p gsk3 β  (Proteintech)

Journal: Cell Death Discovery

Article Title: Mitophagy associated self-degradation of phosphorylated MAP4 guarantees the migration and proliferation responses of keratinocytes to hypoxia

doi: 10.1038/s41420-023-01465-3

Figure Lengend Snippet: A , B Representative bands and corresponding quantification of p-MAP4, p-MAP4 (S737), and p-MAP4(S760) in the skin tissues of MAP4 KI mice. *** p < 0.001 versus the WT group. n = 5. C Representative bands and corresponding statistical analysis of LC3 in skin tissues. ** p < 0.01 versus the WT group. n = 5. D , E Representative images and corresponding quantifications of autophagosomes. Yellow arrows pointed to autophagosomes. Scale bar = 0.5 μm. *** p < 0.001 versus WT group. n = 5. F Representative bands and corresponding statistical analysis of LC3 in primary MKs. ** p < 0.01 versus the WT group. n = 5. G , H Representative bands and corresponding quantifications of autophagosomes in primary MKs. Yellow arrows pointed to autophagosomes/mitophagosomes. Scale bar = 0.5 μm. *** p < 0.001 versus WT group. n = 5. I , J Representative images and corresponding quantifications of GFP-LC3 positive keratinocytes. *** p < 0.001 versus WT group. n = 5. K , L Representative images and corresponding quantifications of autophagosome or autolysosome in keratinocytes measured by mRFP-GFP-LC3. Scale bar = 10 μm. * p < 0.05 and *** p < 0.001 versus WT group. n = 5. M Representative images of co-staining p-MAP4 with mitophagosomes. Scale bar = 10 μm. n = 5. N , O Representative bands and corresponding quantifications of p-MAP4 in keratinocytes treated by hypoxia with or without 30 min pre-treatment of 40 μM MDIVI-1. * p < 0.05 and *** p < 0.001 versus Con group. ### p < 0.001 versus Hypoxia (24 h) group. n = 5.

Article Snippet: MAP4 (1:1000, Affinity, USA), p-MAP4 (1:1000, GL Biochem, CHN), p-MAP4 (S760) (1:1000, GL Biochem, CHN), p-MAP4 (S737) (1:1000, GL Biochem, CHN), LC3 (1:1000, sigma, USA), p62 (1:1000, sigma, USA), Beclin1 (1:1000, CST, USA), Bcl-2 (1:1000, CST, USA), VDAC1 (1:1000, Millipore, USA), Tomm20 (1:1000, Affinity, USA), TIM23 (1:1000, Affinity, USA), GM130 (1:1000, Affinity, USA), Calnexin (1:1000, Affinity, USA), HA-tag (1:1000, proteintech, USA) and β-Actin (1:1000, proteintech, USA).

Techniques: Staining

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: TAOK1 promotes the interaction between TBK1 and IRF3 by trafficking TBK1 along microtubules. a HeLa cells co-transfected with Flag-IRF3 and Myc-TAOK1 plasmids, then pretreated with DMSO or colchicine (10 μm) for 1 h, followed by infection with or without VSV for 4 h. Confocal microscopy of co-localization between Flag-IRF3 (green) and endogeneous TBK1 (red). DAPI served as a marker of nuclei (blue). Scale bar, 10 μm. b Confocal microscopy of HeLa cells transfected with Myc-TAOK1, Myc-TAOK1 K57A, Myc-TAOK1C, and Myc-TAOK1N plasmids (green) followed by VSV infection for 4 h α-tubulin (red) was used to probe the microtubules. DAPI served as a marker of nuclei (blue). Scale bar, 10 μm. c Immunoblot analysis of phosphorylated and total MAP4 proteins in lysates of PMs obtained from WT (TAOK1 f/f ) and TAOK1cko (Lyz2 + TAOK1 f/f ) mice infected with VSV for indicated time. d Mouse PMs were pretreated with DMSO or colchicine (10 μm) for 1 h and then infected with VSV for 4 h. Immunoblot analysis of phosphorylated and total IRF3 and MAP4 proteins in cell lysates. e Mouse PMs were pretreated with DMSO or colchicine (10 μM) for 1 h and then infected with VSV for 4 h. Immunoblot analysis of endogenous signal adapters immunoprecipitated with antibody to TAOK1. * p < 0.05, ** p < 0.01. Data are representative of three independent experiments with similar results. ns, not significant; WCL, whole-cell lysates.

Article Snippet: Anti-p-MAP4 (Ser696) (#PA5-64526) was purchased from Invitrogen (Thermo Fisher Scientific, Scotts Valley, CA, USA).

Techniques: Transfection, Infection, Confocal Microscopy, Marker, Western Blot, Immunoprecipitation

Journal: Journal of Innate Immunity

Article Title: Ste20-Like Kinase TAOK1 Positively Regulates Antiviral Responses by Controlling the TBK1-IRF3 Signaling Axis

doi: 10.1159/000526324

Figure Lengend Snippet: A schematic diagram revealing the proposed mechanism by which TAOK1 positively regulates antiviral immune responses. (1) TAOK1 promoted virus-induced MAP4 phosphorylation and microtubule detachment; (2) TAOK1 bound more dynein instead of MAP4 upon virus infection and promoted the perinuclear transport of TBK1 along microtubules; (3) TAOK1 positively regulated antiviral signaling by promoting the TBK1-IRF3 interaction and IRF3 phosphorylation.

Article Snippet: Anti-p-MAP4 (Ser696) (#PA5-64526) was purchased from Invitrogen (Thermo Fisher Scientific, Scotts Valley, CA, USA).

Techniques: Infection

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.

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: 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.

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: 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.

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: 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.

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: 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.

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: 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.

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: Migration, Activation Assay

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.

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: 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.

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: 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.

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: 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.

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: 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.

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: 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.

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: Migration, Activation Assay