sirna plasmids for control  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Diagram of the Canonical and Non-canonical STING signaling (created with BioRender.com ). B. Constitutive STAT3 and STAT1 activation in SAVI iECs. Phospho-STAT3 Y705 and phospho-STAT1 Y701 (normalized to total protein) were measured in iECs from 3 HC and 3 SAVI donors across passages 1, 3, and 5. SAVI iECs showed increased baseline STAT3 activation (mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05, unpaired t-test). C. STING/TBK1 inhibition blocks acute cGAMP-induced STAT3 activation in HC iECs. iECs from 3 HC donors were stimulated with 2’3’-cGAMP (20µg/ml) for 30 min–24 h. STING inhibitor IFM35883 (STINGi, 2.5 μM) and TBK1 inhibitor MRT67307 (TBK1i, 5 μM) were pre-treated one hour before cGAMP. STAT3 activation at 4h was prevented by STING/TBK1 inhibition (mean ±SEM, ***p < 0.001, **p < 0.01, Mann-Whitney test). D. STING/TBK1 inhibition reduces constitutive STAT3 activation in SAVI iECs. SAVI iECs were treated with IFM35883 (2.5 μM) from P2–P5 or with TBK1i (5 μM) for 48 h. Quantification is from four experiments in iEC line from patient SAVI1 (mean ±SEM, ***p < 0.001, unpaired t-test). E. STAT3 shRNA knockdown (KD), not STAT1 KD preserves CD144 (VE-cadherin) in SAVI iECs. iECs from 3 individual SAVI patients infected with control, STAT3, or STAT1 shRNA at P2 were analyzed by flow cytometry at P5 (mean ±SEM, **p < 0.01, paired t-test). Representative flow cytometry profiles are shown in Figure S4C. F. Constitutive protein expression of SLUG/ SNAI2 is elevated in SAVI iECs and suppressed by STING inhibition. Protein from 3 HC, 3 SAVI, and 3 iso-SAVI iECs at P5 showed increased SLUG (normalized by GAPDH) in SAVI; IFM35883 (2.5 μM) treatment from P2–P5 prevented SLUG upregulation in SAVI iECs (SAVI 1) (mean ±SEM, ***p < 0.001, **p < 0.01, unpaired t-test). See also Figure S4E. G. STAT3 shRNA knockdown reduces SLUG mRNA expression (normalized by internal control 18S) in SAVI iECs at P3. Data are summarized from 3 individual SAVI patients iEC lines. *p < 0.01 as determined by two-tailed unpaired t-test.

Article Snippet: siRNA plasmids for control (sc-37007), STAT3 (sc-29493), and STAT1 (sc-44123) were purchased from Santa Cruz.

Techniques: Activation Assay, Inhibition, MANN-WHITNEY, shRNA, Knockdown, Infection, Control, Flow Cytometry, Expressing, Two Tailed Test

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Preproghrelin, and thus obestatin, rescues autophagy flux in DMD cells. (A) Left panel : Immunofluorescence detection of MHC in human DMD cell models, DMD6 and DMD14, under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM) 72 h post stimulation. Right panel : Immunoblot analysis of p62, LC3, and Cathepsin L, in human DMD6 and DMD14 myotubes at the 72‐h point under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM). (B) DMD myotubes were transfected with siRNA targeting preproghrelin or siRNA‐scramble prior to induction of myogenesis for 96 h. The expression of expansion (p21, Ki‐67, fast‐MHC, slow‐MHC, myogenin), autophagy (p62, LC3, Beclin1, Bnip3, VPS34, AMBRA1) and mitochondrial homeostasis (DRP1, Parkin, TFAM, Mfn2) markers were evaluated by immunoblot analysis ( n = 3). Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM obtained from intensity scans of independent experiments. * p < 0.05. (C) Immunoblot analysis of p62, LC3, Cathepsin L, pAMPK(T172), AMPK, pmTOR(S2448), mTOR, and MHC in human DMD myotubes at the 24‐h point under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM). Cells were treated with the autophagy inhibitor chloroquine (20 µM) 6 h before collection. Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM obtained from intensity scans of three independent experiments *,# p < 0.05. (D) Left panel : Immunofluorescence detection of microtubule‐associated protein 1 light chain 3 isoform B (LC3B) in human DMD cell model under DM (control), DM + obestatin (10 nM), or DM + insulin (1.72 µM) at the 24 h point after stimulation. Cells were treated with the autophagy inhibitor chloroquine. Right panel : the changes in LC3 puncta/area from DMD myotube cells are shown. Data were expressed as mean ± SEM ( n = 3 per group). * p < 0.05. (E) Immunoblot analysis of DRP1, TFAM, Mfn2, and Pink1 in human DMD myotubes at the 24 h point under obestatin treatment (10 nM). Cells were treated with chloroquine before collection. Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM ( n = 3 per group; *,# p < 0.05). (F) Quantification of ROS levels in DMD myotube cells using CellROX TM fluorescent dye. DMD myotubes were treated with obestatin (10 nM), Dexa (1 µM), or vehicle (control) for 24 h. Results are represented as variation of MFI between control and obestatin‐ or Dexa‐treated cells. Data were expressed as mean ± SEM ( n = 3 per group). * p < 0.05.

Article Snippet: An ON‐TARGETplus nontargeting siRNA (Dharmacon) or control nontargeting shRNA plasmid (sc‐108060; Santa Cruz Biotechnology) was used as a control for siRNA or shRNA experiments.

Techniques: Immunofluorescence, Control, Western Blot, Transfection, Expressing

Journal: bioRxiv

Article Title: PIGBOS-CLCC1 Interaction Shapes Cellular Calcium Dynamics and Energy Metabolism

doi: 10.64898/2026.01.08.697870

Figure Lengend Snippet: (A) Western blot showing reduced PIGBOS expression following siRNA-mediated knockdown in HEK293T cells. VDAC was used as a loading control. Quantification from three independent experiments indicates an approximately 70% decrease in PIGBOS protein levels. (B) Knockdown of PIGBOS attenuates the cytosolic Ca 2+ response to histamine stimulation. Bar graph shows the mean fluorescence ratio (F/F 0 ) from 64-66 cells measured using jGCaMP7s. (C) Representative confocal images showing reduced basal ER Ca 2+ levels in PIGBOS knockdown cells compared with scramble siRNA controls. The violin plot (right) summarizes data from 550-635 cells measured using G-CEPIA1-SNAP ER . (D) Histamine-induced ER Ca 2+ release is diminished in PIGBOS knockdown cells ( n = 80) relative to scramble controls ( n = 114). (E) Mitochondrial Ca 2+ uptake following histamine stimulation is reduced in PIGBOS knockdown cells ( n = 63) compared with scramble controls ( n = 86), measured using G-CEPIA1-2mt. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired t test).

Article Snippet: For knockdown experiments, CLCC1 shRNA (Sigma-Aldrich #TRCN0000257146) and a scrambled shRNA control (Addgene #1864) were used.

Techniques: Western Blot, Expressing, Knockdown, Control, Fluorescence

Journal: bioRxiv

Article Title: PIGBOS-CLCC1 Interaction Shapes Cellular Calcium Dynamics and Energy Metabolism

doi: 10.64898/2026.01.08.697870

Figure Lengend Snippet: (A) Schematic of the experimental protocol for assessing ER-independent mitochondrial Ca 2+ uptake in digitonin-permeabilized cells. Sequential steps ( – ): thapsigargin (TG) inhibits SERCA, triggering ER Ca 2+ release through leak channels ; plasma membrane permeabilization allows EGTA to chelate residual cytosolic Ca 2+ ; reintroduction of Ca 2+ -containing buffer enables mitochondrial uptake; mitochondrial Ca²⁺ rise is measured using the targeted indicator GCEPIA1-2mt. (B) Mitochondrial Ca 2+ uptake from the extracellular buffer is unchanged upon PIGBOS overexpression ( n = 16 cells). (C) ER membrane depolarizes more rapidly after histamine stimulation in PIGBOS-overexpressing cells. t 1/2 indicates the time to 50% reduction in fluorescence. Right, quantification of ER membrane depolarization (PIGBOS-overexpressing, n = 60; control, n = 77). (D) Histamine-induced ER membrane depolarization is attenuated in PIGBOS-depleted cells ( n = 45) relative to scrambled controls ( n = 56). (E) FCCP-induced mitochondrial depolarization monitored by TMRE fluorescence. PIGBOS-overexpressing cells ( n = 59) show greater FCCP-induced decay than controls ( n = 117), indicating higher basal mitochondrial membrane potential. (F) PIGBOS knockdown decreases mitochondrial depolarization compared with scrambled siRNA controls (PIGBOS-depleted, n = 76; control, n = 60), suggesting reduced mitochondrial membrane potential. Data are mean ± SD. n.s., not significant; ***P < 0.001 (unpaired t test).

Article Snippet: For knockdown experiments, CLCC1 shRNA (Sigma-Aldrich #TRCN0000257146) and a scrambled shRNA control (Addgene #1864) were used.

Techniques: Clinical Proteomics, Membrane, Over Expression, Fluorescence, Control, Knockdown

Journal: bioRxiv

Article Title: PIGBOS-CLCC1 Interaction Shapes Cellular Calcium Dynamics and Energy Metabolism

doi: 10.64898/2026.01.08.697870

Figure Lengend Snippet: (A) Representative western blot showing reduced CLCC1 protein expression relative to vinculin following shRNA-mediated knockdown. The accompanying bar graph shows the densitometric quantification of CLCC1 protein levels ( N = 3). (B) ER Ca 2+ release following histamine stimulation. CLCC1 knockdown decreases histamine-induced ER Ca 2+ release compared with control cells. The bar graph summarizes data from 39 control and 50 knockdown cells. (C) Basal ER Ca 2+ levels imaged with GCEPIA1-SNAP ER . Representative confocal images of scramble shRNA-transfected control and CLCC1-knockdown cells are shown. The violin plot summarizes quantified fluorescence intensity from 387-419 cells of control and knock-down cells, respectively, indicating a significant reduction in basal ER Ca 2+ content in CLCC1-depleted cells. (D) ER membrane potential changes in response to histamine. CLCC1-deficient cells ( n = 32) show a smaller histamine-induced ER voltage change than scramble controls ( n = 25), as summarized in the bar graph. (E) Mitochondrial Ca 2+ dynamics following histamine treatment. Histamine-induced mitochondrial Ca 2+ increase is significantly lower in CLCC1-knockdown cells ( n = 25) compared with control cells ( n = 31). Data are presented as mean ± SD. Statistical significance was determined using an unpaired t-test: n.s., not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: For knockdown experiments, CLCC1 shRNA (Sigma-Aldrich #TRCN0000257146) and a scrambled shRNA control (Addgene #1864) were used.

Techniques: Western Blot, Expressing, shRNA, Knockdown, Control, Transfection, Fluorescence, Membrane