Journal: BMC cancer

Article Title: 17β-estradiol induces stearoyl-CoA desaturase-1 expression in estrogen receptor-positive breast cancer cells.

doi: 10.1186/s12885-015-1452-1

Figure Lengend Snippet: Fig. 4 ERα silencing and treatment with 4-OH Tamoxifen blocks the 17β-estradiol induction of SCD-1 levels and activity. (a, b) MCF-7 cells were incubated for 7 days in phenol red-free media supplemented with charcoal-stripped FBS (starved), followed by a 5 day incubation period in the same media that was supplemented with 2nM 17β-ED, 10nM of 4-OH tamoxifen (OH-Tam), a combination of both, or their vehicle controls (Ctrl). (a) Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SCD-1 expression was performed using actin as loading control. The graph shows densitometry quantification of the SCD-1 blots. (b) Cellular lipids were extracted, hydrolyzed, transmethylated, and quantified by GC/FID and the indicated (MUFA/SFA) ratios were calculated. (c) MCF-7 cells were starved for 5 days as above and were then subjected to electroporation in the presence of three different ERα-targeting siRNA (ERα-si1, ERα-si2, Erα-si3), or a non-targeting duplex (non-silencing) and were incubated in starvation medium for an additional 3 days. Cellular proteins were then separated by SDS-PAGE and immunoblot analysis of ERα was performed, using actin as loading control. The graph shows densitometry quantification of the ERα blots. (d) Starved cells were transfected with ERα-targeting siRNA or the non-silencing control as in (c) above, and were then incubated in starvation medium containing 2nM 17β-ED or its vehicle control (ctrl) for 3 days, as indicated. Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SCD-1 was performed, using actin as loading control. The graph shows densitometry quantification of the SCD-1 blots. All immunoblots are representative of 3 independent experiments. Data are means ± SEM, n= 3 independent experiments. Values with a different superscript are significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment

Article Snippet: MCF-7 cells (2 × 106 cells) that had been starved as above for 5 days were resuspended in 200 μl phenol red-free RPMI to which was added 4 μl of siRNA targeting ERα (Cat#301461), SCD-1 (Cat # SR-304248), or SREBP-1 (Cat# SR-304579) from OriGene for a final concentration of 100nM or a nontargeting duplex of the same length as negative control (Cat # SR-30004).

Techniques: Activity Assay, Incubation, SDS Page, Western Blot, Expressing, Control, Electroporation, Transfection

Journal: BMC cancer

Article Title: 17β-estradiol induces stearoyl-CoA desaturase-1 expression in estrogen receptor-positive breast cancer cells.

doi: 10.1186/s12885-015-1452-1

Figure Lengend Snippet: Fig. 7 17β-estradiol induces SREBP-1C expression and activation which depend on the IGF-1 pathway. MCF-7 cells were incubated for 7 days in phenol red-free media supplemented with charcoal-stripped FBS (starved). (a and b) Starved cells were then incubated in the same media that was supplemented with 50 ng/ml of IGF-1, 2nM of 17β-ED, a combination of 17β-ED, and 10 μM of the IGF-1R antagonist Ag1024, or their vehicle controls (Ctrl) for 5 days. (a) RNA was extracted from cells and reverse transcribed into cDNA. Relative qPCR for SREBP-1C was performed using HPRT as reference gene. (b) Cellular proteins were separated by SDS-PAGE and immunoblot analysis of the precursor (P) and mature (M) SREBP-1 was performed, using actin as loading control. The graph show densitometry quantification of the SREBP-1 (M) blots. (c) Starved cells were then transfected with anti-SREBP-1 siRNA or its non-silencing control, and incubated with 17β-ED, or its vehicle control as indicated 3 days. Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SREBP-1 (P) and SCD-1 were performed, using actin as loading control. The graphs show densitometry quantification of the SREBP-1 and SCD-1 blots. All immunoblots are representative of 3 independent experiments. Data are means ± SEM, n = 3 or 4 independent experiments. Values that have a different superscript are significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment

Article Snippet: MCF-7 cells (2 × 106 cells) that had been starved as above for 5 days were resuspended in 200 μl phenol red-free RPMI to which was added 4 μl of siRNA targeting ERα (Cat#301461), SCD-1 (Cat # SR-304248), or SREBP-1 (Cat# SR-304579) from OriGene for a final concentration of 100nM or a nontargeting duplex of the same length as negative control (Cat # SR-30004).

Techniques: Expressing, Activation Assay, Incubation, Reverse Transcription, SDS Page, Western Blot, Control, Transfection

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Analysis of proteasome core complex (α1-7 and β1-7) protein label-free quantification (LFQ) intensity values from mass spectrometry experiments in WT and Ump1 -/- yeast strains. The proteasome core complex proteins were significantly upregulated in the absence of Ump1 -/- . **p≤0.01, Welch two-tailed t-test. Boxplots show the median (line), interquartile range (box), and 1.5×IQR whiskers. Individual data points shown represent the mean intensity of the individual CP components. (B) Analysis of proteasome core complex (PSMA1-7 and PSMB1-7) protein label-free quantification (LFQ) intensity values from mass spectrometry experiments in HEK293 cells treated with a POMP siRNA or a scrambled control siRNA (Scrm). The proteasome core complex proteins were significantly downregulated following POMP knockdown. Individual data points shown represent the mean intensity of the individual CP components. **p≤0.01, Welch two-tailed t-test. Boxplots show the median (line), interquartile range (box), and 1.5×IQR whiskers. Individual data points shown represent the mean intensity of the individual CP components. (C) Analysis of the transcription factor Rpn4 protein label-free quantification (LFQ) log 2 -scaled intensity values from mass spectrometry experiments in WT and Ump1 -/- yeast strains. Rpn4 was significantly upregulated in the absence of Ump1 -/- . *p≤0.05, two-tailed Wilcoxon rank-sum test, n=4. Boxplots show the median (line), interquartile range (box), and 1.5×IQR whiskers. Individual data points shown represent biological replicates. (D) Analysis of Nrf1/TCF11 protein label-free quantification (LFQ) log 2 -scaled intensity values from mass spectrometry experiments in HEK293 cells treated with a POMP siRNA or a scrambled control siRNA (Scrm). Nrf1/TCF11 protein was significantly upregulated following POMP knockdown. *p≤0.05, two-tailed Wilcoxon rank-sum test, n=3 (Scrm) and 5 (POMP siRNA). Boxplots show the median (line), interquartile range (box), and 1.5×IQR whiskers. Individual data points shown represent biological replicates. (E) POMP mRNA levels in HEK293 cells following 5 hr treatment with each of three different proteasome inhibitors (Carfilzomib, Epoxomicin and MG132) or DMSO. ****p≤ 0.0001, one-way ANOVA and post-hoc Dunnett’s multiple comparisons test, n=6, mean±SD, FC=fold change. (F) Representative Western Blot analysis from HEK293 cells treated for 5 hr with each of three different proteasome inhibitors (Carfilzomib, Epoxomycin or MG132) or DMSO, assessing proteasome activity, POMP and ACTB loading control levels. (G) Analysis of the Western blot experiments as shown in F. Shown are POMP protein levels relative to the DMSO control. ACTB was used as a loading control. ** = p<0.01, ***p≤0.001, RM one-way ANOVA and post-hoc Dunnett’s multiple comparisons test on non-normalised data, n=8, mean±SD, FC=fold change. (H) Representative Western blot time course from HEK293 cells treated with each of three different proteasome inhibitors (Epoxomicin, Bortezomib, or MG132) or DMSO for 1, 2.5 and 5 hr assessing the levels of TCF11/NRF1, POMP, PSMB5 and, as a loading control, LaminB1. (I) Analysis of the Western blot time course as shown in H. Reported are LaminB1-normalised protein level fold changes relative to T = 0 hr in response to treatment with DMSO, Epoxomycin, Bortexomib, or MG132 for POMP, TCF11/NRF1, PSMB5 and its precursor form pro-PSMB5 (upper band). *p ≤0.05, one-way ANOVA and post-hoc Dunnett’s multiple comparisons test on the 5 h time point, n=4, mean±SEM, FC=fold change. (J) Representative Western Blot analysis of HEK293 cells transfected with two different siRNAs against TCF11/NRF1 or Scrm control, and at 72 hr post-transfection treated with the proteasome inhibitor MG132 for 5 hr. Shown are proteasome activity levels and protein levels for TCF11/NRF1, POMP and the loading control GAPDH. (K) Analysis of the Western blot experiment as shown in J. Reported are POMP protein level changes in response to 5 hr MG132 treatment in TCF11/NRF1 knock-down and Scrm control cells. The early induction of POMP protein is not affected by TCF11/NRF1 knock-down. ns=p > 0.05, one-way ANOVA and post-hoc Dunnett’s multiple comparisons test, n=3, mean±SD, FC=fold change. (L) Schematic illustration of the HSF1 transcription factor activation mechanism. Under basal conditions, HSF1 is kept inactive in the cytoplasm by interaction with heat-shock proteins (Hsps). In response to stress HSF1 gets phosphorylated, released by the Hsps and translocates into the nucleus to form transcriptionally-competent trimers that activate the stress response. (M) Representative Western Blot analysis of HEK293 cells transfected with one siRNA against HSF1 or Scrm control, and at 72 hr post-transfection treated with three different proteasome inhibitors (Epoxomicin, Bortezomib, MG132) for 5 hr. Shown are proteasome activity and protein levels for HSF1, POMP and ACTB as a loading control. (N) Analysis of the Western blot experiment as shown in M. Shown are ACTB-normalised POMP protein level fold changes in response to a 5 hr treatment with Epoxomicin, Bortezomib or MG132 in HSF1 knock-down and Scrm control cells. HSF1 knock-down significantly reduced POMP induction in response to proteasome inhibition. *p≤0.05, unpaired two-tailed t-tests, n=3, mean±SD, FC=fold change. (O) Representative Western blot analysis of HEK293 cells treated with DMSO or the HSF1 activator HSF1B for 7 hr. Shown are proteasome activity levels (Activity) and protein levels for POMP, Hsp70, PSMB5, PSMA1-7, and ACTB. (P) Analysis of the Western blot experiment as shown in O. Shown are ACTB-normalised protein level fold changes in response to treatment with the HSF1 activator. HSF1 activation significantly elevated both POMP and Hsp70 levels, but did not significantly affect proteasome levels.**p≤0.01, ***p≤0.001, paired two-tailed t-tests, n=7 (POMP), 6 (Hsp70, PSMA1-7, Activity), 5 (pro-PSMB5), mean±SD, FC=fold change.

Article Snippet: Pre-designed siRNA oligo duplex sets against POMP (SR309742), TC11/NRF1 (SR303155), HSF1 (SR320556), PSMB1 (SR303824) and PSMB7 (SKU SR303830) were purchased from OriGene.

Techniques: Mass Spectrometry, Two Tailed Test, Control, Knockdown, Western Blot, Activity Assay, Transfection, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Western blot analysis of POMP and TCF11/NRF1 protein levels following siRNA-mediated knockdown of POMP using two independent siRNAs (A and C) in HEK293 cells. Scrambled siRNA (Scr) served as control. Tubulin was used as a loading control.The full-length (f) and cleaved (c) forms of TCF11/NRF1 are indicated next to the blot. (B) Analysis of the experiment shown in A. POMP knockdown leads to a significant increase in the levels of cleaved-TCF11/NRF1. ***p<0.001, unpaired two-tailed t-test, n= 3 (Scrm) and 6 (siRNA POMP), mean±SD, FC = fold change. (C) Representative Western blot time course from HEK293 cells treated with one each one of three different proteasome inhibitors (Epoxomicin, Bortezomib, or MG132) or DMSO for 1, 2.5 and 5 hr assessing the levels of PSMA1-7 and Lamin B1, as a loading control. (D) Analysis of the Western blot time course as shown in C. Reported are Lamin B1-normalised PSMA1-7 protein level fold changes relative to T = 0 hr in response to treatment with DMSO, Epoxomycin, Bortexomib, or MG132. ns=p >0.05, one-way ANOVA and post-hoc Dunnett’s multiple comparisons test on the 5 hr time point, n=4, mean±SEM, FC=fold change. (E) UCSC genome browser view of the POMP locus (chr13: 28,658,045-28,661,232), displaying RefSeq genes (1), OMIM annotations (2), ENCODE candidate regulatory elements (cCREs) (3), H3K27Ac chromatin marks (4), and JASPAR transcription factor binding sites (5) tracks. In the JASPAR transcription factor binding sites (5) track HSF1/2 transcription factor binding sites are shown. (F) Western blot analysis of HEK293 cells treated with proteasome inhibitors (Epoxomicin, Bortezomib, MG132) or DMSO for 5 hr, probed for the heat shock response transcription factor HSF1, its phosphorylated active form (P-HSF1), their transcriptional target Hsp70 and ACTB, as loading control. Proteasome activity changes in response to proteasome inhibition was assayed by ABP in-gel fluorescence. (G) Analysis of experiments like the one shown in F. Treatment with proteasome inhibitors leads to a significant activation of HSF1, measured as an increase in P-HSF1, and its transcriptional downstream target Hsp70. *p≤0.05, **p<0.01, ***p<0.001, paired two-tailed t-tests, n=3 (P-HSF1) and 5 (Hsp70) biological replicates, mean±SD, FC=fold change. (H) Western blot analysis of HEK293 transfected with either one of two different siRNAs targeting HSF1 or scrambled control. The cells were analysed 72 hrs after transfection and blots were probed for HSF1, POMP and GAPDH, as loading control. Transfection of siRNAs leads to strong depletion of HSF1 and a reduction in POMP levels. (I) Analysis of the experiment shown in H. Transfection of HSF1 siRNAs leads to a significant reduction in HSF1 and POMP levels. ***p<0.001, ****p<0.0001, one-way ANOVA and post-hoc Dunnett’s test, n=4 biological replicates, mean±SD, FC=fold change. (J) Western blot analysis HEK293 cells treated simultaneously with KNK-437 and either one of three different proteasome inhibitors (Bortezomib, Epoxomicin, MG132) or DMSO for 5h. The samples were assayed for HSF1, P-HSF1, POMP and GAPDH, as loading control. Changes in proteasome activity were measured by in-gel ABP fluorescence. (K) Analysis of P-HSF1 levels from (H), normalized to total HSF1 and expressed as fold change (FC) relative to DMSO control. KNK-437 significantly reduces phosphorylation and activation of HSF1. **p<0.01, unpaired two-tailed t-test, n=3, mean±SD. (L) Analysis of experiments like the one shown in H. Simultaneous treatment with KNK-437 and proteasome inhibitors significantly reduces the POMP increase induced by proteasome inhibition. RM one-way ANOVA with post-hoc Dunnett’s test. n=7 (DMSO-PI) and 5 (KNK-437-PI), mean±SD, FC=fold change.

Article Snippet: Pre-designed siRNA oligo duplex sets against POMP (SR309742), TC11/NRF1 (SR303155), HSF1 (SR320556), PSMB1 (SR303824) and PSMB7 (SKU SR303830) were purchased from OriGene.

Techniques: Western Blot, Knockdown, Control, Two Tailed Test, Binding Assay, Activity Assay, Inhibition, Fluorescence, Activation Assay, Transfection