Journal: Frontiers in Medicine

Article Title: Downregulation of RORα by alcohol promotes TGFβ and α-SMA expression in mouse lung fibroblasts

doi: 10.3389/fmed.2026.1719787

Figure Lengend Snippet: Chronic ethanol ingestion altered the oscillation patterns of key circadian signaling molecules in mouse lungs. Graphs summarize expression profiles of key circadian regulators in mouse lungs at 4 h intervals over 24 h for ethanol-fed (EtOH, red) and control-fed (CON, blue) groups. Panels show expressions of (A) Bmal1 , (B) Clock , (C) Rorα , and (D) Rev-erbα analyzed by qPCR. N = 6–9 lungs per group. The circadian oscillation patterns of the four genes from ethanol-fed mouse lungs significantly changed compared to lungs from control-fed mice. Data are presented as mean ± SE. * Indicates change with p < 0.05 compared to control-fed (CON) group at the same time point.

Article Snippet: The membranes were blocked with 5% milk/TBS with 0.1% Tween 20 then incubated with antibodies for α-SMA (ab5694 at 1:3000, Abcam Waltham, MA), TGFβ (555,052 at 1:500, BD Pharmingen), fibronectin (sc-9068 at 1:1000, Santa Cruz Biotechnology), BMAL1 (MA5-25133 at 1:500, Invitrogen), CLOCK (5,157 at 1:1000, Cell Signaling), RORα (82930-1-RR at 1:1000, Proteintech), REV-ERBα (14506-1-AP at 1:1000, Proteintech), Per1 (sc-398890 at 1:500, Santa Cruz Biotechnology), Per2 (PA5-100107 at 1:500, Invitrogen), or GAPDH (G9545 at 1:50,000, Sigma-Aldrich) respectively, then incubated with an appropriate secondary antibody and exposed to Clarity Western ECL substrate (Bio-Rad Laboratories).

Techniques: Expressing, Control

Journal: Frontiers in Medicine

Article Title: Downregulation of RORα by alcohol promotes TGFβ and α-SMA expression in mouse lung fibroblasts

doi: 10.3389/fmed.2026.1719787

Figure Lengend Snippet: Chronic ethanol ingestion disrupts temporal protein expression of key circadian signaling molecules in the mouse lungs. Protein levels from mouse lungs collected at ZT0 and ZT12 were analyzed for (A) BMAL1 (75 kD), (B) CLOCK (100 kD), (C) RORα (60 kD), and (D) REV-ERBα (75 kD) and normalized with GAPDH (36 kD) levels in the same blot by western analysis. All data reported as fold-change compared to control-fed at ZT0 (CON) group. Representative western immunoblots are shown above the corresponding graphs. N = 6 per group. Data are presented as mean ± SE. # indicates change with p < 0.005 compared with ZT0 CON group and * indicates changes with p < 0.05 compared with ZT12 CON group.

Article Snippet: The membranes were blocked with 5% milk/TBS with 0.1% Tween 20 then incubated with antibodies for α-SMA (ab5694 at 1:3000, Abcam Waltham, MA), TGFβ (555,052 at 1:500, BD Pharmingen), fibronectin (sc-9068 at 1:1000, Santa Cruz Biotechnology), BMAL1 (MA5-25133 at 1:500, Invitrogen), CLOCK (5,157 at 1:1000, Cell Signaling), RORα (82930-1-RR at 1:1000, Proteintech), REV-ERBα (14506-1-AP at 1:1000, Proteintech), Per1 (sc-398890 at 1:500, Santa Cruz Biotechnology), Per2 (PA5-100107 at 1:500, Invitrogen), or GAPDH (G9545 at 1:50,000, Sigma-Aldrich) respectively, then incubated with an appropriate secondary antibody and exposed to Clarity Western ECL substrate (Bio-Rad Laboratories).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Medicine

Article Title: Downregulation of RORα by alcohol promotes TGFβ and α-SMA expression in mouse lung fibroblasts

doi: 10.3389/fmed.2026.1719787

Figure Lengend Snippet: Ethanol exposure in vitro inhibited key molecules in the circadian signaling pathway. Gene expression levels from synchronized and unsynchronized murine primary lung fibroblasts (mPLFs) ± ethanol (EtOH, 60 mM; 24 h) were analyzed for: (A,B) Bmal1 , (C,D) Clock , (E,F) Rorα , and (G,H) Rev-erbα mRNA expression. Ethanol exposure significantly inhibited BMAL1, RORα, and Rev-erbα gene expressions while upregulating Clock gene expression. N = 6–10 per group. Data are presented as mean ± SE. * Indicates change with p < 0.05 compared to control (CON) group.

Article Snippet: The membranes were blocked with 5% milk/TBS with 0.1% Tween 20 then incubated with antibodies for α-SMA (ab5694 at 1:3000, Abcam Waltham, MA), TGFβ (555,052 at 1:500, BD Pharmingen), fibronectin (sc-9068 at 1:1000, Santa Cruz Biotechnology), BMAL1 (MA5-25133 at 1:500, Invitrogen), CLOCK (5,157 at 1:1000, Cell Signaling), RORα (82930-1-RR at 1:1000, Proteintech), REV-ERBα (14506-1-AP at 1:1000, Proteintech), Per1 (sc-398890 at 1:500, Santa Cruz Biotechnology), Per2 (PA5-100107 at 1:500, Invitrogen), or GAPDH (G9545 at 1:50,000, Sigma-Aldrich) respectively, then incubated with an appropriate secondary antibody and exposed to Clarity Western ECL substrate (Bio-Rad Laboratories).

Techniques: In Vitro, Gene Expression, Expressing, Control

Journal: Frontiers in Medicine

Article Title: Downregulation of RORα by alcohol promotes TGFβ and α-SMA expression in mouse lung fibroblasts

doi: 10.3389/fmed.2026.1719787

Figure Lengend Snippet: Ethanol treatment inhibited core circadian signaling molecules in murine lung fibroblasts. Protein levels from synchronized and unsynchronized murine primary lung (mPLFs) ± ethanol (EtOH, 60 mM; 72 h) were analyzed for: (A,B) BMAL1 (75 kD), (C,D) CLOCK (100 kD), (E,F) RORα (60 kD), and (G,H) REV-ERBα (75 kD) were normalized to GAPDH (36 kD) levels from the same blot and reported as fold-change compared to the untreated (CON) group. Ethanol treatment significantly decreased the BAML1 and RORα protein levels in both synchronized and unsynchronized conditions, decreased REV-ERBα in the unsynchronized condition while it did not change the CLOCK protein expression in either condition. Representative western immunoblots are shown above the graphs. N = 8–15 per group. Data are presented as mean ± SE. * Indicates change with p < 0.05 compared to control (CON) group.

Article Snippet: The membranes were blocked with 5% milk/TBS with 0.1% Tween 20 then incubated with antibodies for α-SMA (ab5694 at 1:3000, Abcam Waltham, MA), TGFβ (555,052 at 1:500, BD Pharmingen), fibronectin (sc-9068 at 1:1000, Santa Cruz Biotechnology), BMAL1 (MA5-25133 at 1:500, Invitrogen), CLOCK (5,157 at 1:1000, Cell Signaling), RORα (82930-1-RR at 1:1000, Proteintech), REV-ERBα (14506-1-AP at 1:1000, Proteintech), Per1 (sc-398890 at 1:500, Santa Cruz Biotechnology), Per2 (PA5-100107 at 1:500, Invitrogen), or GAPDH (G9545 at 1:50,000, Sigma-Aldrich) respectively, then incubated with an appropriate secondary antibody and exposed to Clarity Western ECL substrate (Bio-Rad Laboratories).

Techniques: Expressing, Western Blot, Control

Journal: Biomolecules

Article Title: Vitamin B12 Protects Against Early Diabetic Kidney Injury and Alters Clock Gene Expression in Mice

doi: 10.3390/biom15121689

Figure Lengend Snippet: Effect of B12 on circadian gene rhythmicity in cultured BU.MPT cells. Cells were treated with B12 (+B12) or the vehicle (-B12) and gene expression was assessed by RT-qPCR across the circadian cycle. Control cells displayed strong oscillations in ( A ) Cry1 , ( B ) Nampt , ( C ) Elmo1 , ( D ) Bmal1 , ( E ) Per1 , and ( F ) Dbp , whereas B12 treatment altered the phase and amplitude of these rhythms. Data were normalized to β-actin expression, analyzed using the 2 −ΔΔCt method, and expressed as linear expression ratios, with the mean expression at 36H of the individual gene illustrated without B12 set to 1.0. Total protein extracts from BU.MPT cells synchronized with dexamethasone were collected at selected time points and analyzed by SDS–PAGE followed by ( H ) Western blotting for Bmal1 and Per1. ( G ) Graphs show the band intensity ratios normalized to β-actin. The experiment was performed once, and band quantification was carried out using Fiji ImageJ 1.54p (Wayne Rasband and contributors, National Institute of Health, Bethesda, MD, USA). Original figures can be found in .

Article Snippet: Membranes were incubated overnight at 4 °C with primary antibodies against Bmal1 (1:1000, #14020, Cell Signaling) and Per1 (1:1000, #13463-1-AP, Proteintech, Rosemont, IL, USA), followed by a 30 min incubation with HRP-linked anti-rabbit IgG secondary antibody (1:1000, #7074, Cell Signaling).

Techniques: Cell Culture, Gene Expression, Quantitative RT-PCR, Control, Expressing, SDS Page, Western Blot