Table 1.
Primer sequences of all the genes investigated in this study.
Table 2.
List of primary and secondary antibodies used in this study.
Fig 1.
Differentiated HC11 cells displayed increased milk proteins and lipid levels.
HC11 cells were seeded in base media for 24 hours (UD). HC11 cells were seeded and supplemented with EGF and INS for 3 days (UD+EGF). Finally, HC11 cells were differentiated in base media containing PRL, INS and DEX, and allowed to proceed for 4 days in the absence (DF) or presence of the vehicle (MeOH). Cells from all conditions were processed for total RNA or protein isolation, as described in methods. The relative (compared to RPL0 and RPL8) mRNA levels of CSN2 (A) and WAP (B) were determined. Gene expression analysis was repeated with 6 distinct replicates. Protein expression of β-casein was assessed, as described in methods, using 15μg of total protein for each sample. (C) A representative blot of β-casein expression is shown along with a bar graph quantifying its expression, normalized to β-actin, for undifferentiated (UD), EGF supplemented (UD+EGF), differentiated (DF) and differentiated in the presence of vehicle (MeOH), based on 4 replicates. Lipid levels were assessed in undifferentiated cells (UD) and differentiated cells (DF), by conducting an Oil Red O (ORO) assay, as described in methods. Representative images of ORO-stained undifferentiated HC11 cells (UD) and differentiated cells (DF), taken with a phase-contrast microscope (20x magnification). (F) Lipid levels were quantified in cells, as described in the methods, from 6 distinct replicates of undifferentiated HC11 cells (UD) and differentiated cells (DF). Results were plotted as mean ± SEM and compared using either a student’s t-test (for groups ≤2), or one-way ANOVA (for groups ≥ 3). Statistically significant changes were represented by distinct letters on bar graphs.
Fig 2.
Differentiation increases the expression of mRNA for markers of lipid synthesis and alters mRNA levels of key ECS markers in HC11 cells.
HC11 cells were seeded in base media for 24 hours (UD) and collected for RNA isolation. HC11 cells were seeded in base media for 24 hours, supplemented with EGF and INS for 3 days, and differentiated in base media containing PRL, INS and DEX 4 days (DF). Undifferentiated and differentiated cells were processed for total RNA, as described in methods. The relative mRNA levels of FASN (A), FABP4 (B), GLUT1 (C), HK2 (D), PLIN2 (E), LPL (F), CNR2 (G), NAPE-PLD (H) and FAAH (I) were determined. Gene expression analysis was repeated with 6 distinct replicates. Results were represented as mean ± SEM and compared using a t-test. Statistically significant changes were represented by distinct letters on the bar graphs.
Fig 3.
High concentrations of THC and CBD reduced proliferation and viability of HC11 cells.
HC11 cells were seeded in base media for 24 hours, supplemented with EGF and INS for 3 days, and differentiated in base media containing PRL, INS and DEX, as described in methods, and treated with vehicle control (MeOH), or 0.01, 0.1, 1, 10, 20, 30, 100μM THC (A and C) or CBD (B and D) for 4 days. Changes in cellular proliferation and toxicity were evaluated using a MTS assay and LDH assay, respectively, as described in methods. (A-B) Changes in absorbances were recorded following a MTS assay and results were normalized to vehicle control (MeOH) as a percentage. (C-D) The relative cytotoxicity of THC and CBD was assessed using a LDH assay and percent cytotoxicity (compared to the vehicle control) was plotted. All experiments were repeated with 6 distinct replicates. Results were represented as mean ± SEM and compared using one-way ANOVA. Statistically significant changes were indicated by distinct letters.
Fig 4.
10μM THC and 10μM CBD reduced levels of milk proteins in HC11 cells.
HC11 cells were seeded in base media for 24 hours (UD) and processed for total RNA or protein, as described in the methods. HC11 cells were differentiated in base media containing PRL, INS and DEX, as described in methods, and treated with vehicle control (MeOH), 10μM THC or 10μM CBD for 4 days, and processed for total RNA or protein. The relative mRNA levels of CSN2 (A) and WAP (B) were determined. Gene expression analysis was repeated with 6 distinct replicates. Milk protein expression was assessed, as described in the methods, using 15μg of total protein for each sample. Representative blots of β-casein (C) and WAP (B) are shown along with a bar graph quantifying the protein expression, normalized to β-actin, for undifferentiated cells (UD), vehicle control (MeOH), 10μM THC and 10μM CBD treated HC11 cells, based on 4 replicates. Results were plotted as mean ± SEM and compared using one-way ANOVA. Statistically significant changes were represented by distinct letters.
Fig 5.
10μM THC and 10μM CBD reduced lipid levels and mRNA levels of associated markers in HC11 cells.
HC11 cells were seeded in base media for 24 hours (UD) and processed for total RNA isolation, as described in the methods. HC11 cells were differentiated in base media containing PRL, INS and DEX, as described in methods, and treated with vehicle control (MeOH), 10μM THC or 10μM CBD for 4 days, and processed for total RNA isolation. The relative mRNA levels of FASN (A), FABP4 (B), GLUT1 (C), HK2 (D), PLIN2 (E) and LPL (F) were determined. Gene expression analysis was repeated with 6 distinct replicates. Lipid levels in cells were quantified by conducting an Oil Red O (ORO) assay, as described in methods. Representative images of ORO-stained undifferentiated (UD) cells (G), and cells differentiated with vehicle control (MeOH) (H), 10μM THC (I), and 10μM CBD (J), were taken using a phase-contrast microscope (20x magnification). (K) Lipid levels were quantified in undifferentiated (UD) cells, and cells treated with vehicle control (MeOH), 10μM THC or 10μM CBD, from 6 distinct replicates. Results were plotted as mean ± SEM and compared using one-way ANOVA. Statistically significant changes were represented by distinct letters on bar graphs.
Fig 6.
10μM THC and 10μM CBD altered mRNA levels of ECS markers in HC11 cells.
HC11 cells were seeded in base media for 24 hours (UD) and processed for total RNA or protein isolation, as described in the methods. HC11 cells were differentiated in base media containing PRL, INS and DEX, and treated with vehicle control (MeOH), 10μM THC or 10μM CBD for 4 days. Treated cells were processed for total RNA or protein isolation, as described in methods. The relative mRNA levels of CNR2 (A), NAPEPLD (B) and FAAH (C) were determined. Gene expression analysis was repeated with 6 distinct replicates. Milk protein expression was assessed, as described in methods, using 15μg of total protein for each sample. (D) A representative blot of CB2 expression is shown along with a bar graph quantifying its expression, normalized to β-actin, for undifferentiated (UD), and differentiated cells in the presence of vehicle (MeOH), 10μM THC, or 10μM CBD, based on 4 replicates. Results were plotted as mean ± SEM and compared using one-way ANOVA. Statistically significant changes were represented by distinct letters on bar graphs.
Fig 7.
AM630 and JWH133 rescued the effect of THC and CBD, respectively, in HC11 cells.
HC11 cells were seeded in base media for 24 hours (UD) and processed for total RNA isolation, as described in the methods. HC11 cells were differentiated in base media containing PRL, INS and DEX, as described in methods, and treated with vehicle controls (MeOH, DMSO), 10μM THC, 1μM AM630, and 1μM AM630 + 10μM THC for 4 days, during cellular differentiation and processed for total RNA isolation. The relative mRNA levels of CSN2 (A), HK2 (B) and FABP4 (C) were determined. Next, the HC11 cells were differentiated in base media containing PRL, INS and DEX, and treated with vehicle controls (MeOH, DMSO), 10μM CBD, 10μM JWH133, and 10μM JWH133 + 10μM CBD for 4 days, and processed for total RNA isolation. The relative mRNA levels of CSN2 (D), HK2 (E) and FABP4 (F) were quantified. Gene expression analysis was repeated with 6 distinct replicates, as described in methods. Results were represented as mean ± SEM, and compared using one-way ANOVA. Statistically significant changes were indicated by distinct letters on the bar graphs.