Electronic Supplemental Material (ESM)

ESM Table 1 Primer sequences for quantitative RT-PCR and siRNA sequences

Primer sequences
Gene / Forward (5′–3′) / Reverse (5′–3′)
Human spliced/active XBP1 / CCGCAGCAGGTGCAGG / GAGTCAATACCGCCAGAATCCA
Human CHOP / Quiagen quantitect primer 0002278
Human BIP / Quiagen quantitect primer 00096404
Human GAPDH / CAGCCTCAAGATCATCAGCAA / TGTGGTCATGAGTCCTTCCA
Rat CHOP / CCAGCAGAGGTCACAAGCAC / CGCACTGACCACTCTGTTTC
Rat GADD34 / AGGGATGTGGAGAAGCAGAG / AAAGATCTGAGCCGCTTCTG
Rat NOS2 / GGGAGCCAGAGCAGTACAAG / GGCTGGACTTCTCACTCTGC
Rat FAS / GGAGGAGTACACGGACAGGA / TTTCTTTGCACCTGCACTTG
Rat CCL5 / CCAGAGAAGAAGTGGGTTCA / AGCAAGCAATGACAGGAAAG
Rat CXCL10 / GGGTAAAGGGAGGTGGAGAG / GGGTAAAGGGAGGTGGAGAGA
Rat IL15 / ATGTAAGATACGATCTGGAG / AGTCATGTTACTGTACTCGTG
Rat IRF7 / GGCAAGTGCAAGGTGTACTG / GCCCAAAACCCAGGTAGA
Rat Bcl-2 / CATGCGACCTCTGTTTGA / GTTTCATGGTCCATCCTTG
Rat Mcl-1 / CCTCCAGCCACCAACTACAT / CCACTTTCTTTCTGCCGTGTTA
Rat Gapdh / AGTTCAACGGCACAGTCAAG / TACTCAGCACCAGCATCACC
Rat_USPX9 / GAAGGGGTGCCTACCTCAA / GCCTTCTACACCTGGCTGAC
Rat_FBXW11 / GGTTCCAGATGCTGGGTAGA / CTCGCAGGGATGAGCTAAGA
Rat βTRC
/ TCAGGCTGTGGGACATAGAG
/ GGTCCAGAGCAGCCATAAGA
Rat-FBW7 / GCTGGAGTGGACCAGAGAAG / GGGGAGCAAGGAGATGAAGT
Rat-MULE / GCCCTTCGCTTCCTTGTAG / GCACAGAATGGCCAGCATAG
Rat A20 / TGCTACGACACTCGGAACTG / GGGTCTTCTGAGGATGTTGC
RatPUMA / AGTGCGCCTTCACTTTGG / CAGGAGGCTAGTGGTCAGGT
RatDP5 / GCCGTGGTGTTACTTGGA / GATTGTGCCAGAGCTTCACA
RatBim / GTCTTCCGCCTCTCGGTAAT / AGAGATACGGATCGCACAGG
siRNAs sequences
rat CHOP Ambion SMARTpool siRNA#1 / GGAAGAACUAGGAAACGGA
rat CHOP Ambion SMARTpool siRNA#2 / GGGCUCUGAUCGACCGCAU
rat CHOP Ambion SMARTpool siRNA#3 / CUGAAGAGAACGAGCGGCU
rat CHOP Ambion SMARTpool siRNA#4 / ACGAGGAAAUCGAGCGCCU
rat CHOP Silencer® Select pre-designed siRNA Ambion / GGAAGAACUAGGAAACGG
Human CHOP siRNA#1 Ambion s3995 / GUCCUGUCUUCAGAUGAAtt
Human CHOP siRNA#1 Ambion s225792 / GCACAGCUAGCUGAAGAGtt

ESM Table 2: IL-1β+IFNγ stimulate the mRNA expression of the E3 ubiquitin ligases FBW7, FBW11, β-TRC and USP9X in INS-1E cells. Knocking-down CHOP has no impact on the mRNA expression of those genes.

INS-1E cells were transfected with siCtrl or siCHOP#1 and treated for 15 h with IL-1β+IFN-γ or TNF-α+IFN-γ. Real-time PCR analyses of FBW7, Mule, βTRC and USP9X over GAPDH mRNA expression. Data are means ± SEM of 3-4 independent experiments. *P<0.05 vs. control (ctrl).

INS-1E cells / FBW 7 / FBW 11 / Mule / β-TRC / USP9X
15h / Control / siCTRL / 0.59±0.09 / 0.37±0.08 / 0.61±0.11 / 0.44±0.05 / 0.42±0.09
siCHOP / 0.42±0.06 / 0.43 ±0.11 / 0.38±0.03 / 0.34±0.02 / 0.38±0.03
IL-1β+IFNγ / siCTRL / 0.79±0.06** / 0.88±0.06** / 0.70±0.07 / 0.88±0.07* / 0.83±0.06*
siCHOP / 0.65±0.10 / 0.62±0.12 / 0.57±0.05 / 0.82±0.08 / 0.61±0.08
TNFα+IFNγ / siCTRL / 0.86±0.11 / 0.65±0.11 / 0.87±0.07 / 0.81±0.07* / 0.84±0.08
siCHOP / 0.70±0.23 / 0.69±0.09 / 0.68±0.22 / 0.51±0.21 / 0.70±0.23

Figure S1. CHOP silencing prevents cytokine-induced expression of CHOP and the CHOP target gene GADD34.

(A) Time-course experiment of CHOP protein expression in INS-1E cells treated with IL-1β+IFN-γ (black diamonds) or TNF-α+IFN-γ (white squares). Data are means ± SEM of at least 5 independent Western blots. (B) Quantitative assessment of 5 independent Western blots of CHOP over tubulin expression in INS-1E cells untransfected (NT) or transfected with a control siRNA (siCtrl) or two different CHOP siRNA (siCHOP#1 and #2) and treated or not (black bars) for 15 h with CPA (white bars). (C-D) INS-1E cells were transfected with siCtrl or siCHOP#1 and treated for 15 h with IL-1β+IFN-γ or TNF-α+IFN-γ. Real-time PCR analyses of CHOP (C) and GADD34 (D) over GAPDH mRNA expression. Data are means ± SEM of 4 independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control (ctrl). # P<0.05, ##P<0.01 vs. respective untransfected and siCtrl-transfected condition.

Figure S2. TNF-α+IFN-γ induces a stronger ER stress response in human islets than IL-1β+IFN-γ.

(A-B) Time-course analyzes of BiP (A) and XBP-1s (B) in human islets exposed to TNF-α+IFN-γ (squares) or IL-1β+IFN-γ (circles). Data are means ± SEM of 3 to 5 independent experiments. *P<0.05, **P<0.01 vs. control (ctrl).

Figure S3. CHOP knock down does not prevent late cytokine-induced β-cell apoptosis.

(A) INS-1E cells were untransfected (NT, black bars), transfected with siCtrl (stripe bars), siCHOP#1 (white bars), or siCHOP#2 (dotted bars) and treated for 24 h with IL-1β+IFN-γ or TNF-α+IFN-γ. (B) FACS-purified primary β-cells transfected with the siCtrl (black bars) or siCHOP#1 (white bars) and treated for 48 h with IL-1β+IFN-γ or TNF-α+IFN-γ. (A-B) Prevalence of apoptosis was evaluated by HO-PI staining. Data are means ± SEM of at least 4 independent experiments expressed as the percentage of apoptotic cells over the total number of cells counted. *P<0.05, **P<0.01, ***P<0.001 vs. control (Ctrl).

Figure S4. Cytokine-induced CHOP overexpression is partly JNK-dependent but CHOP knockdown has no impact on JNK activity.

(A) Western blot analysis of CHOP and tubulin in INS-1E cells treated or not (0) for 8, 16 or 24h with cytokines (IL-1β+IFN-γ), in presence of the JNK inhibitor SP600125 (SP600). Upper panel: representative WB. Lower panel: Quantitative assessment of 4 independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. control (ctrl). # P<0.05 vs. respective non-treated (no JNKi) condition. (B) INS-1E cells were transfected with siCtrl or siCHOP#1 and treated or not (0) for 15min, 1h, 4h, 8h or 16h with cytokines (IL-1β+IFN-γ). Western blot analysis of P-JNK, total JNK, CHOP and tubulin. Upper left panel: representative WB. Other panels: Quantitative assessment of 4 independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. respective non-treated condition. ### P<0.001 vs. respective siCtrl transfected condition.

Figure S5. CHOP knockdown does not modify β-cell function.

(A) INS-1E cells were transfected or not (NT) with a control siRNA (siCtrl) or the CHOP siRNA #1 (siCHOP#1). Insulin secretion (expressed in µg/103 cells) was measured by ELISA 48 h later in presence of 1.67mM (black bars) or 16.7mM (white bars) glucose. (B) Transfected INS-1E cells were treated or not for 15 h with IL-1β+IFN-γ prior to the insulin secretion test at 1.67mM (black bars) or 16.7mM glucose (white bars). Data are expressed as the secreted over insulin content. (A-B) Data are means ± SEM of 4 independent experiments. *P<0.05; **P<0.01; ***P<0.001 vs. respective basal secretion level at 1.67mM glucose. # P<0.01 vs. respective non-treated condition.

Figure S6. CHOP knockdown has no effect on the mRNA expression of the Bcl-2 family genes.

INS-1E cells were transfected with siCtrl or siCHOP#1 and treated for 8 (A, C, E) or 15 h (B, D, F, G-H) with IL-1β+IFN-γ or TNF-α+IFN-γ, as indicated. Real-time PCR analyses of BIM (A-B), DP-5 (C-D), PUMA (E-F), Mcl-1 (G) and Bcl-2 (H) over GAPDH mRNA expression. Data are means ± SEM of 4 independent experiments.

Figure S7. CHOP knockdown does not modify eIF2α phosphorylation.

INS-1E cells were transfected with siCtrl or siCHOP#1 and treated for 15 h with IL-1β+IFN-γ, TNF-α+IFN-γ, or the chemical ER stressors CPA and thapsigargin. WB analyses of CHOP, P-eIF2α and tubulin reveals that knocking-down CHOP doesn’t affect cytokine-induced P-eIF2α. A: representative WB. B: quantitative assessment of 4 independent experiments.

Figure S8. CHOP knockdown does not modify the 20s proteasome activity

INS-1E cells were transfected with siCtrl or siCHOP#1 and treated for 8 (A) or 15 h (B) with IL-1β+IFN-γ or TNF-α+IFN-γ, as indicated. Data are means ± SEM of 3 independent experiments.

FigureS9. CHOP knockdown mitigates cytokine-induced expression of NF-κB dependent genes and NO release in INS-1E cells.

(A-D) INS-1E cells were transfected with siCtrl or siCHOP#1 and treated for 8 with IL-1β+IFN-γ or TNF-α+IFN-γ. Real-time PCR analyzes of NOS2 (A), FAS (C) andCCL5 (D)over GAPDH mRNA expression. (B) NO release was quantified in the supernatant of the INS-E cells. Data are means ± SEM of 4 independent experiments. # P<0.01, ##P<0.001 vs. respective siCtrl-transfected condition.