collagen 3  (Proteintech)

Journal: Cell Cycle

Article Title: M2 macrophages promote PKM2 production in fibroblasts to alleviate UVB-induced photoaging

doi: 10.1080/15384101.2025.2514988

Figure Lengend Snippet: M2 macrophages alleviate the aging extent of photoaged L929 cells. (a, b, c) RAW264.7 cells exhibit morphological changes and differential expression of iNOS, Arg-1, CD86, and CD206 after treatment with LPS + IFN-γ (M1) or IL-4 + IL-13 (M2). (d) Photoaged cells treated with M2 macrophage CM show significantly enhanced viability compared to those treated with M1 macrophage CM. (e) M2 macrophages CM reduces the proportion of SA-β-GAL positive cells. (f) M2 macrophage CM increases the synthesis of COL-1, COL-3, and PKM2 in photoaged cells, with a decrease in P16, P21, P53, Caspase-3 and ATM production. (g) Immunofluorescence staining shows that photoaged cells treated with M2 macrophages CM produce more PKM2. (h) Cell survival is assessed after different treatments. M1 macrophage CM results in increased cell death, whereas M2 macrophage CM and control group show no significant differences in cell survival. If the data were normally distributed with equal variances, one-way ANOVA was performed. If the data were normally distributed but with unequal variances, Welch’s one-way ANOVA was used. For non-normally distributed data, the Kruskal–Wallis test was conducted. n ≥ 3 (biological replicates).

Article Snippet: Membranes were blocked with 5% Bovine Serum Albumin (Solarbio, China) diluted in TBST (0.1% Tween 20 in Tris-buffered saline) for 1 h, then incubated overnight at 4°C with primary antibodies against collagen type 1 (COL-1) (66761–1-Ig, 1:2000, Proteintech, China), collagen type 3 (COL-3) (22734–1-AP, 1:1000, Proteintech, China), P16 (sc -74,400, 1:500, Santa Cruz Biotechnology, USA), P21 (28248–1-AP, 1:1000, Proteintech, China), P53 (32532S, 1:1000, Cell Signalling Technology, USA), PKM2 (15822–1-AP, 1:2000, Proteintech, China), Caspase-3 (19677–1-AP, 1:1000, Proteintech, China), ATM (A19650, 1:1000, ABclonal, China), TGF-β1 (BY0105, 1:1000, Abways, China), SMAD2 (ab33875, 1:1000, Abcam, UK), p-SMAD2 (3108T, 1:1000, Cell Signaling Technology, USA), CCL1 (YP-Ab -06,187, 1:1000, Research Cloud Biology, China), CCR8 (A4288, 1:1000, ABclonal, China), ACTIN (AC026, 1:100000, ABclonal, China), GAPDH (10494–1-AP, 1:10000, Proteintech, China) and VINCULIN (CY5164, 1:5000, Abways, China).

Techniques: Quantitative Proteomics, Immunofluorescence, Staining, Control

Journal: Cell Cycle

Article Title: M2 macrophages promote PKM2 production in fibroblasts to alleviate UVB-induced photoaging

doi: 10.1080/15384101.2025.2514988

Figure Lengend Snippet: PKM2 overexpression alleviates senescence in photoaged L929 cells. (a) PKM2 is stably overexpressed in L929 cells. (b) PKM2 OE cells exhibit greater resistance to photoaging, with fewer senescent cells compared to controls. (c) After UVB treatment, PKM2 OE cells retain more cell viability. (d) PKM2 OE cells produce more COL-1 and COL-3 compared to NC cells, with lower levels of P16, P21, P53, Caspase-3, and ATM proteins. (e) mRNA analysis shows that PKM2 OE cells have increased transcription of PKM2, COL-1, and COL-3, consistent with the Western blot results. There is no significant difference in P53 and P21 levels between PKM2 OE and NC cells. (f) Treatment with PKM2-IN-1 induces a transition from normal cells to a senescent phenotype. (g) PKM2-IN-1 reduces the cell viability of L929 cells If variances were equal, comparisons between two groups were conducted using the unpaired t-test, and comparisons among multiple groups were performed using one-way ANOVA. If variances were unequal, Welch’s correction was applied to reanalyze the data. For data that did not meet the assumption of normality, the Mann–Whitney U-test was used for two-group comparisons, and the Kruskal–Wallis test was applied for comparisons among multiple groups. n ≥ 3 (biological replicates).

Article Snippet: Membranes were blocked with 5% Bovine Serum Albumin (Solarbio, China) diluted in TBST (0.1% Tween 20 in Tris-buffered saline) for 1 h, then incubated overnight at 4°C with primary antibodies against collagen type 1 (COL-1) (66761–1-Ig, 1:2000, Proteintech, China), collagen type 3 (COL-3) (22734–1-AP, 1:1000, Proteintech, China), P16 (sc -74,400, 1:500, Santa Cruz Biotechnology, USA), P21 (28248–1-AP, 1:1000, Proteintech, China), P53 (32532S, 1:1000, Cell Signalling Technology, USA), PKM2 (15822–1-AP, 1:2000, Proteintech, China), Caspase-3 (19677–1-AP, 1:1000, Proteintech, China), ATM (A19650, 1:1000, ABclonal, China), TGF-β1 (BY0105, 1:1000, Abways, China), SMAD2 (ab33875, 1:1000, Abcam, UK), p-SMAD2 (3108T, 1:1000, Cell Signaling Technology, USA), CCL1 (YP-Ab -06,187, 1:1000, Research Cloud Biology, China), CCR8 (A4288, 1:1000, ABclonal, China), ACTIN (AC026, 1:100000, ABclonal, China), GAPDH (10494–1-AP, 1:10000, Proteintech, China) and VINCULIN (CY5164, 1:5000, Abways, China).

Techniques: Over Expression, Stable Transfection, Western Blot, MANN-WHITNEY