Cell Signaling Technology Cat# 3195, RRID:AB_2291471, 1:2000), Calnexin (Abcam Cat# ab22595, RRID:AB_2069006, 1:4000), Histone H3 (Bioss Inc. Cat# bs-0349R-HRP, RRID:AB_11112443, 1:1000), transcription factor GATA-4 (GATA4) (Abcam Cat# ab84593, RRID:AB_10670538, 1:2000).

Techniques: Derivative Assay, MANN-WHITNEY, Two Tailed Test

Genetic mutations of phosphorylation sites of MAP4. (a) Schematic of MAP4 KI mice. CRISPR/Cas9 technique was used to generate mouse strain with mutated MAP4 by knocking in the coding sequence (S667A, S737E and S760E) in the ninth exon of MAP4 gene by homologous recombination. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats-associated protein 9. (b) Representative polymerase chain reaction identification showing the HO, HE and WT genotype mice. HO, homozygote; HE, heterozygote. (c, d) Representative WB (c) and quantitative analysis (d) showing phosphorylation of MAP4 in MAP4 KI mice aged at 10 to 14 weeks. The graph showed the mean ± SEM (n = 6). ***P < 0.001, ns, not statistically significant. P values were derived from two-tailed Student's t-test.

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: Genetic mutations of phosphorylation sites of MAP4. (a) Schematic of MAP4 KI mice. CRISPR/Cas9 technique was used to generate mouse strain with mutated MAP4 by knocking in the coding sequence (S667A, S737E and S760E) in the ninth exon of MAP4 gene by homologous recombination. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats-associated protein 9. (b) Representative polymerase chain reaction identification showing the HO, HE and WT genotype mice. HO, homozygote; HE, heterozygote. (c, d) Representative WB (c) and quantitative analysis (d) showing phosphorylation of MAP4 in MAP4 KI mice aged at 10 to 14 weeks. The graph showed the mean ± SEM (n = 6). ***P < 0.001, ns, not statistically significant. P values were derived from two-tailed Student's t-test.

Techniques: CRISPR, Sequencing, Homologous Recombination, Polymerase Chain Reaction, Derivative Assay, Two Tailed Test

MAP4 phosphorylation induces pathological cardiac hypertrophy. (a, b, c) Heart weight (HW), HW normalized by body weight (HW/BW) and HW normalized by tibia length (HW/TL) of two mouse models were determined. n = 10. (d) Heart histological sections with haematoxylin-eosin staining. Bar, 500 μm. n = 6. (e, f) FITC-WGA staining of LV heart sections, and cross sectional areas were analyzed in two mouse models. n = 6 (100 cells counted/animal). Bar, 20 μm. (g, h) Protein markers of hypertrophy in LV heart tissues were detected using WB analysis. n = 6. (i) Blood markers of cardiac damage from two mouse models were showed by cTnT and CK-MB. n = 9–11. (j) Representative TEM images showing ultrastructure of LV heart sarcomere (black circle represented myofilaments) between two mouse models. Bar, 2 μm. n = 6. (k, l) Protein markers of hypertrophy in CMs from two mouse models were detected with or without MAP4(Ala) transfection. n = 5. The experiment was conducted 5 times. (m) Cross sectional areas of CMs were analyzed by cTnI and FITC-WGA co-staining. n = 5. The experiment was conducted 5 times. ANP, atrial natriuretic peptide; MYH7, myosin heavy chain 7; BNP, brain natriuretic peptide; CMs, cardiomyocytes. Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not statistically significant. P values were derived from one-way ANOVA with Bonferroni's post-test.

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: MAP4 phosphorylation induces pathological cardiac hypertrophy. (a, b, c) Heart weight (HW), HW normalized by body weight (HW/BW) and HW normalized by tibia length (HW/TL) of two mouse models were determined. n = 10. (d) Heart histological sections with haematoxylin-eosin staining. Bar, 500 μm. n = 6. (e, f) FITC-WGA staining of LV heart sections, and cross sectional areas were analyzed in two mouse models. n = 6 (100 cells counted/animal). Bar, 20 μm. (g, h) Protein markers of hypertrophy in LV heart tissues were detected using WB analysis. n = 6. (i) Blood markers of cardiac damage from two mouse models were showed by cTnT and CK-MB. n = 9–11. (j) Representative TEM images showing ultrastructure of LV heart sarcomere (black circle represented myofilaments) between two mouse models. Bar, 2 μm. n = 6. (k, l) Protein markers of hypertrophy in CMs from two mouse models were detected with or without MAP4(Ala) transfection. n = 5. The experiment was conducted 5 times. (m) Cross sectional areas of CMs were analyzed by cTnI and FITC-WGA co-staining. n = 5. The experiment was conducted 5 times. ANP, atrial natriuretic peptide; MYH7, myosin heavy chain 7; BNP, brain natriuretic peptide; CMs, cardiomyocytes. Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not statistically significant. P values were derived from one-way ANOVA with Bonferroni's post-test.

Techniques: Staining, Transfection, Derivative Assay

In vivo cardiac function of WT and MAP4 KI mice at different ages

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: In vivo cardiac function of WT and MAP4 KI mice at different ages

Techniques: In Vivo

In vivo blood pressure of WT and MAP4 KI mice at different ages.

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: In vivo blood pressure of WT and MAP4 KI mice at different ages.

Techniques: In Vivo

The effect of MAP4 phosphorylation on cardiomyocyte apoptosis. (a, b) WB analysis displayed protein level of caspase-3 (cleaved) in two mouse models. n = 6. (c, d) Representative images and quantitative analysis depicted LV cardiac apoptosis using TUNEL staining. n = 6. (e, f) Detection of mitochondrial Cyt-c release by WB. n = 6. (g) Representative TEM images showing ultrastructure of CMs apoptosis and mitochondrial disruption (black square represented mitochondria, black arrows indicated chromatin margination and mitochondrial myelin-like structure). Bar, 2 μm. n = 6. (h, i) Caspase-3 and t-caspase-3 in CMs from two mouse models with or without MAP4(Ala) were detected by WB. n = 5. The experiment was conducted 5 times. (j, k) Detection of mitochondrial Cyt-c release in CMs from two mouse models with or without MAP4(Ala) transfection. n = 5. The experiment was conducted 5 times. (l, m) Detection of CMs apoptosis by cTnI and fluorescein-TUNEL co-staining. Bar, 10 μm. n = 5. The experiment was conducted 5 times. (n) Observation of CMs mitochondria with or without MAP4(Ala) transfection by TEM (Black square represented mitochondria). Bar, 1 μm. The experiment was conducted 5 times. t-caspase-3, total caspase-3; Cyt-c, cytochrome c ; mito, mitochondria. Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: The effect of MAP4 phosphorylation on cardiomyocyte apoptosis. (a, b) WB analysis displayed protein level of caspase-3 (cleaved) in two mouse models. n = 6. (c, d) Representative images and quantitative analysis depicted LV cardiac apoptosis using TUNEL staining. n = 6. (e, f) Detection of mitochondrial Cyt-c release by WB. n = 6. (g) Representative TEM images showing ultrastructure of CMs apoptosis and mitochondrial disruption (black square represented mitochondria, black arrows indicated chromatin margination and mitochondrial myelin-like structure). Bar, 2 μm. n = 6. (h, i) Caspase-3 and t-caspase-3 in CMs from two mouse models with or without MAP4(Ala) were detected by WB. n = 5. The experiment was conducted 5 times. (j, k) Detection of mitochondrial Cyt-c release in CMs from two mouse models with or without MAP4(Ala) transfection. n = 5. The experiment was conducted 5 times. (l, m) Detection of CMs apoptosis by cTnI and fluorescein-TUNEL co-staining. Bar, 10 μm. n = 5. The experiment was conducted 5 times. (n) Observation of CMs mitochondria with or without MAP4(Ala) transfection by TEM (Black square represented mitochondria). Bar, 1 μm. The experiment was conducted 5 times. t-caspase-3, total caspase-3; Cyt-c, cytochrome c ; mito, mitochondria. Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Techniques: TUNEL Assay, Staining, Transfection, Derivative Assay

The effects of MAP4 phosphorylation on cardiac fibrosis. (a) Representative TEM images showing ultrastructure of LV cardiac fibrosis (black arrows indicated collagenous fibers). Bar, 2 μm. n = 6. (b, c) LV heart histological sections with Masson's trichrome staining in two mouse models. Bar, 50 μm. n = 6. (d, e) WB analysis showing protein levels of collagen (COL) 1A1, 1A2, 3A1 and fibronectin (FN) in two mouse models. n = 6. (f, g) WB analysis showing protein levels of α-SMA in two mouse models. n = 6. (h, i) Detection of α-SMA by histochemical staining (black arrows indicated α-SMA) in the LV heart tissue of WT and MAP4 KI mice. Bar, 200 μm. n = 6. (j, k) ECM protein markers were detected with or without MAP4(Ala) transfection in primary neonatal mouse myocardial fibroblasts. n = 5. The experiment was conducted 5 times. α-SMA, α-smooth muscle actin. Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: The effects of MAP4 phosphorylation on cardiac fibrosis. (a) Representative TEM images showing ultrastructure of LV cardiac fibrosis (black arrows indicated collagenous fibers). Bar, 2 μm. n = 6. (b, c) LV heart histological sections with Masson's trichrome staining in two mouse models. Bar, 50 μm. n = 6. (d, e) WB analysis showing protein levels of collagen (COL) 1A1, 1A2, 3A1 and fibronectin (FN) in two mouse models. n = 6. (f, g) WB analysis showing protein levels of α-SMA in two mouse models. n = 6. (h, i) Detection of α-SMA by histochemical staining (black arrows indicated α-SMA) in the LV heart tissue of WT and MAP4 KI mice. Bar, 200 μm. n = 6. (j, k) ECM protein markers were detected with or without MAP4(Ala) transfection in primary neonatal mouse myocardial fibroblasts. n = 5. The experiment was conducted 5 times. α-SMA, α-smooth muscle actin. Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Techniques: Staining, Transfection, Derivative Assay

$MAP4 phosphorylation induces MT disassembly and mitochondrial translocation of p-MAP4. (a, b) WB analysis was used to detect polymerized (poly) or free tubulin in two mouse models. VDAC and GAPDH were used as the internal controls for poly and free tubulin, respectively. n = 6. (c) Determination of MAP4 binding to tubulin in two mouse models by immunoprecipitation (IP). n = 6. (d, e) WB analysis showing the levels of p-M in mitochondria fractions and fractions apart from mitochondria in two mouse models. n = 6. (f, g) Poly/free tubulin in CMs were detected by WB. n = 5. The experiment was conducted 5 times. (h) Representative confocal immunofluorescence images showing the MTs organization of the CMs. Bar, 10 μm. The experiment was conducted 5 times. The inserts showed high magnification images of the representative MT network. (i, j) Detection of p-M translocation to mitochondria with or without MAP4(Ala) transfection in CMs. n = 5. The experiment was conducted 5 times. The Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.$

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: MAP4 phosphorylation induces MT disassembly and mitochondrial translocation of p-MAP4. (a, b) WB analysis was used to detect polymerized (poly) or free tubulin in two mouse models. VDAC and GAPDH were used as the internal controls for poly and free tubulin, respectively. n = 6. (c) Determination of MAP4 binding to tubulin in two mouse models by immunoprecipitation (IP). n = 6. (d, e) WB analysis showing the levels of p-M in mitochondria fractions and fractions apart from mitochondria in two mouse models. n = 6. (f, g) Poly/free tubulin in CMs were detected by WB. n = 5. The experiment was conducted 5 times. (h) Representative confocal immunofluorescence images showing the MTs organization of the CMs. Bar, 10 μm. The experiment was conducted 5 times. The inserts showed high magnification images of the representative MT network. (i, j) Detection of p-M translocation to mitochondria with or without MAP4(Ala) transfection in CMs. n = 5. The experiment was conducted 5 times. The Data were shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Techniques: Translocation Assay, Binding Assay, Immunoprecipitation, Immunofluorescence, Transfection, Derivative Assay

Role of p38/MAPK activation in MAP4 phosphorylation-induced cardiac hypertrophy, apoptosis and fibrosis. (a) Detection of p-M and p-P38 with or without SB203580 (SB) (5 μM) in CMs. n = 5. (b) Protein markers of hypertrophy and p-P38 were detected using WB. n = 5. (c) Cross sectional areas of CMs were analyzed using cTnI and FITC-WGA co-staining. n = 5. (d and e) CMs apoptosis was detected by cTnI and fluorescein-TUNEL co-staining. Bar, 10 μm. n = 5. (f) Protein levels of caspase-3 and t-caspase-3 were analyzed using WB. n = 5. (g) Detection of Cyt-c release from mitochondria. n = 5. (h) CMs mitochondria observed by TEM (black square indicated representative mitochondria). Bar, 1 μm. (i) Markers of ECM were examined using WB. n = 5. (j) Poly/free tubulin was analyzed using WB. n = 5. (k) Representative confocal immunofluorescence images showing the MTs organization of the CMs. Bar, 10 μm. The inserts showed high magnification images of the peripheral MT network. (l) Detection of p-M translocation to mitochondria with or without SB. n = 5. (m and n) CMs apoptosis was detected by cTnI and fluorescein-TUNEL co-staining after the cells were transiently transfected with MAP4(Ala), MKK6(Glu) or both. Bar, 10 μm. n = 5. (o) Representative confocal immunofluorescence images showing the MTs organization of the CMs after the cells were transiently transfected with MAP4(Ala), MKK6(Glu) or both. Bar, 10 μm. The inserts showed high magnification images of the peripheral MT network. (p) CMs mitochondria observed by TEM (black square indicated representative mitochondria) after the cells were transiently transfected with MAP4(Ala), MKK6(Glu) or both. Bar, 1 μm. Data were shown as mean ± SEM. The experiment was conducted 5 times. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Journal: EBioMedicine

Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

doi: 10.1016/j.ebiom.2018.10.017

Figure Lengend Snippet: Role of p38/MAPK activation in MAP4 phosphorylation-induced cardiac hypertrophy, apoptosis and fibrosis. (a) Detection of p-M and p-P38 with or without SB203580 (SB) (5 μM) in CMs. n = 5. (b) Protein markers of hypertrophy and p-P38 were detected using WB. n = 5. (c) Cross sectional areas of CMs were analyzed using cTnI and FITC-WGA co-staining. n = 5. (d and e) CMs apoptosis was detected by cTnI and fluorescein-TUNEL co-staining. Bar, 10 μm. n = 5. (f) Protein levels of caspase-3 and t-caspase-3 were analyzed using WB. n = 5. (g) Detection of Cyt-c release from mitochondria. n = 5. (h) CMs mitochondria observed by TEM (black square indicated representative mitochondria). Bar, 1 μm. (i) Markers of ECM were examined using WB. n = 5. (j) Poly/free tubulin was analyzed using WB. n = 5. (k) Representative confocal immunofluorescence images showing the MTs organization of the CMs. Bar, 10 μm. The inserts showed high magnification images of the peripheral MT network. (l) Detection of p-M translocation to mitochondria with or without SB. n = 5. (m and n) CMs apoptosis was detected by cTnI and fluorescein-TUNEL co-staining after the cells were transiently transfected with MAP4(Ala), MKK6(Glu) or both. Bar, 10 μm. n = 5. (o) Representative confocal immunofluorescence images showing the MTs organization of the CMs after the cells were transiently transfected with MAP4(Ala), MKK6(Glu) or both. Bar, 10 μm. The inserts showed high magnification images of the peripheral MT network. (p) CMs mitochondria observed by TEM (black square indicated representative mitochondria) after the cells were transiently transfected with MAP4(Ala), MKK6(Glu) or both. Bar, 1 μm. Data were shown as mean ± SEM. The experiment was conducted 5 times. *P < 0.05, **P < 0.01, ***P < 0.001. P values were derived from one-way ANOVA with Bonferroni's post-test.

Techniques: Activation Assay, Staining, TUNEL Assay, Immunofluorescence, Translocation Assay, Transfection, Derivative Assay

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