Journal: Lipids in Health and Disease

Article Title: Butyrate attenuates lipolysis in adipocytes co-cultured with macrophages through non-prostaglandin E2–mediated and prostaglandin E2–mediated pathways

doi: 10.1186/s12944-016-0387-0

Figure Lengend Snippet: Treatment with NC, GPR41, and 109A siRNA and detection of the expression of GPR41 and 109A RNA in 3T3-L1 cells ( a ). Effect of butyrate, PGE2, and NC, GPR41, and 109A siRNA treatment on lipolysis assayed in TNF-α–stimulated 3T3-L1 cells after 24 h. Concentrations of FFAs ( b ) and free glycerol ( c ) in the medium were determined using an assay kit. As the control, the concentrations of FFAs and free glycerol were determined in the medium of untreated 3T3-L1 cells. The effects of butyrate, PGE2, and NC, GPR41, and 109A siRNA treatment on cAMP accumulation in TNF-α–stimulated 3T3-L1 cells after 24 h ( d ). The concentration of intracellular cAMP was measured using an assay kit. As the control, the concentration of intracellular cAMP was determined in untreated 3T3-L1 cells. Values from five independent experiments are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 versus TNF-α–stimulated 3T3-L1 cells, as determined by ANOVA and the Tukey-Kramer test; and # p < 0.05 and ## p < 0.01 versus TNF-α–stimulated 3T3-L1 cells treated with butyrate (0.5 mmol/L), as determined by ANOVA and the Tukey-Kramer test; † p < 0.05 for TNF-α–stimulated 3T3-L1 cells treated with GPR41 siRNA and butyrate (+) versus 3T3-L1 cells treated with GPR109A siRNA and butyrate (+), as determined by paired t -test

Article Snippet: Mouse GPR41 siRNA duplex and negative control (NC) siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Concentration Assay

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: Skeletal muscle p53 expression under metabolic stress and generation of an inducible skeletal muscle-specific p53 knockout model. (A–B) p53 protein expression in the gastrocnemius muscle of male mice fed a high-fat diet (HFD) for 12 weeks ( n = 6 per group). (C–D) p53 protein expression in the deltoid muscle of patients with type 2 diabetes and non-diabetic individuals ( n = 5-7 per group). (E) Schematic diagram illustrating the generation of the inducible skeletal muscle-specific p53 knockout (iMp53 KO) mouse model. (F) Experimental timeline depicting doxycycline administration and dietary intervention. GAPDH was used as a loading control. All samples were biologically independent. Data are presented as mean ± SE. Statistical analysis was performed using a two-tailed Student's t -test for comparisons between two groups. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Abbreviations: Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Expressing, Knock-Out, Control, Two Tailed Test

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency does not alter insulin sensitivity under metabolically normal conditions. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing chow diet for 19 weeks. (A) p53 gene expression in the gastrocnemius muscle ( n = 5 per group). (B) Average daily calorie intake ( n = 3 per group). (C) Accumulated food intake during the experimental period ( n = 3 per group). (D) Initial baseline body weight, final body weight, and weight gain during the experimental period ( n = 9–10 per group). (E) Weights of skeletal muscle, epididymal fat tissue, and liver ( n = 4 per group). (F–G) Blood glucose levels and area under the curve (AUC) during the glucose tolerance test ( n = 9–10 per group). (H–I) Plasma insulin levels and AUC during the glucose tolerance test ( n = 9–10 per group). (J–K) Percent reduction in blood glucose from baseline and AUC during the insulin tolerance test ( n = 9–10 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t-test for comparisons between two groups. ∗∗∗∗ p < 0.0001. Abbreviations: BW, body weight; Con, control; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Metabolic Labelling, Control, Gene Expression, Clinical Proteomics, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency improves insulin resistance in HFD-fed obese mice. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Data shown in panels H–K were collected after 10 weeks of HFD feeding, while data in panels L and M were obtained after 19 weeks of HFD feeding. (A) p53 gene expression in the gastrocnemius muscle ( n = 5 per group). (B–C) p53 protein expression in the gastrocnemius muscle ( n = 7 per group). (D) Average daily calorie intake ( n = 3 per group). (E) Accumulated food intake during the experimental period ( n = 3 per group). (F) Initial baseline body weight, final body weight, and weight gain during the experimental period ( n = 7 per group). (G) Weights of skeletal muscle, epididymal fat tissue, and liver ( n = 7 per group). (H–I) Blood glucose levels and area under the curve (AUC) during the glucose tolerance test ( n = 7 per group). (J–K) Plasma insulin levels and AUC during the glucose tolerance test ( n = 7 per group). (L–M) Percent reduction in blood glucose from baseline and AUC during the insulin tolerance test ( n = 7 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was assessed using a two-tailed Student's t -test for comparisons between two groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Abbreviations: BW, body weight; Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Control, Gene Expression, Expressing, Clinical Proteomics, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency enhances insulin-stimulated glucose disposal and signaling in skeletal muscle of HFD-fed obese mice. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Following HFD feeding, a hyperinsulinemic-euglycemic clamp test was performed to assess whole-body and tissue-specific insulin sensitivity (A–C; n = 7 per group), including whole-body glucose turnover (A), glucose uptake in the soleus muscle (B), and hepatic glucose production (C). To evaluate insulin signaling, mice were fasted overnight and administered insulin (1.5 U/kg) intraperitoneally; gastrocnemius muscles were harvested 10 min post-injection, and insulin signaling proteins were analyzed by Western blot (D–J; n = 7 per group). GAPDH was used as a loading control, and phosphorylated proteins were normalized to their respective total protein levels. Representative blots of insulin signaling proteins (D), phosphorylated Akt (pAkt; Ser473) levels (E), total Akt (F), phosphorylated AS160 (pAS160) levels (G), total AS160 (H), phosphorylated GSK3β (pGSK3β) levels (I), and total GSK3β (J). All samples were biologically independent. Data are presented as mean ± SE, and statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Abbreviations: AS160, Akt substrate of 160 kDa; Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK3β, glycogen synthase kinase 3β; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Control, Muscles, Injection, Western Blot, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency improves mitochondrial membrane potential and respiratory capacity. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Protein levels of mitochondrial electron transport chain complexes and PGC1α in the gastrocnemius muscle were analyzed by Western blot (A–F; n = 7 per group), with GAPDH used as a loading control. Representative Western blots (A) quantification of PGC1α (B) and quantification of mitochondrial electron transport chain complex proteins (C–F). In vitro , C2C12 myoblasts were transfected with shCon or shp53, induced to differentiate, and then treated with 0.25 mM palmitic acid for 12 h (G–J). Differentiated myotubes were incubated with 10 μM JC-1 dye for 1 h; fluorescence images were captured using a fluorescence microscope, and signal intensities were quantified using a fluorescence microplate reader (G–H). Mitochondrial membrane potential was assessed by JC-1 fluorescence; representative images are shown (G) and quantification analysis of JC-1 signal (H; n = 3 per group). Mitochondrial oxygen consumption rate in C2C12 myotubes was measured using the Seahorse system (I–J; n = 3 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups and one-way ANOVA followed by Tukey's post hoc test for comparisons among four groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Abbreviations: Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, inducible skeletal muscle-specific p53 knockout; PA, palmitic acid; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; shCon, control shRNA-transfected C2C12 cells; shp53, p53 shRNA-transfected C2C12 cells.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Membrane, Control, Western Blot, In Vitro, Transfection, Incubation, Fluorescence, Microscopy, Two Tailed Test, Knock-Out, shRNA

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 deficiency reduces the area of mitochondria-associated membranes in skeletal muscle of HFD-fed obese mice. Eight-week-old control and iMp53 KO male mice were fed a doxycycline-containing high-fat diet (HFD) for 19 weeks. Transmission electron microscopy was used to visualize mitochondria in the tibialis anterior muscle at magnifications of ×15,000 and × 60,000 (inset) (A). Mitochondrial density (number per μm 2 ; B) and mitochondrial area (C) were quantified (B–C; n = 3 per group). The ratio of mitochondria-associated membrane (MAM) length relative to mitochondrial perimeter was also quantified (D; n = 5 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups. ∗∗ p < 0.01. Abbreviations: Con, control; KO, inducible skeletal muscle-specific p53 knockout.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Control, Transmission Assay, Electron Microscopy, Membrane, Two Tailed Test, Knock-Out

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: Skeletal muscle p53 interacts with mitochondria-associated membrane (MAM) components and regulates mitochondrial calcium loading. Eight-week-old male mice were fed either a chow diet or a high-fat diet (HFD) for 12 weeks, after which expression levels of MAM-related proteins and SERCA1 in the tibialis anterior muscle were analyzed by Western blot (A–B; n = 6 per group). Eight-week-old control and iMp53 KO male mice were then fed a doxycycline-containing HFD for 19 weeks, and expression levels of MAM components and SERCA1 were again assessed in the gastrocnemius muscle via Western blot (C–D; n = 7 per group). GAPDH was used as a loading control. GRP75-targeted immunoprecipitation was performed using gastrocnemius muscle lysates from control and p53-deficient mice to assess protein–protein interactions (E–F), and p53-targeted immunoprecipitation was conducted to evaluate its interaction with MAM components (G–H; n = 3 per group). To assess mitochondrial calcium dynamics, C2C12 cells transfected with either shCon or shp53 were treated with 0.25 mM palmitic acid for 1 h or 3 h on day 5 of differentiation. Mitochondrial calcium levels were visualized via confocal microscopy (I) and quantified by flow cytometry (J; n = 6 per group). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t- test for comparisons between two groups and one-way ANOVA followed by Tukey's post hoc test for comparisons among four or more groups. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. Abbreviations: Con, control; KO, inducible skeletal muscle-specific p53 knockout; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP75, glucose-regulated protein 75; IP3R, inositol 1,4,5-trisphosphate receptor; PA, palmitic acid; SERCA1, sarcoplasmic reticulum Ca 2+ ATPase 1; shCon, control shRNA-transfected C2C12 cell; shp53, p53 shRNA-transfected C2C12 cell; VDAC, voltage-dependent anion channel.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Membrane, Expressing, Western Blot, Control, Immunoprecipitation, Protein-Protein interactions, Transfection, Confocal Microscopy, Flow Cytometry, Two Tailed Test, Knock-Out, shRNA

Journal: Redox Biology

Article Title: Inducible skeletal muscle-specific p53 deletion alleviates high-fat diet-induced insulin resistance by modulating mitochondria-associated membrane in obese mice ☆

doi: 10.1016/j.redox.2025.103828

Figure Lengend Snippet: p53 protein expression is positively correlated with mitochondria-associated membrane (MAM)-related proteins in human skeletal muscle. Protein expression levels of IP3R, GRP75, VDAC, and SERCA1 were analyzed in the deltoid muscle samples from non-diabetic and diabetic individuals (A–D; n = 5-7 per group), with GAPDH used as a loading control. Correlations between p53 protein levels and the expression of MAM-related proteins were assessed across all human skeletal muscle samples (E–H; n = 24). All samples were biologically independent. Data are presented as mean ± SE. Statistical significance was determined using a two-tailed Student's t -test for comparisons between two groups. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP75, glucose-regulated protein 75; IP3R, inositol 1,4,5-trisphosphate receptor; SERCA1, sarcoplasmic reticulum Ca 2+ -ATPase 1; VDAC, voltage-dependent anion channel.

Article Snippet: For gene knockdown experiments, C2C12 cells were transfected with small hairpin (sh) RNA plasmids targeting either control (shCon; sc-108060) or p53 (shp53; sc-29436-SH) (Santa Cruz Biotechnology) using Lipofectamine 3000 (Thermo Fisher Scientific), following the manufacturer's protocol.

Techniques: Expressing, Membrane, Control, Two Tailed Test

Journal: Oncogene

Article Title: TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.

doi: 10.1038/onc.2011.102

Figure Lengend Snippet: Figure 4 Activated ATF4 stimulates Redd1 expression in response to 2-DG. H1299, H460 and HeLa cells were transfected with siCTL or siATF4 for 20 h, followed by 30 mM 2-DG for 6 h. Protein and mRNA levels were measured via western blot and RT–PCR analyses.

Article Snippet: TXNIP (#1, SC-44943; #2, J-010814-05-0005), ATF4 (#1, sc-35112; #2, J-005125-10-0005), LKB1 (#1, sc-35816; #2, J-005035-07-0005), AMPKa1/2 (#1, sc-45312; #2, J-005027-06-0005 and J-005361-06-0005) and control siRNAs (#1, sc-37007; #2, D-001810-01-05) were purchased from Santa Cruz Biotechnology and Thermo Scientific Dharmacon (Chicago, IL, USA).

Techniques: Expressing, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1.

doi: 10.1038/onc.2011.102

Figure Lengend Snippet: Figure 3 2-DG induces ATF4 transcriptional activity. (a, b) H1299, H460 and HeLa cells were treated with the indicated concentrations of 2-DG for 6 h. (c) H1299 cells were treated with 30 mM 2-DG for the indicated times. Protein levels were measured using western blot analysis (a, c) and mRNA levels with RT–PCR (b). Experiments were performed in duplicate.

Article Snippet: TXNIP (#1, SC-44943; #2, J-010814-05-0005), ATF4 (#1, sc-35112; #2, J-005125-10-0005), LKB1 (#1, sc-35816; #2, J-005035-07-0005), AMPKa1/2 (#1, sc-45312; #2, J-005027-06-0005 and J-005361-06-0005) and control siRNAs (#1, sc-37007; #2, D-001810-01-05) were purchased from Santa Cruz Biotechnology and Thermo Scientific Dharmacon (Chicago, IL, USA).

Techniques: Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: INS-1 β-cells were treated with D MSO or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) for 16h or 24h ( A ), 16h ( B-E, H ), or 2-48h ( F-G ). A . Cells were processed for TUNEL analyses and mean ± SEMs of %apoptotic cells, relative to total cells, are presented. ( *,† Significantly different from corresponding DMSO group, p < 0.05; † significantly different from corresponding DMSO and cytokine groups, p < 0.05, n=3/group.) B-E, H . Representative blot from cells treated without or with P BA for iPLA 2 β, p65-NFκB, pPERK, and CHOP expression and corresponding densitometries. Tubulin and Oct-1 (nuclear marker) were uses as loading controls in n=3 independent analyses. Cumulative densitometries are presented in . Representative blot of iPLA 2 β expression in cytosolic and nuclear fractions prepared, as described , and cumulative densitometry.

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: TUNEL Assay, Expressing, Marker

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: A . MIN-6 cells were pre-treated with NFκB inhibitor Bay11-7082 (10 µM) or STAT1 inhibitor AZD 1480 (10 µM) for 1h. The cells were then treated with D MSO or c ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) for 16h and processed for p65-NFκB, SpTAT1, iPLA 2 β, pPERK, CHOP, and loading control tubulin immunoblotting analyses. Representative blots after BAY11 treatment ( A ) and corresponding densitometries ( B-F) ). Representative blots after AZD treatment ( G ) and corresponding densitometries ( H-I ). Cumulative densitometries are presented in . Cells treated for 24h were processed for TUNEL analyses. ( a Significantly different from DMSO, p < 0.0001; b Significantly different from CTK+Bay11, p = 0.0007; c Significantly different from CTK+AZD, p = 0.0028.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Control, Western Blot, TUNEL Assay

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: INS-1 β-cells were treated with D MSO or C ytokines, as in , for 2-8h and then processed for ChIP analyses using select antibodies for iPLA 2 β and AcH4 and subsequent qPCR analyses for Pla2g6 promoter region.

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques:

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were pre-treated for 1h with selective inhibitors for iPLA 2 β ( A ) S -B EL (1 µM) or ( B ) FK GK18 (5 x 10 -8 M); iPLA 2 γ ( C ) R -B EL (1 µM); or cPLA 2 α ( D ) Cay10502, (10 µM) and then treated for 16h with DMSO, or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ). The cells were then processed for p65-NFκB, pPERK, CHOP, pSTAT1, and loading control tubulin immunoblotting. Representative blots and densitometries are presented. Cumulative densitometries are presented in .

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Control, Western Blot

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN-6 β-cells were treated with D MSO or cytokines for 24h, as in , in the absence or presence of D MSO, P BA, S -BEL, F KGK18, R -BEL, C ay10502, scr ambled RNA, or si RNA targeting Pla2g6 . Cytosols were then prepared and processed for immunoblotting analyses for p65-NFκB, iPLA 2 β, pSTAT1, pPERK, and CHOP, and IκBα immunoblotting analyses. (Significance differences are presented as inserts.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Western Blot

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were transfected with scr ambled RNA or si RNA targeting Pla2g6 and treated with D MSO or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) and processed for iPLA 2 β, p65-NFκB, IKBα, pSTAT1, pPERK, CHOP, and loading control tubulin immunoblotting. Representative blots ( A ) and corresponding densitometries ( B-G ) are presented. Cumulative densitometries are presented in .

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were transfected with control (G0) or guide RNA constructs (G1 and G2) targeting Pla2g6 . A. Pla2g6 CRIPR-Cas9 gRNAs. B . Genotyping analyses by PCR. C . Predicted deletion region. D, E . Cells were treated with D MSO or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) for 16h and then processed for iPLA 2 β and loading control tubulin immunoblotting. Representative blots and densitometries are presented. F . Cells were treated with D MSO or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) for 24h and then processed for TUNEL analyses. Data are means±SEMs of percent apoptotic cells, relative to total cells. ( †,* Significantly different from G0-DMSO, p<0.00001, p<0.005; # different from G0-CTK p<0.0005, n=3/group). G-I . Cells were treated with D MSO or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) for (16h) and then processed for pPERK, p65-NFκB, and loading control tubulin immunoblotting and representative blots ( G, n=3-4) and cumulative densitometries presented ( H-I ). a,b,c G0 CTK group significantly from GO DMSO group, p<0.0001, p=0.05, p=0.0046, respectively; d G0 CTK group significantly different from G1+G2 CTK group, p<0.0057.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Transfection, Control, Construct, Western Blot, TUNEL Assay

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were transfected with control (G0) or guide RNA constructs (G1 and G2) targeting Pla2g6 ( Top panels ) or were pretreated with S - B EL (10 µM, 1h) ( Bottom panels ) and then treated with D MSO or C ytokines (50 U/ml IL-1β + 150 U/ml IFNγ) for (2h). Subsequently, ChIP was performed using antibody directed against iPLA 2 β and acetylated histone (AcH4) followed by qPCR analyses of Pla2g6 and Nf κ b promoter regions. AcH4 was used to confirm transcriptional activation and IgG as a negative control. A, D. Fold enrichment, relative to IgG, under basal (DMSO) conditions. B, E. Cytokine-induced fold enrichment, relative to IgG. C, F. Cytokine-induced fold enrichment, relative to DMSO: C . a,b G0 C groups significantly different from other groups, p<0.01, p<0.05, respectively; n=3.); F . a,b Cytokine groups significantly different from corresponding DMSO groups, p<0.005, p<0.05, respectively; c C+B significantly different from corresponding C p<0.05; n=3.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Transfection, Control, Construct, Activation Assay, Negative Control

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were treated with C ytokines ± inhibitors as in , or PGs, as in , and processed for iPLA 2 β immunoblotting analyses. Representative blots and corresponding densitometries for CTK ± S -BEL (A), CTK ± FKGK18, CTK ± R -BEL, CTK ± S -BEL, and cytokines and PGs (E) are presented. Cumulate densitometries are presented in and .

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Western Blot

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were treated with D MSO or PGs cocktail for 16h and then processed for iPLA 2 β immunoblotting analyses. ( † Significantly different from D, p < 0.05, 1-tailed test, n=4/group.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: Western Blot

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: MIN6 β-cells were treated with a PGs cocktail, as in , for 4h and processed for ChIP and qPCR analyses. A . Fold enrichment of iPLA 2 β at promoter regions, relative to IgG. B-F . Fold enrichment of iPLA 2 β at promoter regions, relative to DMSO: B , Pla2g6 ; C , NfkB ; D , Stat1 ; E . Gcg ; F . Pepck . ( a,c PGs groups significantly different from corresponding DMSO groups, p < 0.001 and p < 0.01, respectively. b,d PGs + S -BEL groups significantly different from corresponding DMSO groups, p < 0.01 and p < 0.05, respectively.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques:

Journal: bioRxiv

Article Title: ER Stress-Induced β-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Developmen t

doi: 10.64898/2026.03.02.708596

Figure Lengend Snippet: Human islets (3500-5000/condition) from healthy donors were treated with cytokines (100 U/ml IL-1β + 300 U/ml IFNγ for 2h ( A, B ) or a PGs cocktail ( C ) as in for 4h. The islets were then processed for ChIP analyses using antibodies directed against p65-NFκB or iPLA 2 β and subsequent qPCR analyses. D . Human islets (250/condition) from healthy donors were treated with cytokines (100 U/ml IL-1β + 300 U/ml IFNγ or a PGs cocktail for 24h and processed for TUNEL analyses. Data are mean ± SEMs of %apoptotic cells, relative to total cells. ( a,b Significantly different from DMSO group, p < 0.0001 and p = 0.004, respectively.)

Article Snippet: The group VI iPLA 2 siRNA (m) (sc-43820, Santa Cruz, Dallas, TX) duplex solution was then added directly to the dilute Transfection Reagent (sc-36868, Santa Cruz Biotechnology, Dallas, Texas) using a pipette.

Techniques: TUNEL Assay

Journal: Frontiers in molecular neuroscience

Article Title: Pharmacological Stimulation of Phagocytosis Enhances Amyloid Plaque Clearance; Evidence from a Transgenic Mouse Model of ATTR Neuropathy.

doi: 10.3389/fnmol.2017.00138

Figure Lengend Snippet: FIGURE 3 | Expression of phagocytic cell markers in stomach tissue: (A) The expression of CD88 was measured by immunoblotting indicating a decrease in the marker in both the untreated group and the group treated with the PMX53 antagonist, while the two groups treated with the agonists exhibited the greatest level of expression. Similar effects were observed with Neutrophil elastase (B), Ly6G (C) and IL-36γ (D). Immunoblots for the macrophage specific markers F4/80 (E) and CD68 (F) however indicate their overexpression in the group of mice treated with the modified agonist molecule EP67, while the group treated with the PMX53 antagonist exhibited the lowest levels of expression for both markers. n = 6/group data presented as mean ± 1 SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The antibodies used for immunoblotting were against: BiP (anti-rabbit Santa Cruz sc-13968 1/350), C1q (anti-rabbit Santa Cruz sc-27661 1/100), Caspase-3 (anti-rabbit Enzo Life Sciences ALX-210-806-C100 1/1000), CD68 (anti-rabbit Santa Cruz sc-9139 1/150), CD88 (anti-mouse Santa Cruz sc-53795 1/100), ELANE (anti-rabbit Abcam ab68672 1/1000), F4/80 (anti-rabbit Santa Cruz sc-25830 1/100), IL-36γ (anti-goat Santa Cruz sc-168163 1/100), Ly6G (anti-mouse Antibodies online ABIN361224 1/1000) and Properdin (anti-rabbit Santa Cruz sc-68367 1/100).

Techniques: Expressing, Western Blot, Marker, Over Expression

Journal: Frontiers in molecular neuroscience

Article Title: Pharmacological Stimulation of Phagocytosis Enhances Amyloid Plaque Clearance; Evidence from a Transgenic Mouse Model of ATTR Neuropathy.

doi: 10.3389/fnmol.2017.00138

Figure Lengend Snippet: FIGURE 4 | Amyloid plaque infiltration by macrophages and neutrophils: serial sections stomach sections from mice representing the four groups of mice were stained with Thioflavin-S, α-TTR, the pan-macrophage marker α-CD68 and the neutrophil marker α-Neutrophil elastase (ELANE). Immunofluorescence on the stomach tissue from the untreated mouse indicates the complete absence of neutrophils from the plaque, while there is some co-expression with CD68 (A). A similar pattern was observed with the mouse treated with the PMX53 antagonist molecule (B), while complete co-localization with both CD68 and ELANE was observed in the mouse treated with the full agonist molecule (C). Immunofluorescence on the mouse treated with the modified agonist EP67 however revealed complete plaque co-localization with CD68 and the complete absence of ELANE from the region (D). Scale bar = 75 µm.

Article Snippet: The antibodies used for immunoblotting were against: BiP (anti-rabbit Santa Cruz sc-13968 1/350), C1q (anti-rabbit Santa Cruz sc-27661 1/100), Caspase-3 (anti-rabbit Enzo Life Sciences ALX-210-806-C100 1/1000), CD68 (anti-rabbit Santa Cruz sc-9139 1/150), CD88 (anti-mouse Santa Cruz sc-53795 1/100), ELANE (anti-rabbit Abcam ab68672 1/1000), F4/80 (anti-rabbit Santa Cruz sc-25830 1/100), IL-36γ (anti-goat Santa Cruz sc-168163 1/100), Ly6G (anti-mouse Antibodies online ABIN361224 1/1000) and Properdin (anti-rabbit Santa Cruz sc-68367 1/100).

Techniques: Staining, Marker, Expressing

Journal: Frontiers in molecular neuroscience

Article Title: Pharmacological Stimulation of Phagocytosis Enhances Amyloid Plaque Clearance; Evidence from a Transgenic Mouse Model of ATTR Neuropathy.

doi: 10.3389/fnmol.2017.00138

Figure Lengend Snippet: FIGURE 7 | Amyloid plaque co-localization with the lysosomal marker: immunofluorescence of serial stomach sections from the group treated with the full agonist molecule (A) and the PMX53 antagonist (B) show the complete co-expression of the amyloid plaque with Lamp-1 (Ai) in the mouse treated with the agonist as opposed to the antagonist treated mouse which presents complete absence of the lysosomal marker in the vicinity of the plaque (Bi). Sections were co-stained with Thioflavin-S, a-Lamp-1 and a-CD68 (Ai-iv and Bi-iv) and Thioflavin-S, a-hTTR and a-ELANE (Av-viii and Bv-viii). Scale bar = 75 µm.

Article Snippet: The antibodies used for immunoblotting were against: BiP (anti-rabbit Santa Cruz sc-13968 1/350), C1q (anti-rabbit Santa Cruz sc-27661 1/100), Caspase-3 (anti-rabbit Enzo Life Sciences ALX-210-806-C100 1/1000), CD68 (anti-rabbit Santa Cruz sc-9139 1/150), CD88 (anti-mouse Santa Cruz sc-53795 1/100), ELANE (anti-rabbit Abcam ab68672 1/1000), F4/80 (anti-rabbit Santa Cruz sc-25830 1/100), IL-36γ (anti-goat Santa Cruz sc-168163 1/100), Ly6G (anti-mouse Antibodies online ABIN361224 1/1000) and Properdin (anti-rabbit Santa Cruz sc-68367 1/100).

Techniques: Marker, Expressing, Staining