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Journal: Cell Communication and Signaling : CCS
Article Title: ER-phagy mediates the anti-tumoral synergism between HDAC inhibition and chemotherapy
doi: 10.1186/s12964-025-02198-9
Figure Lengend Snippet: ISX and Gemcitabine synergistically induce ER-stress ( A ) Scheme of the IRE1-selective ER-stress reporter. ( B ) Immunofluorescence images of Panc1 cells transiently transfected with IRE1 reporter (green), treated with DMSO, 20 µM ISX, 1.0 µM Gem and the combination ISX/Gem (I/G) for 48h. Nuclei appear blue (DAPI). Scale bar: 100µm. Shown is one representative of n=3. ( C ) Quantification of Panc1 cells transiently expressing IRE1 reporter. Cells were treated as described in ( B ) or with 1.0 μM Thapsigargin (Th) for 48h, analyzed using the number of positive cells per viewing field normalized to the values of the DMSO samples. Each dot represents one image (from n=2 independent experiments; mean ± SD). ( D ) Rel. mRNA expression of ER-stress genes selective for the IRE1 pathway ( XBP1s , DNAJB9 ). Panc1 cells were treated as described in ( C ) (mean of n=3 ± SD). ( E ) Scheme of the PERK-selective ER-stress reporter. ( F ) Immunofluorescence pictures of Panc1 cells transiently expressing PERK reporter (red) and treated as in ( B ). Nuclei appear blue (DAPI). Scale bar: 100µm. Shown is one representative of n=3. ( G ) Quantification of PERK pathway activity in Panc1 cells stably expressing a PERK reporter construct. Treatment as in ( C ). Shown are the numbers of positive cells per viewing field normalized to the DMSO condition. Each dot represents one analyzed viewing field (mean ± SD of n=2). ( H ) Rel. mRNA expression of ER-stress genes selective for the PERK pathway ( CHOP , PERK ) in Panc1 cells treated as described in ( C ) Quantification of ATF6 activity in Panc1 cells transiently expressing an ATF6 luciferase reporter, treated as described in ( C ) (mean of n=3 ± SD). ( K ) Rel. mRNA expression of HERPUD1 , an ER-stress gene selective for the ATF6 pathway (Panc1 cells treated as in ( B ); mean of n=3 ± SD)
Article Snippet: The following plasmids were used:
Techniques: Immunofluorescence, Transfection, Expressing, Activity Assay, Stable Transfection, Construct, Luciferase
Journal: Cell Communication and Signaling : CCS
Article Title: ER-phagy mediates the anti-tumoral synergism between HDAC inhibition and chemotherapy
doi: 10.1186/s12964-025-02198-9
Figure Lengend Snippet: The impact of HDACi/Gem on apoptosis and its link to clinical data. ( A ) Rel. mRNA expression of FAM134B and CHOP (mean of n=3 ± SD). Panc1 cells were treated with DMSO or the combination of 20 µM ISX and 1.0 µM Gem for different time periods. ( B ) Rel. mRNA expression of FAM134B and PERK (mean of n=3 ± SD). Panc1 cells were treated with DMSO, 20 µM ISX, 1.0 µM Gem, dual treatment (I/G), or 1.0 µM Thapsigargin (Th) in calcium-free or calcium-containing DMEM medium for 48h. ( C ) Flow cytometric quantification of IRE1 activity in Panc1 cells stably expressing an IRE1 reporter construct. Treatment as in ( B ) (n=3 ± SD). ( D ) Caspase 3/7 activity in Panc1 cells exposed to ISX (I, 20 µM) and Gem (G, 1.0 µM) for various time periods. Staurosporine (1.0 µM) was included as a positive control. The mean ± SD of two representative experiments measured in triplicates is shown. ( E ) Caspase 3/7 activity in Panc1 cells treated with the indicated drugs for 60h. ISX (I, 20 µM); Gem (G, 1.0 µM); IRE1-inh. 4µ8c (30µM); PERK-inh. ISRIB (1.0µM); ATF6-inh. Ceapin-A7 (6.0µM). Shown is the mean (± SD) of two representative experiments measured in quadruplicate each. Each dot represents one biological sample. ( F ) Western blot analysis of FAM134B protein levels in Panc1 cells treated for 48h with DMSO, 20 µM Loperamide, or 5/10 µM Nelfinavir. Shown is one representative experiment of n=5. β-Actin was used as a loading control. ( G , H , I ) FAM134B mRNA expression in bulk tissue from PDAC patients and corresponding healthy pancreatic tissue. Each dot represents one patient. ( J , K ) Kaplan-Meier plots representing the overall survival of patients with PDAC as a function of FAM134B expression (Scan-split; log-rank p). ( L ) Scheme illustrating the ER-phagy-induced mechanism by which HDACi and Gemcitabine synergize in their anti-tumor effectiveness
Article Snippet: The following plasmids were used:
Techniques: Expressing, Activity Assay, Stable Transfection, Construct, Positive Control, Western Blot, Control