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

Journal: Nutrition & Metabolism

Article Title: A low-protein diet exerts a beneficial effect on diabetic status and prevents diabetic nephropathy in Wistar fatty rats, an animal model of type 2 diabetes and obesity

doi: 10.1186/s12986-018-0255-1

Figure Lengend Snippet: Plasma FGF21 and HMW adiponectin levels and expression of UCP1 in BAT are elevated in LPD-fed Wistar fatty rats. ( a ) Fasting or postprandial plasma FGF21, ( b ) HMW adiponectin, ( c ) and mRNA expression of Ucp1 in BAT after the 24-week dietary intervention ( n = 3 each). ( d ) A representative photograph of immunoblotting for UCP1 in BAT is shown. ( e ) Quantitative ratios of UCP1 to β-actin in BAT after the 24-week dietary intervention ( n = 3 each). The data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 for the indicated comparison. n.s.: not significant

Article Snippet: TaqMan probes for type 3 collagen (Col3) (Product ID: Rn01437681), Cd68 (Rn01495634), interleukin-6 (Il6) (Rn01410330), C-C motif chemokine ligand 2 (Ccl2) (Rn00580555), Toll-like receptor 4 (Tlr4) (Rn00569848), kidney injury molecule-1 (Kim-1) (RN00597703) and uncoupling protein1 (Ucp1) (Rn00562126) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) [ ].

Techniques: Expressing, Western Blot

Journal: Nutrition & Metabolism

Article Title: A low-protein diet exerts a beneficial effect on diabetic status and prevents diabetic nephropathy in Wistar fatty rats, an animal model of type 2 diabetes and obesity

doi: 10.1186/s12986-018-0255-1

Figure Lengend Snippet: The LPD prevents renal injuries, the impairment of autophagy and the activation of mTORC1 in the kidneys of Wistar fatty rats. ( a ) Representative microphotographs of Masson’s trichrome (MT) staining of glomeruli (scale bar: 100 μm) and the tubulo-interstitial area (scale bar: 1 mm) after the 24-week dietary intervention. ( b ) Glomerular fibrotic score ( n = 3) and ( c ) tubulo-interstitial fibrotic score ( n = 3), assessed using MT staining after the 24-week dietary intervention. mRNA expression of Col3 ( d ), Kim-1 ( e ), Cd68 ( f ), Ccl12 ( g ), Tlr4 ( h ) and Il6 ( i ) in the renal cortex, adjusted to 18 s levels, after the 24-week dietary intervention ( n = 6 each). ( j ) Representative photographs of immunoblotting for p62, β-actin, p-S6RP and S6RP in the renal cortex after the 24-week dietary intervention ( n = 3). Quantitative ratios of p62 to β-actin ( k ) and p-S6RP to S6RP ( l ) ( n = 3). Col3: type 3 collagen, Kim-1: kidney injury molecule-1, Ccl2: C-C motif chemokine ligand 2, Tlr4: Toll-like receptor 4, Il6: interleukin-6, p-S6RP: phospho-S6 ribosomal protein, S6RP: S6 ribosomal protein. The data are shown as the mean ± SD. * p < 0.05, ** p < 0.01 and *** p < 0.001 for the indicated comparison. n.s.: not significant

Article Snippet: TaqMan probes for type 3 collagen (Col3) (Product ID: Rn01437681), Cd68 (Rn01495634), interleukin-6 (Il6) (Rn01410330), C-C motif chemokine ligand 2 (Ccl2) (Rn00580555), Toll-like receptor 4 (Tlr4) (Rn00569848), kidney injury molecule-1 (Kim-1) (RN00597703) and uncoupling protein1 (Ucp1) (Rn00562126) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) [ ].

Techniques: Activation Assay, Staining, Expressing, Western Blot

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: UVB-treated L929 cells showed senescence and reduced PKM2 levels. (a) L929 cells were exposed to UVB irradiation for 4 days in culture dishes. (b) After UVB treatment, L929 cells exhibited positive SA-β-GAL staining, whereas control cells were negative. (c) UVB exposure led to a marked decrease in cell viability. (d, e) Collagen and PKM2 production were reduced in UVB-treated L929 cells, while the expression of P16, P21, and P53 increased. Additionally, the expression of Caspase-3 and ATM was also elevated. (f) Immunofluorescence staining revealed reduced collagen and PKM2 production following UVB irradiation. If the data were normally distributed with equal variances, unpaired t-test was performed. If the data were normally distributed but with unequal variances, Welch’s t-test was used. For non-normally distributed data, the Mann–Whitney U-test was applied. 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: Irradiation, Staining, Control, Expressing, Immunofluorescence, MANN-WHITNEY

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

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: CCL1 secreted by M2 macrophages enhances PKM2 production in photoaged L929 cells. (a) Comparison of CCL1 levels in M1/M2 macrophages and their supernatants. (b) Immunofluorescence staining shows that M2 macrophages produce higher levels of CCL1. (c,d) Western blot and immunofluorescence staining reveal that both L929 and photoaged L929 cells express CCR8. (e) Exogenous CCL1 reduces the proportion of SA-β-GAL positive senescent cells. (f, g) Exogenous CCL1 increases the production of collagen and PKM2, while reducing the activation of P16, P21, P53, Caspase-3, and ATM in photoaged L929 cells. (h, i) The CCR8 antagonist R243 partially reverses the effects of M2 macrophage CM on photoaged 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: Comparison, Immunofluorescence, Staining, Western Blot, Activation Assay, MANN-WHITNEY